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Aromatic Hydrocarbon Receptor(AhR)⅐AhR NuclearTranslocator-and p53-mediated Induction of theMurine Multidrug Resistance mdr1Gene by3-Methylcholanthrene and Benzo(a)pyrene in Hepatoma Cells*Received for publication,September18,2000,and in revised form,November10,2000Published,JBC Papers in Press,November28,2000,DOI10.1074/jbc.M008495200Marie-Claude Mathieu,Isabelle Lapierre,Karine Brault‡,and Martine Raymond§From the Institut de Recherches Cliniques de Montre´al,Montre´al,Que´bec H2W1R7,CanadaThe mouse multidrug resistance gene family consists of three genes(mdr1,mdr2,and mdr3)encoding P-gly-coprotein.We show that the expression of mdr1is in-creased at the transcriptional level upon treatment of the hepatoma cell line Hepa-1c1c7with the polycyclic aromatic hydrocarbon3-methylcholanthrene(3-MC). This increase is not observed in the aromatic hydrocar-bon receptor(AhR)-defective TAOc1BP r c1and the AhR nuclear translocator(Arnt)-defective BP r c1variants, demonstrating that the induction of mdr1by3-MC re-quires AhR⅐Arnt.We show that the mdr1promoter (؊1165to؉84)is able to activate the expression of a reporter gene in response to3-MC in Hepa-1c1c7but not in BP r c1cells.Deletion analysis indicated that the re-gion from؊245to؊141contains cis-acting sequences mediating the induction,including a potential p53bind-ing sequence.3-MC treatment of the cells increased the levels of p53and induced p53binding to the mdr1pro-moter in an AhR⅐Arnt-dependent manner.Mutations in the p53binding site abrogated induction of mdr1by 3-MC,indicating that p53binding to the mdr1promoter is essential for the induction.Benzo(a)pyrene,a polycy-clic aromatic hydrocarbon and AhR ligand,which,like 3-MC,is oxidized by metabolizing enzymes regulated by AhR⅐Arnt,also activated p53and induced mdr1tran-scription.2,3,7,8-Tetrachlorodibenzo-p-dioxin,an AhR ligand resistant to metabolic breakdown,had no effect. These results indicate that the transcriptional induc-tion of mdr1by3-MC and benzo(a)pyrene is directly mediated by p53but that the metabolic activation of these compounds into reactive species is necessary to trigger p53activation.The ability of the anticancer drug and potent genotoxic agent daunorubicin to induce mdr1independently of AhR⅐Arnt further supports the proposition that mdr1is transcriptionally up-regulated by p53in response to DNA damage.Multidrug resistance(MDR)1is characterized by cross-resis-tance of the cells to a large number of structurally and func-tionally unrelated cytotoxic agents used in chemotherapy.In cultured cells,MDR is frequently caused by the overexpression of P-glycoprotein(Pgp),an integral membrane protein belong-ing to the ATP-binding cassette superfamily of transporters and which functions as an energy-dependent efflux pump of cytotoxic drugs(1,2).Pgp is encoded by a small family of genes with two members in humans(MDR1and MDR2/MDR3)and three in rodents(mdr1/mdr1b,mdr2,and mdr3/mdr1a)(1,2). Only one human gene(MDR1)and two rodent genes(mdr1/ mdr1b and mdr3/mdr1a)can confer MDR upon overexpression in drug-sensitive cells(1,2).The different mdr genes and Pgp isoforms are expressed in a tissue-specific manner(1,2).In the mouse,mdr1is expressed mostly in the adrenal cortex,kidney,and pregnant uterus, mdr2in the liver at the canalicular face,and mdr3in the intestine and to a lesser extent in the heart,liver,lung,and capillaries of the brain(3).Pgps are localized on the apical membrane of epithelial cells lining luminal spaces,suggesting that they function in normal tissues as transporters of toxic substances and/or specific endogenous cellular products(4). Knockout mice experiments have demonstrated a role for the mdr3gene in the maintenance of the blood-brain barrier and drug elimination and for the mdr2gene in the transport of phospholipids in the bile(5,6).No physiological function has been attributed to the mouse mdr1gene so far,since knockout mdr1(Ϫ/Ϫ)mice display no obvious physiological abnormali-ties(7).However,different experimental evidence indicates that Pgp encoded by mdr1can serve in the transport of steroids(8).A number of factors have been found to modulate the level of mdr gene expression in the liver.For example,high levels of MDR1RNA have been found in human hepatocarcinomas,and overexpression of the mdr1isoforms has also been observed in rodent liver during cholestasis,during regeneration following partial hepatectomy,during chemically induced hepatocarcino-genesis,and following administration of various natural and synthetic xenobiotics(1,2).In particular,it has been shown that expression of the rat mdr1b gene is increased in liver cells in response to treatment with various polycyclic aromatic hy-*This work was supported by a research grant from the Cancer Research Society Inc.(to M.R).The costs of publication of this article were defrayed in part by the payment of page charges.This article must therefore be hereby marked“advertisement”in accordance with18 U.S.C.Section1734solely to indicate this fact.‡Supported by a studentship from the Medical Research Council ofCanada.Present address:Dept.of Biological Sciences,Bio-Mega Re-search Division,Boehringer Ingelheim(Canada)Ltd.,Laval,Que´bec H7S2G5,Canada.§Supported by a scholarship from Le Fonds de la recherche en sante´du Que´bec.To whom correspondence should be addressed:Institut de recherches cliniques de Montre´al,110Pine Ave.W.,Montre´al,Que´bec H2W1R7,Canada.Tel.:514-987-5770;Fax:514-987-5764;E-mail: raymonm@ircm.qc.ca.1The abbreviations used are:MDR,multidrug resistance;Pgp,P-glycoprotein;3-MC,3-methylcholanthrene;B(a)P,benzo(a)pyrene; TCDD,2,3,7,8-tetrachlorodibenzo-p-dioxin;DN,daunorubicin;CAT, chloramphenicol acetyl transferase;AhR,aromatic hydrocarbon recep-tor;Arnt,AhR nuclear translocator;EMSA,electrophoretic mobility shift assay;DME,drug metabolizing enzymes;PAH polycyclic aromatic hydrocarbon;XRE,xenobiotic response element;bp,base pair(s);kb, kilobase pair(s).T HE J OURNAL OF B IOLOGICAL C HEMISTRY Vol.276,No.7,Issue of February16,pp.4819–4827,2001©2001by The American Society for Biochemistry and Molecular Biology,Inc.Printed in U.S.A.This paper is available on line at 4819 at ZHEJIANG UNIVERSITY, on November 21, Downloaded fromdrocarbon(PAH)compounds,including3-methylcholanthrene (3-MC),and that this increased expression occurs at the tran-scriptional level(9–11).However,the precise molecular mech-anisms involved in mdr1b regulation in response to3-MC are still unknown.PAHs are carcinogenic compounds arising from the incom-plete combustion of organic matter and are widespread in the environment,including tobacco smoke and tar.PAHs such as 3-MC and benzo(a)pyrene(B(a)P)as well as halogenated aro-matic hydrocarbons such as2,3,7,8-tetrachlorodibenzo-p-di-oxin(TCDD)are specific inducers of genes coding for drug-metabolizing enzymes(DME),including cyp1a1and cyp1a2, that code for cytochromes P450involved in metabolic oxidation (12).PAHs and TCDD bind in the cytoplasm to the aromatic hydrocarbon receptor(AhR),a member of the bHLH-PAS(basic helix-loop-helix Per-Arnt-Sim)family of transcription factors (12,13).The ligand-bound AhR translocates to the nucleus, where it binds as a heterodimer with the AhR nuclear trans-locator(Arnt;another bHLH-PAS protein)to specific cis-acting regulatory DNA sequences located in the promoter of its tar-gets(known as AH-,dioxin-,or xenobiotic-responsive elements (or AHRE,DRE,or XRE,respectively))to enhance their tran-scription(12,13).Given that mdr1b expression is increased in liver cells in response to treatment with various PAHs,it was postulated that mdr1b may be under the control of the AhR(9). However,studies failing to show mdr1induction in the liver of mice treated with TCDD,one of the most potent agonists of the AhR,suggested that mdr1expression was not regulated by AhR(14).The involvement of AhR in the regulation of mdr1 has so far remained controversial.The mouse hepatoma cell lines Hepa-1c1c7(wild type), TAOc1BP r c1(AhR-defective),and BP r c1(Arnt-defective)con-stitute a powerful experimental system to investigate the tran-scriptional regulation of different AhR⅐Arnt targets in response to xenobiotics(12).The two mutant cell lines were derived as B(a)P-resistant variants of Hepa-1c1c7and were identified based on their inability to induce aryl hydrocarbon hydroxylase activity in response to TCDD treatment(15).TAOc1BP r c1cells have a decreased level of AhR(ϳ10%of wild-type cells)and therefore decreased induction of the cyp1a1promoter and lower aryl hydrocarbon hydroxylase activity in response to TCDD and other AhR ligands(15–18).BP r c1cells have a nor-mal cytosolic AhR,which fails to accumulate in the nucleus because of a defective Arnt(15).They have virtually no basal or inducible levels of cyp1a1expression and aryl hydrocarbon hydroxylase activity(15–17).In the present report,we have used this panel of cell lines to investigate the transcriptional regulation of the murine mdr1 gene by3-MC and other xenobiotic compounds.Our results demonstrate that mdr1is transcriptionally induced by3-MC and B(a)P and that this induction is mediated by p53but also requires AhR⅐Arnt.A model for the AhR⅐Arnt-and p53-medi-ated transactivation of mdr1in response to genotoxic stress is proposed.EXPERIMENTAL PROCEDURESCell Culture—Wild-type Hepa-1c1c7and Hepa1–6,AhR-defective TAOc1BP r c1,and Arnt-defective BP r c1cells were obtained from the American Type Culture Collection(ATCC;Manassas,VA)and main-tained in culture under the conditions recommended by the ATCC. Chinese hamster ovary LR73cell lines stably transfected with plasmid constructs carrying full-length cDNAs for the mouse mdr1,mdr2,or mdr3genes(LR73mdr1,LR73mdr2,and LR73mdr3,respectively;a gift from Dr.Philippe Gros,McGill University,Montre´al,Canada)were grown as described elsewhere(19,20).For inductions,cells atϳ50% confluence were exposed to different concentrations of xenobiotics for various periods of time(the exact conditions for each experiment are indicated in the figure legends).3-MC,B(a)P,and daunorubicin were obtained from Sigma,and TCDD was obtained from the Centre d’expertise en analyze environnementale du Que´bec(Laval,Canada).Stock solutions of3-MC(5m M)and B(a)P(25m M)were prepared in Me2SO,and the stock solutions of daunorubicin(1mg/ml)were pre-pared in water.TCDD was obtained in n-nonane at a concentration of 50␮g/ml and was stored at room temperature.Stock solutions of3-MC, B(a)P,and daunorubicin were stored atϪ80°C.RNA Preparation—Total RNA was prepared from3-MC-treated and untreated hepatocytes as well as from the LR73mdr1,LR73mdr2,and LR73mdr3cell lines by homogenizing the cells in a solution containing guanidium hydrochloride(6M)followed by sequential ethanol precipi-tation,as described previously(21).RNase Protection Assay—The plasmid constructed to detect the mdr1 RNA consisted of a165-bp Bam HI fragment isolated from the mdr1 cDNA(positions1926–2090relative to the ATG initiation codon(22)), blunt-ended with T4DNA polymerase,and cloned into plasmid pGEM-7Z(Promega,Madison,WI)at the Sma I site,giving plasmid pmdr1-G7.This plasmid was linearized with Eco RI and used as a template to synthesize an antisense mdr1probe using SP6RNA polym-erase(Amersham Pharmacia Biotech).The pKX10–3Z plasmid consist-ing of an Xba I–Kpn I mouse␤-actin cDNA fragment(positions724–969 in the␤-actin cDNA)cloned into pGEM-3Z at the Xba I and Kpn I sites (kindly provided by Dr.Rashmi Kothary,Institut du cancer de Mon-tre´al,Montre´al,Canada)was used to generate a control actin probe. pKX10–3Z was linearized with Xba I and used to synthesize an anti-sense actin RNA probe with T7RNA polymerase.The riboprobes were synthesized in the presence of[␣-32P]UTP,and the RNase protection assay was performed according to standard protocols(23).Nuclear Run-on Transcription Assay—The run-on experiment was performed essentially as described by Fisher et al.(24).Nuclei wereisolated from Hepa-1c1c7cells treated with Me2SO or with3-MC(5␮M) for48h and were used to label nascent RNAs with[␣-32P]UTP.Plas-mids pVT101-U/mdr1,carrying the full-length mouse mdr1cDNA(25); pmP1450–3Ј,carrying a1.2-kb Pst I cDNA fragment overlapping part of the mouse cyp1a1cDNA(26)(obtained from the ATCC);and pKX10–3Z were linearized with Stu I,Bam HI,and Xba I,respectively.The linear-ized plasmids were denatured,immobilized in duplicate onto a nylon membrane,and hybridized with the[␣-32P]UTP-labeled RNAs for48h at65°C.The membranes were washed and exposed for7days with two intensifying screens.Slot Blot Analyses—Slot blotting was performed as previously de-scribed(21).RNA samples(10␮g)were denatured in7ϫSSC-7.5% formaldehyde for15min at65°C and applied to a nylon membrane (Zeta-Probe).Detection of specific RNAs was performed by hybridiza-tion at65°C in0.5M NaPO4,pH7.2,1m M EDTA,7%SDS,1%bovine serum albumin,and100␮g/ml salmon sperm DNA with32P-labeled DNA probes.The mdr1probe was a4.2-kb Sph I–Eco RI fragment over-lapping the full-length mouse mdr1cDNA,isolated from plasmid pGEM7/mdr1(a gift from Dr.Philippe Gros,McGill University,Mon-tre´al);the cyp1a1probe was a 1.2-kb Pst I fragment isolated from plasmid pmP1450–3Ј;and the actin probe was a245-bp Xba I–Kpn I fragment isolated from pKX10–3Z.The membranes were washed twiceat65°C with a solution containing40m M NaPO4,pH7.2,5%SDS,1 m M EDTA,0.5%bovine serum albumin and twice with a solutioncontaining40m M NaPO4,pH7.2,5%SDS,and1m M EDTA before autoradiography.Chloramphenicol Acetyl Transferase(CAT)Expression Plasmids—Plasmid pMcat5.9consists of a482-bp DNA fragment containing the dioxin-responsive elements of the cyp1a1gene cloned upstream of the mouse mammary tumor virus promoter and the CAT gene(24)(kindly provided by Dr.Allan Okey,University of Toronto).Plasmids pmdr1, p-452,p-245,p-141,and p-93(previously referred to as pSacICAT, pExo6CAT,pExo2CAT,pExo1CAT,and pAluCAT,respectively)have been described elsewhere(27).The mdr1promoter sequence in these constructs ends at positionϩ84with respect to the transcription start site(27).To produce the p53mutant constructs,pM1and pM2,plasmid pSBM13was used.This plasmid consists of a1.2-kb Sac I–Hin dIII mdr1 promoter fragment(positionsϪ1165toϩ84)cloned into M13mp18. Single-stranded DNA was prepared from pSBM13and used as a tem-plate to perform site-directed mutagenesis of the p53binding site,using the mutant oligonucleotides M15Ј-TACCTGAA T AC A TAAAGACA and M25Ј-CGTAAAGA T AA A TCTATGTA(the base changes are shown in boldface type).The resulting M1and M2mdr1promoter fragments were then excised from pSBM13with Sac I and Hin dIII,blunt-ended with T4DNA polymerase,and cloned into plasmid pCAT at the Hin dIII site also blunt-ended with T4DNA polymerase,yielding plasmids pM1 and pM2.The presence of the mutations in the resulting constructs was confirmed by DNA sequencing.Transient Transfections and CAT Assays—Cells were plated at aInduction of the Mouse mdr1Gene by PAHs4820at ZHEJIANG UNIVERSITY, on November 21, Downloaded fromdensity of 8ϫ105/60-mm plate and transfected on the following day with 10␮g of plasmid DNA,using a standard calcium phosphate pre-cipitation method (28).After incubation with the DNA precipitate for 16h,the cells were washed twice with phosphate-buffered saline and supplied with fresh medium containing the different xenobiotics.After 48h,the cells were collected.Cell extracts were prepared,and protein concentrations were determined by the Bradford method (29).CAT activities were assayed by standard protocols as described previously,using 2␮g of proteins (27).Preparation of Nuclear Extracts—Nuclear extracts were prepared according to Schreiber et al .(30),with some modifications.Cells were harvested in cold phosphate-buffered saline,0.6m M EDTA and col-lected by centrifugation.The cell pellets were resuspended in 400␮l of ice-cold buffer A (10m M Tris,pH 8.0,10m M KCl,0.1m M EDTA,0.1m M EGTA,1m M dithiothreitol)containing 0.5m M phenylmethylsulfonyl fluoride,10␮g/ml aprotinin,1␮g/ml pepstatin,and 5␮g/ml leupeptin and swelled on ice for 15min.Subsequently,25␮l of 10%Nonidet P-40were added,and the tubes were vortexed vigorously.The nuclear pellets were collected by centrifugation and resuspended in 100␮l of cold buffer C (20m M Tris,pH 8.0,400m M NaCl,1m M EDTA,1m M EGTA,1m M dithiothreitol)in the presence of protease inhibitors.The suspen-sions were shaken vigorously at 4°C for 1h and centrifuged for 15min at 4°C,and the supernatants were frozen in aliquots at Ϫ80°C.Proteinconcentrations were determined by the Bradford method (29).ElectrophoreticMobility Shift Assay—Oligonucleotides overlapping the potential p53binding site in the mdr1promoter (5Ј-GAACACGTA-AAGACAAGTCTAT)and the p53consensus sequence in the p21waf1/cip1promoter (5Ј-GAACATGTCCCAACATGTTGAG)(31)were end-labeled with ␥-32P using T4polynucleotide kinase and annealed to their respec-tive in a M M 2.5m M dithiothreitol,4%Ficoll,1␮g of poly(dI-dC),and 20,000cpm of radiolabeled probe.The binding reactions were carried out at room temperature for 15min.Where needed,1␮g of the monoclonal anti-p53antibody pAb421(32)(Calbiochem)or of the polyclonal anti-Jun or anti-Skn-1antibodies (Santa Cruz Biotechnology,Inc.,Santa Cruz,CA)was added,and the incubation was continued for an additional 15min.The complexes were separated on 5%nondenaturing polyacrylamide gels in 1ϫTBE (90m M Tris,65m M boric acid,2.5m M EDTA,pH 8.0)at 200V.The gels were exposed to XAR films (Eastman Kodak Co.)for 16h with two intensifying screens at Ϫ80°C.Western Blotting—Total proteins from 3-MC-or Me 2SO-treated Hepa-1c1c7and BP r c1cells were extracted in ice-cold buffer (10m M Tris-HCl,pH 8.0,150m M NaCl,1m M EDTA,1%Nonidet P-40,and 1%sodium deoxycholate)containing 10␮g/ml leupeptin,10␮g/ml aproti-nin,1␮M sodium orthovanadate,and 1m M phenylmethylsulfonyl flu-oride.Total proteins (75␮g/sample)or nuclear extracts (30␮g/sample)were separated by SDS-polyacrylamide gel electrophoresis on a 10%acrylamide gel,transferred to a nitrocellulose membrane,and analyzed with the monoclonal anti-p53antibody pAb421(32)(Calbiochem)at a concentration of 5␮g/ml.Immune complexes were revealed by incuba-tion with a goat anti-mouse IgG antibody coupled to alkaline phospha-tase (Bio-Rad)and developed with 5-bromo-4-chloro-3-indolylphosphate p -toluidine salt and nitro blue tetrazolium chloride substrates as rec-ommended by the manufacturer (Life Technologies,Inc.).RESULTSTranscriptional Induction of the Mouse mdr1Gene by 3-MC in Hepatoma Cells—We have used an RNase protection assay to study the expression of mdr1in the hepatoma cell line Hepa-1c1c7upon exposure to 3-MC (Fig.1).An mdr1-specific riboprobe was prepared by cloning into pGEM7-Zf a mouse mdr1cDNA fragment overlapping the linker region of the protein,this domain displaying the lowest sequence homology among the three mouse mdr cDNAs (21).When tested with RNA prepared from LR73stable transfectants expressing each of the three mouse mdr cDNAs,the mdr1riboprobe was found to recognize the mdr1RNA but not the mdr2or mdr3RNA,thus confirming its specificity (Fig.1,top right ).The mdr1probe was then used with RNA from Hepa-1c1c7cells treated or not with 3-MC (Fig.1,top left ).This experiment showed that the amount of mdr1RNA detected is very low in untreated cells but is strongly increased in 3-MC-treated cells,demonstrating that expression of the mouse mdr1gene is induced by 3-MCtreatment.The use of an actin probe confirmed that equal quantities of RNA were used in the assay (Fig.1,bottom ).A similar experiment performed with mdr2-and mdr3-specific riboprobes showed that the expression of these genes is not induced under such conditions,demonstrating that the induc-tion of mdr1expression by 3-MC is isoform-specific (data not shown).A nuclear run-on experiment was performed to determine whether mdr1induction by 3-MC occurs at the transcriptional level (Fig.2).In addition to the mouse mdr1cDNA,cDNAs for the mouse cyp1a1gene (known to be transcriptionally regu-lated by 3-MC (12))and for the actin gene were also included as positive and negative controls,respectively.The data in Fig.2show that 3-MC induces an increase in the rate of mdr1mRNA synthesis,indicating that 3-MC acts at the transcriptional level to induce mdr1gene expression in Hepa-1c1c7cells.AhR ⅐Arnt-dependent Induction of mdr1Expression by 3-MC—To determine whether the increase in mdr1expression in response to 3-MC exposure is AhR ⅐Arnt-mediated,we ana-lyzed the mdr1RNA levels upon 3-MC treatment in two wild-type hepatoma cell lines Hepa-1c1c7and Hepa 1–6and in two variant cell lines derived from Hepa-1c1c7,TAOc1BP r c1(AhR-defective)and BP r c1(Arnt-defective)(15)(Fig.3).As controls,we also analyzed the level of cyp1a1and actin expression under the same conditions (Fig.3,middle and right ,respectively).This experiment showed that mdr1is expressed at low levels in the four cell lines in the absence of 3-MC induction (Fig.3,left panel ).Upon 3-MC treatment,the expression of mdr1is in-duced in the two wild-type hepatoma cell lines (by ϳ5-fold),this induction being completely abrogated in the AhR-defective or in the Arnt-defective variants (Fig.3,left panel ).The actin control probe confirmed that equal amounts of RNA had been applied to the membrane (Fig.3,right panel ).These data clearly demonstrate that the induction of mdr1in response to 3-MC requires an intact AhR ⅐Arnt complex,like cyp1a1(Fig.3,middle )(12).The Mouse mdr1Promoter Confers 3-MC-regulated Expres-sion in an AhR ⅐Arnt-dependent Manner—To determine if reg-ulatory sequences responsible for mdr1induction by 3-MC are present in the promoter region of the gene,plasmid pmdr1,consisting of a 1.2-kb Sac I–Hin dIII DNA fragment overlapping the mdr1promoter region (positions Ϫ1165to ϩ84with respect to the transcription start site (27))fused to the CAT reporter gene,was analyzed in transient transfection experiments.Plasmid pMcat5.9,which consists of a 482-bp fragment derived from the cyp1a1promoter fused to the mouse mammary tumorF IG .1.Increased mdr1expression in Hepa-1c1c7upon 3-MC treatment.The expression of mdr1was analyzed by RNase protection assay.Total RNAs (45␮g)from Hepa-1c1c7cells treated with 5␮M 3-MC (ϩMC )or with Me 2SO (ϪMC )for 56h and from the control cell lines LR73/mdr1,LR73/mdr2,and LR73/mdr3were analyzed with an mdr1riboprobe,which protects a 169-nt fragment within the mdr1transcript,or with a ␤-actin riboprobe,which protects a 245-nt actin transcript fragment.Autoradiography was for 15h with two intensify-ing screens (mdr1)or for 5h without intensifying screens (actin ).Induction of the Mouse mdr1Gene by PAHs4821at ZHEJIANG UNIVERSITY, on November 21, 2012 Downloaded fromvirus promoter and to the CAT gene (24),as well as the empty pCAT vector were also included as positive and negative con-trols,respectively.The three plasmids were transiently trans-fected into Hepa-1c1c7and BP r c1cells.The cells were treated with 3-MC or with Me 2SO for 48h,and the cellular extracts were prepared and assayed for CAT activity.This experiment showed that the mdr1promoter is transcriptionally active in Hepa-1c1c7cells and BP r c1cells,since it can drive the expres-sion of the CAT gene in both cell lines,albeit at low levels (Fig.4).This result is consistent with the basal level of expression of mdr1detected by slot blot analysis in these cells (Fig.3).3-MC treatment of the Hepa-1c1c7cells transfected with pmdr1re-sulted in a 10-fold induction in CAT activity as compared with untreated cells,reaching levels of CAT activity similar to those detected in the Hepa-1c1c7pMcat5.9transfectants upon 3-MC treatment.However,this induction was completely abrogated in BP r c1cells (Fig.4),consistent with the lack of mdr1induc-tion at the RNA level observed in the slot blot assay (Fig.3).Similar results were obtained upon transfection in TAOc1BP r c1cells (data not shown).These results,showing that the mdr1promoter is able to activate the expression of the reporter gene in response to 3-MC in Hepa-1c1c7but not in BP r c1and TAOc1BP r c1cells,demonstrate that (i)the mdr1promoter is able to confer 3-MC-mediated transcriptional acti-vation;(ii)this activation requires a functional AhR ⅐Arnt com-plex;and (iii)the sequences mediating this induction are lo-cated between positions Ϫ1165and ϩ84in the mdr1promoter.Two Putative XREs Located in the mdr1Promoter Are Dis-pensable for the Induction of mdr1by 3-MC—The AhR ⅐Arnt transcriptional complex binds to a specific DNA sequence,5Ј-(A/T)NGCGTG,known as an XRE to activate transcription (12).XREs render heterologous promoters responsive to xeno-biotics and function in a position-and orientation-independent manner (33,34).Examination of the mdr1promoter sequence indicated the presence of two potential XREs in an inverted orientation in the distal portion of the promoter at positionsϪ1129and Ϫ620(5Ј-CACGCAT and 5Ј-CACGCAA,respective-ly).To identify the cis -acting sequences responsible for the induction of mdr1by 3-MC and to investigate the possible involvement of these putative XREs,we analyzed the tran-scriptional activity of a series of mdr1promoter 5Ј-deletion CAT constructs after transient transfection into Hepa-1c1c7and treatment of the resulting transfectants with 3-MC (Fig.5A ).3-MC treatment of Hepa-1c1c7cells transfected with plas-mids p-452or p-245resulted in a level of CAT induction similar to that observed in cells transfected with plasmid pmdr1car-rying the full-length promoter,indicating that sequences lo-cated within positions Ϫ1165to Ϫ245are dispensable for the induction of mdr1by 3-MC,including the two putative XREs as well as a potential AP-1binding site (5Ј-TGACTCA;positions Ϫ265to Ϫ255(35))(Fig.5,B and C ).However,further deletion of a 104-bp region down to position Ϫ141(p Ϫ141)was found to greatly diminish the induction of CAT activity by 3-MC (Fig.5,B and C ),demonstrating that sequences important for the induction are located between positions Ϫ245and Ϫ141.CAT activity in the absence of 3-MC was reduced in the p Ϫ141transfectants when compared with the p Ϫ245transfectants,indicating that sequences between positions Ϫ245and Ϫ141are also involved in the basal transcriptional activity of the mdr1promoter in hepatoma cells.Finally,we found that alowF IG .2.Nuclear run-on experiment.Nuclei were isolated from Hepa-1c1c7cells treated with 5␮M 3-MC (ϩMC )or with Me 2SO (ϪMC )for 48h.Nascent RNAs were radiolabeled with [␣-32P]UTP and used to probe duplicate nylon membranes on which denatured cDNAs for mdr1,cyp1a1,and actin had been immobilized.The membranes were washed and exposed for 7days with two intensifyingscreens.F IG .3.AhR ⅐Arnt-dependent induction of mdr1expression by 3-MC.Total RNAs (10␮g)from wild-type Hepa-1c1c7and Hepa 1–6,AhR-defective TAOc1BP r c1,and Arnt-defective BP r c1cells treated (ϩMC )or not treated (ϪMC )with 3-MC at 5␮M for 56h were applied onto a nylon membrane.The membrane was hybridized sequentially with an mdr1(left ),a cyp1a1(middle ),and a ␤-actin (right )probe.Autoradiography was for 18h (mdr1and cyp1a1)or for 2h (actin)with two intensifyingscreens.F IG .4.AhR ⅐Arnt-dependent induction of the mdr1promoter by 3-MC.Plasmids pCAT (no promoter),pmdr1(mdr1promoter from position Ϫ1165to ϩ84),and pMcat5.9(pMcat;482-bp fragment from the cyp1a1promoter fused to the mouse mammary tumor virus pro-moter)were transiently transfected into Hepa-1c1c7and BP r c1cells by the calcium phosphate method.The cells were then treated with 3-MC (5␮M )or Me 2SO for 48h.Total cellular extracts were prepared,and equal quantities of proteins (2␮g)were assayed for CAT activity.A ,autoradiogram of a representative CAT assay,showing the activity of plasmids pCAT,pmdr1and pMcat in Hepa-1c1c7and BP r c1cells treated (ϩ)or not treated (Ϫ)with 3-MC (MC ).The position of the [14C]chloramphenicol (CM )and of its acetylated products (AcCM )is indicated on the left .B ,quantitative analysis of CAT activities.The percentage of conversion of [14C]chloramphenicol to its acetylated de-rivatives was quantitated by liquid scintillation counting.Open bars ,ϪMC ;filled bars ,ϩMC .The results presented are the averages of three independent transfections performed in duplicate.S.D.values are rep-resented by the bars .Induction of the Mouse mdr1Gene by PAHs4822 at ZHEJIANG UNIVERSITY, on November 21, 2012 Downloaded from。

promega celltiter-fluor cell viability assay protocol

Promega Corporation ·2800 Woods Hollow Road ·Madison, W I 53711-5399 USA Toll Free in USA 800-356-9526·Phone 608-274-4330 ·Fax 608-277-2516 ·1.Description ..........................................................................................................12.Product Components and Storage Conditions ............................................43.Reagent Preparation and Storage ...................................................................54.Protocols for the CellTiter-Fluor™ Cell Viability Assay ..........................5A.Determining Assay Sensitivity, Method 1........................................................6B.Determining Assay Sensitivity, Method 2........................................................7C.Example Viability Assay Protocol.....................................................................8D.Example Multiplex Assay Protocol (with luminescent caspase assay).......9E.Recommended Controls (10)5.General Considerations ..................................................................................106.References .........................................................................................................117.Related Products ..............................................................................................121.DescriptionThe CellTiter-Fluor™ Cell Viability Assay (a)is a nonlytic, single-reagent-addition fluorescence assay that measures the relative number of live cells in a culture population after experimental manipulation (Figures 1 and 2). The CellTiter-Fluor™ Cell Viability Assay measures a conserved and constitutive protease activity within live cells and therefore serves as a marker of cell viability (1). Results obtained using the CellTiter-Fluor™ Cell Viability Assay correlate well with other established methods of determining cell viability(Figure 3). The live-cell protease activity is restricted to intact viable cells and is measured using a fluorogenic, cell-permeant, peptide substrate (glycyl-phenylalanyl-aminofluorocoumarin; GF-AFC). The substrate enters intact cells where it is cleaved by the live-cell protease activity to generate a fluorescent signal proportional to the number of living cells (Figure 4). This live-cell protease becomes inactive upon loss of cell membrane integrity and leakage into the surrounding culture medium.The CellTiter-Fluor™ Cell Viability Assay also can be used in a single-well,sequential, multiplex format with other downstream chemistries to normalize data by cell number. Data from the assay can serve as an internal control andCellTiter-Fluor™ Cell Viability AssayAll technical literature is available on the Internet at: /protocols/ Please visit the web site to verify that you are using the most current version of this Technical Bulletin. Please contact Promega Technical Services if you have questions on useof this system. E-mail: techserv@allow identification of errors resulting from cell clumping or compoundcytotoxicity. The CellTiter-Fluor™ Cell Viability Assay is compatible with most Promega luminescence assays or spectrally distinct fluorescence assay methods,such as assays measuring caspase activation, reporter gene expression or orthogonal measures of viability. However, some P450-Glo™ multiplexprotocols may require removing culture supernatant to a separate assay well before performing the assay because of isoform-specific competitive inhibition of the cytotochrome P450 enzymes by the coumarin product of the CellTiter-Fluor™ Cell Viability Assay reaction.Figure 1. Schematic diagram of the CellTiter-Fluor™ Cell Viability Assay.Promega Corporation ·2800 Woods Hollow Road ·Madison, W I 53711-5399 USA Toll Free in USA 800-356-9526·Phone 608-274-4330 ·Fax 608-277-2516 ·6868M ACellTiter-Fluor™ ReagentAdd GF-AFC Substrate to Assay Buffer to create the CellTiter-Fluor™ Reagent.MeasureAssay BenefitsMeasure the Relative Number of Live Cells in Culture: Nonlytic, single-reagent-addition, homogeneous, “add-mix-measure” protocol.Get More Data from Every Well: The CellTiter-Fluor™ Cell Viability Assay can be performed in multiplex with most Promega luminescence assays.Normalize Data for Cell Number: Normalizing data for live-cell number makes results more comparable well-to-well, plate-to-plate, day-to-day.Figure 3. The CellTiter-Fluor™ Cell Viability Assay shows strong correlation with established methods for measuring viability. Panel A. The GF-AFC Substrate signal from serial dilutions of live cells plotted against results from the CellTiter-Glo ®Luminescent Cell Viability Assay (Cat.# G7570), which measures cellular ATP.Panel B.The GF-AFC Substrate signal from serial dilutions of live cells plottedagainst results achieved using the CellTiter-Blue ®Cell Viability Assay (Cat.# G8080).Promega Corporation ·2800 Woods Hollow Road ·Madison, W I 53711-5399 USA Toll Free in USA 800-356-9526·Phone 608-274-4330 ·Fax 608-277-2516 ·6867M AA.B.R e s o r u f i n F l u o r e s c e n c e (R F U )2,5002,6002,7002,8002,9003,0003,100GF-AFC Fluorescence (RFU)A T P -B a s e d A s s a y L u m i n e s c e n c e (R L U )0GF-AFC Fluorescence (RFU)LIVE-CELL SUBSTRATE:cell-permeant fluorogenic substrate for the live-cellprotease (Gly-Phe-AFCoumarin)NucleusLive CellLive-cell protease substrate can cross the cell membrane.Active Live-CellProteaseO CF 3GF–N HOGFOCF 3OH 2N6995M AFigure 2. CellTiter-Fluor™ Cell Viability Assay chemistry. The cell-permeant substrate enters the cell, where it is cleaved by the live-cell protease activity to produce the fluorescent AFC. The live-cell protease is labile in membrane-compromised cells and cannot cleave the substrate.Figure 4. The CellTiter-Fluor™ Cell Viability Assay signal derived from viable cells (untreated) is proportional to cell number. Dead cells (treated) do not contribute appreciable signal in the assay.2.Product Components and Storage ConditionsProductSize Cat.#CellTiter-Fluor™ Cell Viability Assay10mlG6080Cat.# G6080 contains sufficient reagents for 100 assays at 100µl/assay in a 96-well plate format or 400 assays at 25µl/assay in a 384-well plate format. Includes:• 1 × 10ml Assay Buffer• 1 × 10µl GF-AFC Substrate (100mM in DMSO)ProductSize Cat.#CellTiter-Fluor™ Cell Viability Assay5 × 10mlG6081Cat.# G6081 contains sufficient reagents for 500 assays at 100µl/assay in a 96-well plate format or 2,000 assays at 25µl/well in a 384-well format. Includes:• 5 × 10ml Assay Buffer• 5 × 10µl GF-AFC Substrate (100mM in DMSO)ProductSize Cat.#CellTiter-Fluor™ Viability Assay2 × 50mlG6082Cat.# G6082 contains sufficient reagents for 1,000 assays at 100µl/assay in a 96-well plate format or 4,000 assays at 25µl/well in a 384-well format. Includes:• 2 × 50ml Assay Buffer• 2 × 50µl GF-AFC Substrate (100mM in DMSO)Storage Conditions: Store the CellTiter-Fluor™ Cell Viability Assaycomponents at –20°C. See product label for expiration date.Promega Corporation ·2800 Woods Hollow Road ·Madison, W I 53711-5399 USA Toll Free in USA 800-356-9526·Phone 608-274-4330 ·Fax 608-277-2516 ·6866M ACells or Cell Equivalents/WellA F C F l u o r e s c e n c e (R F U )3.Reagent Preparation and Storagepletely thaw the CellTiter-Fluor™ Cell Viability Assay components in a37°C water bath. Vortex the GF-AFC substrate to ensure homogeneity, thenbriefly centrifuge for complete substrate volume recovery.2.Transfer the GF-AFC Substrate (10µl for Cat.# G6080 and G6081; 50µl for Cat.#G6082) into the Assay Buffer container (10ml for Cat.# G6080 and G6081; 50mlfor Cat.# G6082) to form a 2X Reagent. Mix by vortexing the contents until thesubstrate is thoroughly dissolved.Note: The solution may initially appear “milky” when the GF-AFC substrate isdelivered to the buffer. This is normal. The substrate will dissolve withvortexing. The CellTiter-Fluor™ Reagent may be scaled to accommodate thevolumes required for downstream multiplexes. To do this, use 1/5 the volumeof buffer when you prepare the reagent (i.e., 10µl of the GF-AFC Substrate in2ml of Assay Buffer). Be sure to label the bottle to indicate that this is a moreconcentrated reagent, suitable for multiplex assays. Add the reagent at 1/5 thevolume of the cell culture.Storage: The CellTiter-Fluor™ Viability Reagent should be used within 24hours if stored at room temperature. Unused GF-AFC Substrate and AssayBuffer can be stored at 4°C for up to 7 days with no appreciable loss ofactivity.4.Protocols for the CellTiter-Fluor™ Cell Viability AssayMaterials to Be Supplied by the User•96-, 384-, or 1536-well opaque-walled tissue culture plates compatible with fluorometer (clear or solid bottom)•multichannel pipettor or liquid-dispensing robot•reagent reservoirs•fluorescence plate reader with filter sets for AFC (380–400nm Ex/505Em)•orbital plate shaker•compound known to cause 100% cytotoxicity or lytic detergent (digitonin, Calbiochem Cat.# 300410 or Sigma-Aldrich Cat.# D141 at 20mg/ml in DMSO).If you have not performed this assay on your cell line previously, werecommend determining assay sensitivity using your cells and one of the two methods described below (Section 4.A or 4.B). If you do not need to determineassay sensitivity for your cells, proceed to Section 4.C.Promega Corporation·2800 Woods Hollow Road ·Madison, W I 53711-5399 USA Toll Free in USA 800-356-9526·Phone 608-274-4330 ·Fax 608-277-2516 ·6.Dilute digitonin to 300µg/ml in water. Using a multichannel pipet,carefully add 10µl of the diluted digitonin to all wells of columns 7–12 to lyse cells (treated samples). Add 10µl of water to all wells of columns 1–6to normalize the volume (untreated cells).7.Add 100µl of the CellTiter-Fluor™ Reagent to all wells, mix briefly by orbital shaking and incubate at 37°C for at least 30 minutes.Note:Longer incubations may improve assay sensitivity and dynamic range. However, do not incubate longer than 3 hours, and be sure to shieldplates from ambient light.Promega Corporation ·2800 Woods Hollow Road ·Madison, W I 53711-5399 USA Toll Free in USA 800-356-9526·Phone 608-274-4330 ·Fax 608-277-2516 ·8.Measure resulting fluorescence with a fluorometer (380–400nm Ex /505nm Em )Note: You may need to adjust instrument gains (applied photomultiplier tube energy).9.Calculate the practical sensitivity for your cell type by making a signal-to-noise calculation for each dilution of cells (10,000 cells/well; 5,000cells/well; 2,500 cells/well, etc.).Viability S:N =(Average Untreated – Average Treated)Note: The practical level of assay sensitivity for the assay is a signal-to-noise ratio of greater than 3 standard deviations (derived from reference 1).4.B.Determining Assay Sensitivity, Method 21.Harvest adherent cells (by trypsinization, etc.), wash with fresh medium (to remove residual trypsin) and resuspend in fresh medium.Note:For cells growing in suspension, proceed to Step2.2.Determine the number of viable cells by trypan blue exclusion using a hemacytometer, then adjust the cells by dilution to 100,000 viable cells/ml in at least 20ml of fresh medium.Note:Concentrate the cells by centrifuging and removing medium if the pool of cells is less than 100,000 cells/ml.3.Divide the volume of diluted cells into separate tubes. Subject one tube to "moderate" sonication (empirically determined by postsonicationmorphological examination) to rupture cell membrane integrity and to simulate a 100% dead population. The second tube of untreated cells will serve as the maximum viable population.4.Create a spectrum of viability by blending sonicated and untreatedpopulations in 1.5ml microcentrifuge tubes as described in Table 2.Promega Corporation ·2800 Woods Hollow Road ·Madison, W I 53711-5399 USA Toll Free in USA 800-356-9526·Phone 608-274-4330 ·Fax 608-277-2516 ·4.B.Determining Assay Sensitivity, Method 2 (continued)5.After mixing each blend by gently vortexing, pipet 100µl of each blend into8 replicate wells of a 96-well plate. Add the 100% viable cells to column 1,95% viable to column 2, etc. Add cell culture medium only to column 10 toserve as a no-cell control.6.Add CellTiter-Fluor™ Reagent in an equal volume (100µl per well) to allwells, mix briefly by orbital shaking, then incubate for at least 30 minutesat 37°C.Note:Longer incubations may improve assay sensitivity and dynamicrange. However, do not incubate longer than 3 hours, and be sure to shieldplates from ambient light.7.Measure resulting fluorescence with a fluorometer (380–400nm Ex/505nm Em).Note: You may need to adjust instrument gains (applied photomultipliertube energy).8.Calculate the practical sensitivity for your cell type by making a signal-to-noise calculation for each blend of cell viability (X = 95, 90%, etc.).Viability S:N = (Average 100% – Average X%)Standard Deviation of 0% (viable cells)Note: The practical level of assay sensitivity for the assay is a signal-to-noise ratio of greater than 3 standard deviations (derived from reference 1).4.C.Example Viability Assay Protocol1.Set up 96-well assay plates containing cells in culture medium at desireddensity.2.Add test compounds and vehicle controls to appropriate wells so that thefinal volume is 100µl in each well (25µl for a 384-well plate).3.Culture cells for the desired test exposure period.4.Add CellTiter-Fluor™ Reagent in an equal volume (100µl per well) to allwells, mix briefly by orbital shaking, then incubate for at least 30 minutesat 37°C.Note:Longer incubations may improve assay sensitivity and dynamicrange. However, do not incubate more than 3 hours, and be sure to shieldplates from ambient light.5.Measure resulting fluorescence using a fluorometer (380–400nm Ex/505nm Em).Note:You may need to adjust instrument gains (applied photomultipliertube energy).Promega Corporation·2800 Woods Hollow Road ·Madison, W I 53711-5399 USA Toll Free in USA 800-356-9526·Phone 608-274-4330 ·Fax 608-277-2516 ·4.D.Example Multiplex Assay Protocol (with luminescent caspase assay)1.Set up 96-well assay plates containing cells in culture medium at the desired density.2.Add test compounds and vehicle controls to appropriate wells so that the final volume is 100µl in each well (25µl for a 384-well plate).3.Culture cells for the desired test exposure period.Note:Caspase activation is a transient event dictated by compound potency and cell cycle susceptibility. Time course experiments are often useful for defining peak caspase activity and cytotoxicity.4.Add 20µl of CellTiter-Fluor™ Reagent (prepared as 10µl substrate in 2ml Assay Buffer) to all wells, and mix briefly by orbital shaking. Incubate for at least 30 minutes at 37°C.Note: Longer incubations may improve assay sensitivity and dynamic range. However, do not incubate longer than 3 hours, and be sure to shield plates from ambient light.5.Measure resulting fluorescence using a fluorometer (380–400nm Ex /505nm Em ).Note: You may need to adjust instrument gains (applied photomultiplier tube energy).6.Add an equal volume of Caspase-Glo ®3/7 Reagent prepared as described in Technical Bulletin #TB323 to wells (100–120µl per well), incubate for 30minutes, then measure luminescence using a luminometer.Figure 5. Multiplex of CellTiter-Fluor™ Assay and Caspase-Glo ®3/7 Assay.The CellTiter-Fluor™ Reagent was added to wells and viability measured after incubation for 30 minutes at 37°C. Caspase-Glo ®3/7 Reagent was added andluminescence measured after a 30-minute incubation (10,000 cells/well).Promega Corporation ·2800 Woods Hollow Road ·Madison, W I 53711-5399 USA Toll Free in USA 800-356-9526·Phone 608-274-4330 ·Fax 608-277-2516 ·6865M A® 3/7 Assaylog 10[paclitaxel] ML u m i n e s c e n c e (R L U )F l u o r e s c e n c e (R F U )4.E.Recommended ControlsNo-Cell Control: Set up triplicate wells without cells to serve as a control to determine background fluorescence.Untreated Cells Control:Set up triplicate wells with untreated cells to serve as a vehicle control. Add the same solvent used to deliver the test compounds to the vehicle control wells.Optional Test Compound Control: Set up triplicate wells without cells but containing the vehicle and test compound to test for possible interference with the assay chemistry.Positive Control for Viability:Set up triplicate wells containing cells treated with a compound known to be toxic to the cells used in your model system.5.General ConsiderationsOptical Filters and Instrumentation:Fluorogenic dyes exhibit distinct absorption (excitation) and emission profiles when a light energy source is applied. Most fluorometers or multimode instruments contain optical band-pass filters that restrict the wavelengths of light used to excite a fluorophore and the wavelengths passing through to the detector. Note that deviation from the optimal filter set recommendations (Figure 6) may adversely affect assay sensitivity and performance.Figure 6. Optimal excitation and emission spectra for AFC.Background Fluorescence and Inherent Serum Activity:Tissue culturemedium that is supplemented with animal serum may contain detectable levels of the protease marker used to measure live-cells. This protease activity may vary among different lots of serum. To correct for variability, determine background fluorescence using samples containing medium plus serum without cells.Temperature: The generation of fluorescent product is proportional to the live-cell protease activity. The activity of this protease is influenced by temperature.Promega Corporation ·2800 Woods Hollow Road ·Madison, W I 53711-5399 USA Toll Free in USA 800-356-9526·Phone 608-274-4330 ·Fax 608-277-2516 ·6864M AWavelength (nm)F l u o r e s c e n c e (R F U )For best results, we recommend incubating at a constant controlled temperatureto ensure uniformity across the plate. After adding reagent and briefly mixing,we suggest one of two options:1.At 37°C in a water-jacketed incubation module (Me’Cour, etc.).Note:Incubation at 37°C in a CO2culture cabinet may lead to edge-effectsresulting from thermal gradients.2.At room temperature with or without orbital shaking.Note: Assays performed at room temperature may require more than30minutes of incubation for optimal sensitivity. However, do not incubatelonger than 3 hours.Assay Controls: In addition to a no-cell control to establish background fluorescence, we recommend including a maximum viability (untreated cells)and maximum cytotoxicity control in the experimental design. The maximum viability control is established by adding vehicle only (used to deliver the test compound to test wells). In most cases, this consists of a buffer system ormedium and the equivalent amount of solvent added with the test compound.The maximum cytotoxicity control can be determined using a compound thatcauses 100% cytotoxicity or a lytic compound added to compromise viability (digitonin). See Section 4.A.Viability Marker Half-Life:The activity of the protease marker found has nohalf-life in viable cells. Viable cells will process the substrate to liberate the AFC fluorophore. However, when cells lose membrane integrity, the protease activity declines very quickly. Therefore enzymatic instability of the live-cell proteaseoutside of viable cells establishes GF-AFC as a good marker for cell viability.Light Sensitivity: Although the GF-AFC Substrate demonstrates good general photostability, the liberated AFC fluorophore (after contact with protease) can degrade with prolonged exposure to ambient light sources. We recommend shielding the plates from ambient light at all times.Cell Culture Medium:The GF-AFC Substrate is introduced into the test wellusing an optimized buffer system that mitigates differences in pH fromtreatment. In addition, the buffer system supports protease activity in a host of different culture media with varying osmolarity. With the exception of media formulations with either very high serum content or phenol red indicator, no substantial performance differences will be observed among media.6.References1.Niles, A.L. et al. (2007) A homogeneous assay to measure live and dead cells in thesame sample by detecting different protease markers. Anal. Biochem.366, 197–206.2.Zhang, J-H. et al.(1999) A simple statistical parameter for use in evaluation andvalidation of high-throughput screening assays. J. Biomol. Screen. 4, 67–73.Promega Corporation·2800 Woods Hollow Road ·Madison, W I 53711-5399 USA Toll Free in USA 800-356-9526·Phone 608-274-4330 ·Fax 608-277-2516 ·7.Related ProductsCell Viability and CytotoxicityAssays ProductSize Cat.#MultiTox-Fluor Multiplex Cytotoxicity Assay 10ml G9200MultiTox-Glo Multiplex Cytotoxicity Assay 10ml G9270CytoTox-Glo™ Cytotoxicity Assay 10ml G9290CytoTox-Fluor™ Cytotoxicity Assay10ml G9260CellTiter-Glo ®Luminescent Cell Viability Assay 10mlG7570CytoTox-ONE™ Homogeneous Membrane Integrity Assay1,000–4,000 assaysG7891CellTiter-Blue ®Cell Viability Assay20mlG8080Additional Sizes Available.Apoptosis AssaysProductSize Cat.#Caspase-Glo ®2 Assay 10ml G0940Caspase-Glo ®6 Assay 10ml G0970Caspase-Glo ®3/7 Assay 10ml G8091Caspase-Glo ®8 Assay 10ml G8201Caspase-Glo ®9 Assay10ml G8211Apo-ONE ®Homogeneous Caspase 3/7 Assay10ml G7790Additional Sizes Available.Reporter Gene AssaysProductSize Cat.#Bright-Glo™ Luciferase Assay System 10ml E2610Steady-Glo ®Luciferase Assay System10ml E2510Additional Sizes Available.Promega Corporation ·2800 Woods Hollow Road ·Madison, W I 53711-5399 USA Toll Free in USA 800-356-9526·Phone 608-274-4330 ·Fax 608-277-2516 ·(a)Patent Pending.© 2007–2012 Promega Corporation. All Rights Reserved.Apo-ONE, Caspase-Glo, CellTiter-Blue, CellTiter-Glo and Steady Glo are registered trademarks of Promega Corporation.BrightGlo, CellTiter-Fluor, CytoTox-Fluor, CytoTox-Glo, CytoTox-ONE and P450-Glo and are trademarks of Promega Corporation.Products may be covered by pending or issued patents or may have certain limitations. Please visit our Web site for more information.All prices and specifications are subject to change without prior notice.Product claims are subject to change. Please contact Promega Technical Services or access the Promega online catalog for the most up-to-date information on Promega products.。

鸢尾素激活PGC-1α、UCP-1促进噬脂和褐变抑制ACHN细胞增殖和迁移

鸢尾素激活PGC-1α、UCP-1促进噬脂和褐变抑制ACHN细胞增殖和迁移熊绍风;熊小伟;彭国平;王昆;方洋;吕燕妮【期刊名称】《重庆医学》【年(卷),期】2024(53)3【摘要】目的探讨鸢尾素对肾细胞癌的影响及PGC-1α、UCP-1在其中发挥的关键性作用。

方法采用Western blot测定鸢尾素对人肾细胞腺癌细胞(ACHN)PGC-1α、UCP-1蛋白的影响;采用实时荧光定量逆转录PCR(qRT-PCR)方法检测鸢尾素对其自噬相关和褐变相关mRNA的影响;采用油红O染色检测鸢尾素对噬脂的影响;采用凋亡试剂盒检测鸢尾素对细胞凋亡率的影响;采用划痕实验检测鸢尾素对细胞的愈合和修复能力。

结果鸢尾素能够上调棕色化相关蛋白(PGC-1α、UCP-1)及mRNA(PGC-1α、UCP-1、PRDM16)的表达并激活自噬相关mRNA(Beclin-1、LC3Ⅱ、P62)的表达(P<0.05);鸢尾素能够促进噬脂,抑制ACHN细胞增殖、细胞迁移运动与修复能力(P<0.05)。

结论鸢尾素能够通过激活PGC-1α、UCP-1促进噬脂和褐变进而抑制ACHN细胞增殖和迁移。

【总页数】7页(P321-327)【作者】熊绍风;熊小伟;彭国平;王昆;方洋;吕燕妮【作者单位】南昌大学第一附属医院药学部;南昌大学药学院药理教研室;江西应用科技学院【正文语种】中文【中图分类】R737.11【相关文献】1.鸢尾黄酮通过促进自噬抑制结肠癌细胞增殖并诱导其凋亡研究2.机械牵张力和去甲肾上腺素协同激活小鼠血管平滑肌细胞α_1-肾上腺素能受体-ERK1/2信号促进细胞增殖机械牵张力和去甲肾上腺素协同激活小鼠血管平滑肌细胞α1-肾上腺素能受体-ERK1/2信号促进细胞增殖3.大黄酚激活SIRT1/PGC-1α抑制心脏成纤维细胞的增殖、侵袭和迁移及向肌成纤维细胞转化4.白细胞介素9通过激活信号转导子和转录激活子3(STAT3)通路促进胰腺癌细胞的增殖和迁移5.尿石素A(urolithin A)通过激活Nrf2通路和自噬抑制高脂诱导的肝细胞炎症反应及氧化应激因版权原因，仅展示原文概要，查看原文内容请购买。

ruixi-帕他色替 Ipatasertib 棕榈酰溶血卵磷脂 P-lyso-PC

帕他色替Ipatasertib 1001264-89-6 棕榈酰溶血卵磷脂P-lyso-PC帕他色替Ipatasertib (GDC-0068, RG7440)是一种高选择性的pan-Akt抑制剂，靶向作用于Akt1/2/3，在无细胞试验中IC50为5 nM/18 nM/8 nM，比作用于PKA选择性高620倍。

Phase 2。

丙氨酸氨基转移酶(ALT),天门冬氨酸氨基转移酶(AST),γ-谷氨酰基转移酶(GGT),碱性磷酸酶(ALP),乳酸脱氢酶(LDH),α-羟丁酸脱氢酶(α-HBDH),肌酸激酶MB同工酶(CK-MB),肌酸激酶(CK),C-反应蛋白(CRP)和心肌肌钙蛋白T(cTnT)].血清中主要代谢物的代谢组学分析利用高效液相色谱-质谱(HPLC-MS)技术进行.相应数据经统计分析后,筛选并鉴定出有重要变化的代谢物.结果应用HPLC-MS发现4个平均质荷比(m/z)分别为496.33,478.34,184.13和991.71的离子碎片.经鉴定,4个离子碎片均来自于1-棕榈酰溶血磷脂酰胆碱(C16:0).IHD组血清1-棕榈酰溶血磷脂酰胆碱(C16:0)浓度为(76.35±18.28)μmol/L,明显高于正常对照组[(64.24±15.56)μmol/L,P<0.001].IHD组其他传统心肌指标与对照组比较,除AST升高外(P<0.05),其余9项均无明显差异(P>0.05).受试者工作特性(ROC)曲线显示,诊断IHD时1-棕榈酰溶血磷脂酰胆碱(C16:0)的ROC曲线下面积(0.90±0.03)显著高于其他常规生化指标(P<0.05).结论1-棕榈酰溶血磷脂酰胆碱(C16:0)对IHD具有临床价值.产品名称：帕他色替英文名称：IpatasertibCAS:1001264-89-6状态：固体/粉末/溶液产品规格：mg保存：冷藏储藏条件：-20℃储存时间：1年用途：科研温馨提醒：仅供科研，不能用于人体实验产品名称：棕榈酰溶血卵磷脂英文名称：P-lyso-PCCAS：17364-16-8状态：固体/粉末/溶液产品规格：mg保存：冷藏储藏条件：-20℃储存时间：1年用途：科研温馨提醒：仅供科研，不能用于人体实验相关产品：二硬脂酰基磷脂酰乙醇胺-聚乙二醇-Asn-Gly-Arg肽4armPEG20k-Mal4armPEG-Mal四臂聚乙二醇马来酰亚胺Methacrylate-PEG-NHSMW:3400甲基丙烯酸酯-聚乙二醇-活性酯PMMA-PEG-NHSCY3-PEG-cholesterolMW:2K花菁染料-聚乙二醇-胆固醇CY3-PEG-cholDSPE-PEG,MW:10K磷脂-聚乙二醇PEG-二硬脂酰磷脂酰乙醇胺PEG-BMA-PDSMA聚乙二醇-甲基丙烯酸丁酯-吡啶二硫乙基甲基丙烯酰胺瑞禧WFF.2023.3。

NPC1L1_固醇脂质吸收的关键蛋白质

参考文献
［１］ＰｌａｔａｎｉａｓＬＣ．ＮａｔＲｅｖＩｍｍｕｎｏｌ，２００５，５：３７５—３８６［２］ＴａｎｉｇｕｃｈｉＴｅｔａｌ．ＡｎｎｕＲｅｖＩｍｍｕｎｏｌ，２００１，１９：６２３—６５５［３］ＭｏｙｎａｇｈＰＮ．ＴｒｅｎｄｓＩｍｍｕｎｏｌ，２００５，２６：４６９—４７６［４］ＬｅＢｏｎＡｅｔａｌ．ＣｕｒｒＯｐｉｎＩｍｍｕｎｏｌ，２００２，１４：４３２—４３６［５］ＩｗａｓａｋｉＡｅｔａｌ．ＮａｔＩｍｍｕｎｏｌ，２００４，５：９８７—９９５［６］ＳａｔｏＭｅｔａｌ．Ｉｍｍｕｎｉｔｙ，２０００，１３：５３９—５４８［７］Ｏ′ＮｅｉｌｌＬＡ．ＣｕｒｒＯｐｉｎＩｍｍｕｎｏｌ２００６，１８：３—９［８］ＩｗａｓａｋｉＡｅｔａｌ．ＮａｔＩｍｍｕｎｏｌ，２００４，５：９８７—９９５［９］ＦｕｊｉｔａＴｅｔａｌ．ＣｕｒｒＭｅｄＣｈｅｍ，２００５，４：２９—３３［１０］ＴｈｅｏｆｉｌｏｐｏｕｌｏｓＡＮｅｔａｌ．ＡｎｎｕＲｅｖＩｍｍｕｎｏｌ，２００５，２３：３０７—３３６［１１］ＬｏｈｏｆｆＭｅｔａｌ．ＮａｔＲｅｖＩｍｍｕｎｏｌ，２００５，５：１２５—１３５［１２］ＫｌｅｉｎＵｅｔａｌ．ＮａｔＩｍｍｕｎｏｌ，２００６，７：７７３—７８２［１３］ＦｉｌｌａｔｒｅａｕＳｅｔａｌ．ＮａｔＩｍｍｕｎｏｌ，２００６，７：７０４—７０６［１４］ＰｉｎｅＲ．ＪＩｎｔｅｒｆｅｒｏｎＣｙｔｏｋｉｎｅＲｅｓ，２００２，２２：１５—２５［１５］Ｌｉ－ＷｅｂｅｒＭｅｔａｌ．ＮａｔＲｅｖＩｍｍｕｎｏｌ，２００３，３：５３４—５４３

PRRS

Porcine reproductive and respiratory syndrome virus as a vector:Immunogenicity of green ﬂuorescent protein and porcine circovirus type 2capsid expressed from dedicated subgenomic RNAsYanlong Pei a ,Douglas C.Hodgins a ,Jiaqiang Wu a ,1,Siao-Kun W.Welch b ,Jay G.Calvert b ,Gang Li c ,Yijun Du d ,Cheng Song a ,d ,Dongwan Yoo d ,⁎aDepartment of Pathobiology,University of Guelph,Guelph,Ontario,Canada N1G 2W1bP ﬁzer Animal Health,Kalamazoo,MI 49001,USA cInstitute of Animal Health and Husbandry,Chinese Academy of Agricultural Sciences,Beijing,China dDepartment of Pathobiology,University of Illinois at Urbana-Champaign,2001South Lincoln Ave,Urbana,IL 61802,USAa b s t r a c ta r t i c l e i n f o Article history:Received 31January 2009Returned to author for revision 3March 2009Accepted 31March 2009Available online 6May 2009Keywords:PRRSVReverse geneticsForeign gene expression vector Vaccine vector Nidovirus ArterivirusPorcine reproductive and respiratory syndrome virus (PRRSV)is the causative agent of PRRS,which is characterized by late-term abortions in sows and respiratory disease in young ing an infectious cDNA clone of North American PRRSV strain P129,the viral genome was engineered to transcribe an additional subgenomic RNA initiating between non-structural and structural genes.Two unique restriction sites and a copy of the transcription regulatory sequence for ORF6(TRS6)were inserted between ORFs 1b and 2a,yielding a general purpose expression vector.The enhanced green ﬂuorescent protein (GFP)gene was cloned between the unique sites such that the inserted gene was transcribed from TRS2which was located upstream within ORF1b,while the copy of TRS6drives ORF2a/b transcription.Upon transfection of cells with this plasmid,PRRSV infection was initiated and progeny virus “P129-GFP ”was obtained.Cells infected with P129-GFP showed ﬂuorescence and the inserted gene was phenotypically stable for at least 37serial in vitro passages.Subsequently,a capsid (C)protein gene was cloned from porcine circovirus type 2(PCV2)recovered from an outbreak of porcine multisystemic wasting syndrome (PMWS)and inserted into the PRRSV infectious clone vector,generating virus “P129-PCV ”.To determine the immunogenicity of the recombinant viruses,pigs were immunized intramuscularly with P129-WT (wild-type),P129-GFP,or P129-PCV2.By 5weeks post-infection,speci ﬁc antibody responses to GFP and PCV2capsid were elicited.This is the ﬁrst report of foreign gene expression using PRRSV from dedicated subgenomic RNAs and demonstrates the potential use of PRRSV as a vaccine vector for swine pathogens.©2009Elsevier Inc.All rights reserved.IntroductionPorcine reproductive and respiratory syndrome (PRRS)is an emerged and re-emerging disease in pigs.The disease was ﬁrst recognized in Germany and the US almost simultaneously in the late 1980s and has since spread globally to most pork producing countries (Keffaber,1989;Ben ﬁeld et al.,1992;Albina,1997).PRRS is characterized by abortions and mummi ﬁed fetuses in sows,and respiratory distress with poor growth in young pigs.The disease is mild in gilts and boars,but the virus (PRRSV)persists in semen and thus can be transmitted widely by arti ﬁcial insemination.Since its emergence,PRRS has become one of the most economically important diseases in the swine industry.Modi ﬁed-live vaccines are available,but safety and limited ef ﬁcacy areongoing concerns.No speci ﬁc treatment is available for PRRS,and thus the economic losses are enormous.Numerous isolates of PRRSV representing many geographical regions have been sequenced,revea-ling the existence of two distinct genotypes of PRRSV:European (type I)and North American (type II).The two genotypes share overall sequence identity of 63%and differ antigenically as well as genetically (Nelson et al.,1993;Meng et al.,1995;Wootton et al.,2000).PRRS virus is an enveloped,single-stranded,positive-sense RNA virus belonging to the family Arteriviridae and along with the Coronaviridae family,forms the order Nidovirales .The PRRSV genome is approximately 15kb in size and includes the 5′cap structure and 3′polyadenylated tail (Sagripanti et al.,1986;Wootton et al.,2000).The genome consists of nine genes:open reading frames (ORFs)1a,1b,2a,2b,3,4,5,6,and 7.The 5′three-quarters of the genome consists of two slightly overlapping ORFs,1a and 1b,and they are translated directly from the genome-sense RNA.ORF1b is expressed as a fusion protein with ORF1a by a frame-shifting mechanism,and the ORF1a and ORFla/b proteins are auto-cleaved by viral proteases into at least 13cleavage products that are theVirology 389(2009)91–99⁎Corresponding author.E-mail address:dyoo@ (D.Yoo).1Current address:Shandong Key Laboratory of Animal Disease Control and Breeding,Shandong Academy of Agricultural Sciences,Jinan,Shandong,China.0042-6822/$–see front matter ©2009Elsevier Inc.All rights reserved.doi:10.1016/j.virol.2009.03.036Contents lists available at ScienceDirectVirologyj o u r n a l ho m e p a g e :w w w.e l s ev i e r.c o m /l o c a t e /y v i r onon-structural proteins Nsp1α,Nsp1β,and Nsp2through Nsp12.The Nsps are thought to be involved in genome replication and subgenome transcription(van Dinten et al.,1999;van Marle et al.,1999).The viral structural proteins are encoded by ORFs2a,2b,and3through7which are located downstream of ORFs1a and1b.These genes are expressed as a3′-coterminal nested set of subgenomic(sg)RNAs(de Vries et al., 1990).The5′untranslated leader sequences of the sgRNAs are derived from the5′end of the viral genome and fused to the body segments of the sgRNAs at conserved hexanucleotide motifs[5′UCAAC(U/C)3′] located immediate upstream of every transcription unit(de Vries et al., 1990;den Boon et al.,1996).The conserved hexanucleotide motif and poorly conservedﬂanking sequences form secondary structures in the sgRNAs that make up the transcriptional regulatory sequences(TRS)that are necessary for sgRNA formation(Pasternak et al.,2000).With the exception of sgRNA7,sgRNAs are structurally polycistronic but,with the exception of sgRNA2a/b,functionally monocistronic as only the5′most proximal gene of each sgRNA is translated.ORFs2a,2b,and3through7 code for GP2(glycoprotein2),E(envelope),GP3,GP4,GP5,M (membrane)and N(nucleocapsid)proteins,respectively,and they make up virion particles(Meulenberg et al.,1995).The recent development of infectious clones for PRRSV has allowed speciﬁc alterations of viral genomes and generation of mutant viruses(Yoo et al.,2004).However,manipulation of arterivirus genomes is complicated by the condensed organization of the viral genome.In the case of the European genotype of PRRSV,each gene overlaps slightly except for ORFs 1b and2a(Meulenberg et al.,1993),although the overlap of genes seems less important for virus replication and growth for equine arteritis virus (de Vries et al.,2000).For the North American genotype,structuralgenes Fig.1.Genomic organization of P129-WT,P129-GFP,and P129-PCV2.(A)Genomic organization and sequence of the region surrounding the ORF1b/ORF2junction of the unmodiﬁed “wild-type”P129strain of PRRSV(P129-WT).The boxed sequence indicates the core hexanucleotide of TRS2.Amino acids indicate translated sequences of polyprotein1a/b(ORF1b),the GP2protein(ORF2a),and the E protein(ORF2b).The initiation codons are indicated in bold,and the E protein sequence is italicized.Numbers in parenthesis indicate genomic sequence positions.Stars indicate translation stops.(B)P129-GFP.Unique AﬂII and Mlu I sites and a copy of TRS6were introduced into the non-coding region between ORF1b and ORF2a,creating expression vector pCMV-S-P129-1bMCS2.The GFP gene was ampliﬁed by PCR and inserted between the AﬂII and Mlu I restriction sites.(C)P129-PCV2.The PCV2 capsid gene was ampliﬁed by PCR and cloned between the AﬂII and Mlu I restriction sites.92Y.Pei et al./Virology389(2009)91–99also overlap with the exceptions of ORFs 1b and 2a,and ORFs 4and 5(Nelsen et al.,1999;den Boon et al.,1991;Snijder and Meulenberg,1998).Thus,the entire genome of the North American type PRRSV possesses only 4short non-coding regions:191nucleotides of 5′untranslated region (UTR),1nucleotide between ORF1b and ORF2a,10nucleotides between ORF4and ORF5,and 151nucleotides of 3′UTR upstream of the polyadenylation tail.The 5′and 3′UTRs contain genome replication and transcription signals,and therefore offer limited sites for gene insertion and manipulation.The presence of overlapping genes hampers mutational analysis of the N-and C-termini of the structural proteins and also makes it dif ﬁcult to insert heterologous genes into the viral genome.In the present study,we used a genomic cDNA clone of PRRSV (Lee et al.,2005)to generate a vector for foreign gene expression from a dedicated subgenomic transcription unit inserted in the region between the structural and non-structural genes.The vector was used to express GFP in vitro and in vivo .The inserted gene was tolerated by the virus,stable for at least 37passages in cell culture,and induced antibodies to GFP in young pigs.Using this approach,we also generated a recombinant PRRSV expressing the capsid protein gene of porcine circovirus type 2(PCV2).PCV2is a small DNA virus in the Circoviridae family,with a genome ofonly 1.76kb.PCV2ORF1is essential for viral DNA replication (Mankertz et al.,1998;Fenaux et al.,2000),while ORF2encodes the capsid protein containing type-speci ﬁc epitopes that are believed to be important for virus neutralization (Nawagitgul et al.,2000;Fenaux et al.,2004).Accumulating evidence suggests a major role for PCV2in postweaning multisystemic wasting syndrome (PMWS)and porcine dermatitis and nephropathy syndrome (PDNS)(Hasslung et al.,2005).These syndromes cause serious economic impacts in the swine industry today (Chae 2005).We showed the recombinant PRRSV P129-PCV2induced an anti-PCV2antibody response in immunized pigs in the presence of maternal antibodies to PCV2.Our vector construction may be applicable not only for PRRSV,but also for other members of the families Arteriviridae and Coronaviridae .ResultsDevelopment of PRRSV as an expression vector for GFPTo explore the possibility of developing PRRSV as a gene expression vector,the GFP gene was inserted between the stop codon ofORF1bFig.2.(A)Restriction patterns of P129-WT (lane 1),P129-GFP (lane 2),and P129-PCV2(lane 3)genomic clones generated by Sma I digestion.Fragments of 590bp,4736bp and 10,779bp are expected from all three clones.The fourth fragment varies in size depending on the gene inserted at the non-structural and structural gene junction.Relative to the P129-WT fragment (2787bp,lane 1),the GFP-containing fragment from P129-GFP is 766bp larger (3553bp,lane 2)and the PCV2capsid-containing fragment from P129-PCV2is 754bp larger (3541bp,lane 3).(B –E)Recovery of recombinant PRRSV foci from full-length genomic clones.(F)Integration of the GFP gene in P129-GFP viral genome.Genomic RNA was extracted from P129-GFP virus and digested with DNase I prior to reverse transcription.Without reverse transcription,no products were ampli ﬁed from P129-GFP using ORF7-speci ﬁc PCR primers P129-7F and P129-7R (lane 1)or primers P129-1bF and P129-2aR that span the insertion site (lane 2).With reverse transcription,primers P129-7F and P129-7R ampli ﬁed the ORF 7fragment (534bp)from both P129-WT (lane 3)and P129-GFP (lane 4).Using P129-1bF and P129-2aR,a product of 766bp larger (lane 6)than the product from P129-WT (lane 5)was ampli ﬁed from P129-GFP.(G)Integration of the PCV2C gene in PRRSV.Genomic RNA from P129-WT (lanes 9,10,12)and P129-PCV2(lanes 7,8,and 11)was ampli ﬁed using primers P129-1bF and pared to P129-WT (lane 9)a product that is 754bp larger was ampli ﬁed from P129-PCV2(lane 7).Using PCV2C gene speci ﬁc primers PCV2-F and PCV2-R,the expected 497bp product was generated from P129-PCV2(lane 8)but not from P129-WT (lane 10).Using PRRSV ORF4-speci ﬁc primers P129-4F and P129-4R,the expected 567bp product was ampli ﬁed from both P129-PCV2and P129-WT (lanes 11and 12,respectively).93Y.Pei et al./Virology 389(2009)91–99and the start codon of ORF2a (Fig.1A).This non-coding region is extremely short,comprising only one adenosine nucleotide.The TRS associated with ORFs 2a and 2b (TRS2;TGAACC)is positioned 26nucleotides upstream from the start of ORF2a and is embedded in ORF1b.Upon insertion of GFP to the region,TRS2will drive transcription of the GFP gene instead of ORFs 2a and 2b.Thus,a synthetic TRS (TTAACC)with ﬂanking sequences derived from TRS6was introduced 22nucleotides downstream of GFP and 17nucleotides upstream from the ORF2a start (Fig.1B).TRS6was chosen because RNA secondary structure suggested that it was shorter than other PRRSV TRS elements and because the distance between the copy of TRS6driving ORF2a/b and the authentic TRS6ensured that potential intramolecular homologous RNA recombination would result in a non-viable (ORF2-5deleted)virus.The PRRSV genomic clone containing GFP was designated P129-GFP,and the insertion was con ﬁrmed by restriction patterns (Fig.2A,lane 2)and sequencing.MARC-145cells were transfected with P129-GFP and the production of virus was monitored daily for development of cytopathic effect (CPE)(Figs.2B,C,D).CPE was observed 4days post-transfection and the development of CPE was one day slower than for P129-WT.After three consecutive passages,P129-GFP virus was re-examined for GFP sequence integration in the viral genome (Fig.2F).While ORF7ampli ﬁcation products were identical in size for P129-WT (lane 3)and P129-GFP (lane 4),1bF and 2aR primers produced a larger size product from P129-GFP (lane 6)than from P129-WT (lane 5).The larger product was the expected size for correct insertion of GFP gene intothe viral genome.Replication of P129-GFP was slightly slower than that of P129-WT at passages 1to 3.However,plaques were comparable in size and morphology for P129-WT and P129-GFP,and the titers at passage 3were 5×105plaque forming units (PFU)/ml and 1×105PFU/ml,respectively.Fluorescence was evident in P129-GFP-infected cells (Fig.3A),demonstrating the expression of GFP during infection.To examine the genetic stability of recombinant PRRSV,P129-GFP was passaged 37times in MARC-145cells and GFP expression was monitored by ﬂuorescence microscopy.Individual plaques of 20formed by P129-GFP were randomly chosen and examined for ﬂuorescence.The selected plaques were all positive for ﬂuorescence (Table 1),and sequencing of the viral RNA con ﬁrmed stability of the insert (data not shown).This data showed the genetic stability of P129-GFP after serial passages in cell culture and the stable expression of GFP.It also demonstrates that the region between ORF1b and ORF2a is a suitable site for foreign gene insertion for PRRSV.This was the ﬁrst demonstration of the use of a nidovirus as an expression vector wherein the foreign gene is inserted in the region between the non-structural and structural protein codingsequences.Fig.3.Expression of GFP or PCV2capsid protein by P129-GFP and P129-PCV2in MARC-145cells.(A)Live cells infected with P129-GFP passage 3;(B)live cells infected with P129-GFP passage 37;(C)uninfected cells;(D)P129-PCV2passage 3ﬁxed and stained with PCV2-speci ﬁc antibody conjugated with FITC at 48h post-infection;(E and F)P129-PCV2infected cells ﬁxed and co-stained with PCV2-speci ﬁc antibody conjugated with FITC (E)or GP4protein-speci ﬁc monoclonal antibody 169(F).Table 1Stability of GFP expression in P129-GFP virus.Virus Titer (PFU/ml)GFP expressing plaques Passage 38.3×10e620positive/20plaques Passage 372.4×10e720positive/20plaques94Y.Pei et al./Virology 389(2009)91–99Construction of PRRSV expressing PCV2capsidPCV2is associated with porcine multisystemic wasting disease (PMWS,now termed PCVAD [PCV-associated disease])and porcine dermatitis and nephropathy syndrome (PDNS).PCV2is transmitted by the oro-nasal route and shed in the bronchial secretions and feces,thus the transmission route is similar to that of PRRSV.The capsid (C)protein is the major antigen able to elicit protective immunity against PCV2.Thus,using the PRRSV vector system described above,an additional recombinant PRRSV was constructed to carry the PCV2C gene.A 702bp C gene was cloned by PCR from a lung tissue positive for PCV2.Sequencing of the C gene showed 99–100%amino acid identity to published sequences available in the GenBank database.The P129-PCV2clone was constructed by inserting the C gene into PRRSV in the same way as the GFP gene insertion for P129-GFP (Fig.1C).The insertion of the C gene was con ﬁrmed by restriction digestion pattern (Fig.2A,lane 3)and sequencing.The P129-PCV2recombinant virus was recovered from MARC-145cells by transfection (Fig.2E),and the insertion of the C gene in the viral genome was con ﬁrmed by RT-PCR of the viral RNA (Fig.2G).The titer of passage 3virus was 2×105PFU/ml and the plaque morphology was indistinguishable from P129-WT.Infection of cells with P129-PCV2and staining with PCV2-speci ﬁc antibody produced distinct ﬂuorescence (Fig.3E),which shows the expression of the C protein by P129-PCV2.The capsid protein of PCV2is arginine-rich and normally shuttles into the nucleus during PCV2replication (Liu et al.,2001),and similarly,the PRRSV N has also been shown to localize in the nucleus and nucleolus (Lee et al.,2006;Pei et al.,2008).Thus,in cells infected with P129-PCV2,the synthesis of legitimate PCV2capsid should be evident by the translocation of capsid into the nucleus.Thus,the C protein expression by P129-PCV2was con ﬁrmed by co-staining of virus-infected cells with PRRSV N protein-speci ﬁc antibody and PCV2-speci ﬁc antiserum (Fig.4).While the PRRSV N protein was found in the both cytoplasm and the nucleolus as usual (panel A),the PCV2capsid protein was speci ﬁcally localized to the nucleus and nucleolus (panel C)in the same cell,clearly demonstrating the expression of PCV2capsid protein by the recombinant PRRSVP129-PCV2.Fig.4.Co-expression of the PRRSV N protein (green)and the PCV2capsid protein (red)during infection of P129-PCV2.MARC-145cells were infected with P129-PCV2and stained 24h post-infection with PRRSV N-speci ﬁc MAb SDOW-17(A and B)or PCV2-speci ﬁc pig serum (C and D).Arrows indicate the PRRSV N protein in the nucleolus (panel A)in addition to the cytoplasm and the PCV2capsid protein in the nucleus and nucleolus (panel C).Panel E shows the merge of A and C.Panel F shows the merge of B and D.Table 2PCR primers and their genomic Sequence (5′–3′)aGenomic position b PurposePCV2-F cacggatattgtagtcctggt 1093–1114PCV2PCR test PCV2-R ccgcaccttcggatatactgtc1565–1586PCV2PCR testPCV2-orf2F gatgcttaagatgacgtatccaaggtggcg 1715–1734PCV2ORF2ampli ﬁcation PCV2-orf2R gtacacgcgtcattaagggttaagtcccccc 1031–1050PCV2ORF2ampli ﬁcationP129-F1F aacagaagagttgtcgggtccac11,699–11,721P12911,783–12,055,ampli ﬁcation P129-F1R gctttcacgcgtccccacttaagttcaattcaggcctaaagttggttca 12,031–12,055Introduction of A ﬂII and Mlu IP129-F2F gcgacgcgt gttccgtggcaacccctttaaccagagtttcagcggaaga atgaaatggggtctatacaaagcctcttcgaca 12,056–12,089P12912,056–12,697,ampli ﬁcation and introduction of Mlu I and TRS6P129-F2R aacagaacggcacgatacaccacaaa 13,819–13,844P12912,056–12,679,ampli ﬁcation P129-7F tcatccgattgcggcaaatg 14,724–14,743P129ORF7ampli ﬁcation P129-7R agaatgccagcccatca 15,242–15,258P129ORF7ampli ﬁcation P129-4F gtttcacctagaatggctg 13,213–13,231ORF4ampli ﬁcation P129-4R ccccaacatacttgaacattc 13,750–13,770ORF4ampli ﬁcationP129-1bF ggtgaggactgggaggattac 11,921–11,941ORF1b –ORF2a,region ampli ﬁcation P129-2aRcagtacgtagcattggaacc12,758–12,777ORF1b –ORF2a,region ampli ﬁcationa Restriction sites are underlined.TRS6and surrounding sequences are indicated in bold.bGenomic positions for PCV2primers were based on GenBank accession AF027217.Genomic position for P129primers were based on GenBank accession AF494042.95Y.Pei et al./Virology 389(2009)91–99Infection of pigs and antibody responses to GFP and PCV2capsid protein To determine antibody responses to GFP and the PCV2capsid protein,pigs were immunized with the recombinant PRRS viruses.Fifteen PRRSV-free pigs at 4weeks of age were randomly allotted to 3groups of 5pigs each.Animals were immunized twice on days 0and 21by intramuscular injection of 5×105PFU per animal with either P129-WT,P129-GFP,or P129-PCV2.Following inoculation,the animalswere maintained for 5weeks for clinical observation and serum collection.Clinical signs of PRRS were minimal and comparable in all 3groups (data not shown).Mild clinical signs were not unexpected,since the infectious cDNA clone used in these studies was not attenuated.Tonsil samples were collected at necropsy on day 35and assessed by RT-PCR for the presence of PRRSV ORF7using primers P129-7F and P129-7R (Table 2)as well as the GFP and PCV2capsid inserts using primers P129-1bF and P129-2aR.All 15pigs were positive for ORF7,indicating infection and persistence of PRRSV in the tonsils.PCR products from the ORF1b/ORF2a junction were not detected in these tonsil samples,possibly due to the much lower molar ratio of ORF1b-containing RNA template relative to ORF7-containing RNA in infected cells.Alternatively,it is possible that the GFP and PCV2capsid genes might have been unstable in vivo and lost in the inoculated pigs over time.To examine antibody responses in these pigs,ELISAs were conducted for PRRSV,GFP,and PCV2C protein.All pigs produced good levels of antibodies to PRRSV (Fig.5A),and the antibody titers were comparable among groups.P129-GFP elicited speci ﬁc antibodies to GFP,whereas pigs immunized with P129-PCV2or P129-WT were negative for GFP (Fig.5B).The GFP antibodies increased following ﬁrst immunization,and the second immunization at day 21boosted the antibody response somewhat (Fig.5B).Similarly,antibodies for PCV2C protein were detected in pigs immunized with P129-PCV2(Fig.5C).In these pigs,however,PCV2antibodies were detected at day 0in all 3treatment groups and tended to wane over time.These antibodies likely represent maternal antibodies taken up in colostrum shortly after farrowing at the farm of origin and prior to experimental infection.However,an increase in anti-PCV2antibodies was observed at 28days in the P129-PCV2group only (Fig.5C)due to boosting effects from the second immunization at day 21.In contrast,anti-PCV2antibodies in the other two treatment groups waned gradually from day 0until at least until day 35.To further determine the speci ﬁc antibody responses to GFP and the PCV2C protein,Western blots were conducted using sera from these pigs.Serum from the P129-GFP group was reactive with GFP (Fig.6A),consistent with the ELISA data (Fig.5B).Similarly,serum from the P129-PCV2group showed a strong reaction with C pro-tein (Fig.6B).Weaker reactions were observed using sera from the P129-WT and P129-GFP groups,consistent with the presence of maternalantibodies.Fig.5.ELISA showing induction of speci ﬁc antibodies in sera from pigs inoculated with P129-GFP,P129-PCV2,or P129-WT viruses.(A)antibodies to PRRSV N protein;(B)antibodies to GFP;(C)antibodies to PCV2Cprotein.Fig. 6.Western blots showing induction of speci ﬁc antibodies in sera from pigs inoculated with P129-GFP,P129-PCV2,or P129-WT at day 0or day 35post-inoculation.Blots contain GFP protein (A)or PCV2virions (B).Arrows indicate positions of GFP and PCV2capsid protein.C denotes positive control (anti-GFP monoclonal antibody).M indicates molecular weight markers.The virus used to infect pigs that contributed to the serum pools is indicated above each lane.96Y.Pei et al./Virology 389(2009)91–99DiscussionThe primary target cell of PRRSV is the alveolar macrophage,and pigs are the only animal species known to be susceptible to PRRSV infection.Therefore,development of PRRSV as a vaccine vector would be useful for the delivery of porcine pathogen genes to the respiratory tract of the pig.Arterivirus genomes are organized in a complex way. Most genes overlap one another in different reading frames,making it difﬁcult to engineer the genome for foreign gene insertion.An early approach involved engineering the3′terminal region of the N gene (Groot Bramel-Verheije et al.,2000).Modiﬁcation of the3′terminal sequence of N gene was possible and caused only minimal effects on virus replication and growth.However,no more than7amino acids were inserted.More recently,Nsp2,a large product of proteolytic cleavage of the ORF1a and ORF1a/b polyproteins,was found to be remarkably heterogeneous in sequence,with several hypervariable regions.Therefore,the GFP gene was inserted in-frame into or between the hypervariable regions to create nsp2-GFP fusion proteins (Fang et al.,2006;Kim et al.,2007).This approach was successful and allowed for the production of recombinant viruses.However,in all cases the inserted GFP gene was not phenotypically stable and lost greenﬂuorescence after several passages in cell culture(Fang et al., 2006;Kim et al.,2007;Han et al.,2007).In the present study,we inserted GFP and the PCV2capsid genes into the short region separating the non-structural protein genes from the structural protein genes.In contrast to the nsp2-GFP fusion proteins described above,we expressed GFP as a separate transcription unit resulting in an additional sgRNA.This approach has the advantage of eliminating the need to alter the coding sequence of a viral gene,and also minimizes effects on expression and post-translational modiﬁcation of viral gene products.As a result,our recombinant virus was stable for at least37cell culture passages without loss of the gene or the greenﬂuorescent phenotype.PRRSV is known to induce immune suppressive effects in pigs (Charerntantanakul et al.,2006)and can persist up to6months in infected pigs(Wills et al.,1997).For these reasons,co-infection of PRRSV with other pathogen such as PCV2can result in much more severe clinical outcome than either agent alone(Harms et al.,2001; Kim et al.,2003).Therefore,a dual-purpose vaccine capable of protecting pigs against both PRRS and PCV2would be advantageous. Our study demonstrates the potential of PRRSV as a viral vaccine vector.Prior to the purchase of animals for infection studies using P129-PCV2,all pigs were screened for PCV2by PCR.PCR is the gold standard for detection of PCV2,whereas antibody screening is not routinely conducted in diagnostic laboratories due to cross-reactivity between PCV2and PCV1which is ubiquitous and widely distributed in theﬁeld (Magar et al.,2000).Although all pigs entering the present study tested negative for the presence of PCV2by PCR,antibodies were detected in sera collected on day0.These antibodies were most likely of maternal origin,since they decreased in concentration over the duration of the experiment in pigs receiving P129-WT or P129-GFP. Serological studies show that maternal antibodies for PCV2decay during theﬁrst2months of life(Rodríguez et al.,2002;Larochelle et al.,2003).Western blots and ELISA gave comparable results in this regard.In the current study,antibodies to PCV2only increased after day21of the study and did so only in pigs receiving P129-PCV2, suggesting that the increases were most likely speciﬁc responses to P129-PCV2vaccination.At the termination of the study(day35post-infection),tonsil samples in all3groups were positive for the PRRSV N gene by RT-PCR.The presence of the inserted GFP and PCV2genes in these same samples could not be conﬁrmed using primersﬂanking the region of the gene insertion.No products were ampliﬁed,even from pigs infected with the P129-WT virus.Failure to amplify the ORF1b/ORF2a junction is likely the result of template RNA concentra-tions below the level of detection of the PCR assay,and is consistent with the observation that the copy number of ORF1b(present only on genomic RNA)is much lower than the copy number of ORF7(present on all sgRNAs as well as genomic RNA).In conclusion,a PRRSV gene expression vector was generated, capable of expressing a foreign gene from an additional transcription unit located in the region between the non-structural and structural genes of the virus.The recombinant PRRSVs expressing the GFP or PCV2capsid genes were generated and shown to replicate well in cell culture.The addition of766nt(GFP)or754nt(PCV2capsid)of foreign genetic material,representing approximately5%of the PRRSV genome,was tolerated with no evidence of compensatory deletions or rearrangements elsewhere in the genome.Pigs inoculated with these recombinant PRRSVs produced foreign gene speciﬁc antibodies.Our study demonstrates the potential of PRRSV to function as a vector for development of multivalent vaccines against swine diseases.Materials and methodsCells and virusesMARC-145African green monkey kidney cells(Kim et al.,1993) were maintained as previously described(Lee et al.,2003).Dulac porcine kidney cells,kindly provided by L.Babiuk(Vaccine and Infectious Disease Organization,SK,Canada),were grown in Modiﬁed Eagle's Medium(MEM)supplemented with5%fetal bovine serum (FBS;Gibco BRL),penicillin(100U/ml),and streptomycin(50μg/ml). Cells were maintained at37°C with5%CO2.Stocks of recombinant viruses derived from infectious clones were prepared by passaging three times on MARC-145cells.Titers of PRRSV were determined by standard plaque assays on MARC-145cells using6-well plates(35mm diameter)in duplicate.Plaques were stained with0.01%neutral red. For isolation of PCV2,lung tissues were obtained from a PCR-positive pig(Ontario18099)submitted to Animal Health Laboratory of the University of Guelph(Guelph,ON,Canada).The tissues were homogenized in PBS and theﬁltrate was used to infect Dulac cells. At3days post-infection,cells were stained using a porcine circovirus hyperimmune serum(VMRD,Pullman,WA,USA)to conﬁrm infection. On day4post-infection,cells were harvested and freeze–thawed three times.Cell debris was removed by centrifugation at5000×g, and the supernatant was stored at−80°C until use.Construction of a PRRSV expression vector and recombinant PRRSVsFor construction of the PRRSV expression vector,the regions ﬂanking the ORF1b/ORF2a junction were ampliﬁed by PCR using the shuttle plasmid p2-7D4(containing genomic positions11,504to 15,395)as template.Two DNA products corresponding to positions 11,783to12,055and12,056to12,697were ampliﬁed.The primer set P129F1-F(containing an Eco47III site)and P129F1-R(containing AﬂII and Mlu I sites)was used for the upstream product(Table2).The primers set P129F2-F(containing Mlu I site and TRS6)and P129F2-R (containing a Bsr GI site)was used to amplify the downstream product(Table2).The twoﬂanking products were digested with Eco 47III–Mlu I and Mlu I–Bsr GI,respectively,and included in a three-way ligation with Eco47III–Bsr GI-digested full-length genomic cDNA clone pCMV-S-P129(Lee et al.,2003).The resulting construct pCMV-S-P129-1bMCS2contained a complete PRRSV genome with unique AﬂII and Mlu I sites and a copy of TRS6inserted between ORF1b and ORF2a.Transfection of MARC-145cells with this construct produced viable virus that replicated normally(data not shown).For insertion of foreign genes,pCMV-S-P129-1bMCS2was digested with AﬂII–Mlu I and ligated to either the GFP gene or the PCV2capsid gene into which AﬂII and Mlu I sites were introduced during PCR (Fig.1).Recombinant genomic clones were screened by Sma I digestion(Fig.2),and selected clones were sequenced to conﬁrm the presence of insertions.The PCV2capsid protein gene was cloned97Y.Pei et al./Virology389(2009)91–99。

PGC-1α通过上调SIRT3表达发挥心肌保护作用

PGC-1α通过上调SIRT3表达发挥心肌保护作用游嘉;岳中宝;陈少锐;李卓明;刘培庆【摘要】[目的]探讨第Ⅲ类组蛋白去乙酰化酶成员3(SIRT3)在过氧化物酶体增殖物受体γ共激活因子1α(PGC-1α)的调控下发挥改善心肌肥大和心肌能量代谢的作用.[方法]建立血管紧张素Ⅱ(Ang Ⅱ)诱导的心肌肥大模型;利用质粒转染诱导PGC-1α基因过表达、siRNA干扰敲低SIRT3表达;Western blot检测PGC-1α、SIRT3及心肌肥大标志蛋白肌球蛋白重链β(β-MHC)的水平.TMRE染色法测定线粒体膜电位;测定ATP含量;DHE染色法测定活性氧水平;qRT-PCR法检测线粒体编码基因、能量代谢相关基因的表达水平.[结果]过表达PGC-1α能够显著提高SIRT3的蛋白表达水平和酶活性(P＜0.05).过表达PGC-1α抑制Ang Ⅱ诱导的β-MHC上调(P＜0.05),增加ATP水平(P＜0.05),促进线粒体编码基因的转录水平(P ＜0.05),降低细胞内活性氧(P＜0.05),上调脂肪酸氧化磷酸化代谢相关基因的表达(P＜ 0.05).SIRT3干扰可逆转PGC-1α的心肌保护作用(P＜0.05).[结论]PGC-1α通过上调SIRT3发挥调控心肌肥大、线粒体功能、活性氧清除、能量代谢等方面的作用.【期刊名称】《中山大学学报（医学科学版）》【年(卷),期】2016(037)003【总页数】8页(P343-350)【关键词】PGC-1α;SIRT3;心肌肥大;能量代谢;活性氧;线粒体功能【作者】游嘉;岳中宝;陈少锐;李卓明;刘培庆【作者单位】中山大学药学院药理毒理实验室//新药成药性评估与评价国家地方联合工程实验室,广东广州510006;中山大学药学院药理毒理实验室//新药成药性评估与评价国家地方联合工程实验室,广东广州510006;中山大学药学院药理毒理实验室//新药成药性评估与评价国家地方联合工程实验室,广东广州510006;中山大学药学院药理毒理实验室//新药成药性评估与评价国家地方联合工程实验室,广东广州510006;中山大学药学院药理毒理实验室//新药成药性评估与评价国家地方联合工程实验室,广东广州510006【正文语种】中文【中图分类】R966心脏作为高耗能器官，其正常功能的维持依赖于心肌细胞对能量底物的合理利用以及通过线粒体氧化磷酸化途径合成ATP的能力。

pcdna3.1(+)质粒图谱

4. Select a transformant with the correct restriction pattern and use sequencing to confirm that your gene is cloned in the proper orientation.
5. Transfect your construct into the mammalian cell line of interest using your own method of choice. Generate a stable cell line, if desired.
pcDNA™3.1(+) pcDNA™3.1(–)
Catalog nos. V790-20 and V795-20 Version J 5 August 2008 28-0104
User Manual
ii
Table of Contents
Important Information........................................................................................................................v Accessory Products ............................................................................................................................vi
Appendix...................................................................................................................... 10

MCP-1

【关键词ｌ结核性脓胸；ＭＣＰ．１基因；单核苷酸多态性（ＳＮＰ）
ＡｓｓｏｃｉａｔｉｏｎｏｆＭＣＰ－１ｇｅｎｅｐｏｌｙｍｏｒｐｈｉｓｍｔｏｔｈｅｒｉｓｋｏｆｔｕｂｅｒｃｕｌｏｕｓｅｍｐｙｅｍａＸＵＷｅｉ — ｌｅ，ＱＩＨａｉ — ｌｉａｎｇ，ＱＩＮ
ｏｆＢＣＧｖａｃｃｉｎａｔｉｏｎａｎｄＢＭＩｓｔａｔｕｓ，ｔｈｅｉｎｄｉｖｉｄｕａｌｓ，ｅｓｐｅｃｉａｌｌｙｉｎｔｈｅｇｒｏｕｐｗｉｔｈｏｕｔｈｉｓｔｏｙｒｏｆＢＣＧｖａｃｃｉｎａｔｉｏｎｏｒｉｎ
ｗｅｒｅａｎａｌｙｚｅｄｂｙｐｏｌｙｍｅｒａｓｅｃｈａｉｎｒｅａｃｔｉｏｎ — ｒｅｓｔｉｒｃｔｉｏｎｆｒａｇｍｅｎｔｌｅｎｇｔｈｐｏｌｙｍｏｒｐｈｉｓｍ（ＰＣＲ－ＲＦＬＰ）ｉｎ５００ｐａｔｉｅｎｔｓ
ａｎｄ一３６２Ｇ／Ｃｔｏｔｈｅｉｒｓｋｏｆｔｕｂｅｒｃｕｌｏｕｓｅｍｐｙｅｍａ（ＴＥ）．ＭｅｔｈｏｄｓＴｈｅＳＮＰｓｏｆＭＣＰ一１－２５１８Ａ／Ｇａｎｄ一３６２Ｇ／Ｃ

急性冠脉综合征患者共刺激分子PD-1、CD28的表达变化分析

急性冠脉综合征患者共刺激分子PD-1、CD28的表达变化分析魏玮;章树业;张政;陈牧雷;范谦【期刊名称】《中国现代医生》【年(卷),期】2012(50)18【摘要】目的探讨外周血T淋巴细胞共刺激分子,程序性细胞死亡因子-1(PD-1)和CD28在急性冠脉综合征(ACS)疾病进展中的意义.方法选取急性心肌梗死(AMI组)患者25例、不稳定型心绞痛(UAP组)患者25例、稳定型心绞痛(SAP组)患者10例作为研究对象,临床上冠脉造影正常的患者11例作为对照组,采用流式细胞仪测定各组患者CD4+T淋巴细胞上PD-1和CD28的表达情况.结果与SAP组和对照组相比,AMI组及UAP组CD4+、PD-1+双阳性细胞比例增高(P＜0.05),而CD4+、CD28+双阳性细胞比例无明显变化(P＞0.05).结论急性冠脉综合征患者中,作为负调控因子的PD-1表达增强,可能在冠状动脉粥样硬化斑块失稳定和疾病进展中起到作用.【总页数】3页(P58-60)【作者】魏玮;章树业;张政;陈牧雷;范谦【作者单位】浙江医院干部保健科,浙江杭州 310013;解放军第三○二医院生物治疗研究中心,北京 100039;解放军第三○二医院生物治疗研究中心,北京 100039;首都医科大学附属北京朝阳医院心脏中心,北京 100020;首都医科大学附属北京朝阳医院心脏中心,北京 100020【正文语种】中文【中图分类】R543.5【相关文献】1.HBV 慢性患者外周血单个核细胞CD28／B7家族共刺激分子mRNA 表达及其意义 [J], 王琳;赵春楠;史进方;彭群新;顾国浩2.共信号分子CD28、ICOS、PD-1、CTLA-4在系统性红斑狼疮患者外周血中的表达水平及意义 [J], 白冷媚;谢传美3.IRP患者骨髓单个核细胞中CD28/B7家族共刺激分子mRNA表达研究 [J],4.2型糖尿病肾病患者外周血CD28/CTLA4共刺激分子的表达及其意义 [J], 吴昊; 顾敏娟; 夏洪; 黄慧5.冠状动脉粥样硬化性心脏病患者外周血共刺激分子CTLA-4、CD28的表达 [J], 高秀梅;康秀文;王莹因版权原因，仅展示原文概要，查看原文内容请购买。

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