Markers were obtained from Pharmacia (Saclay, France). Polyvinylidene Fluoride (PVDF) membrane for western blotting was obtained from Millipore (Bedford, MA). All other reagents and chemicals were of the highest purity grade available.PlasmidspVP-PXR, pCYP3A4-Luc, pRL-tk and short hairpin RNA (shRNA) construct against the hPXR were kindly provided by Dr. Wen Xie (University of Pittsburg). pCMV-3Xflag was kindly provided by Dr. Richard G. Pestell (Georgetown University). The cDNA of human PXR was subcloned into pCMV-3Xflag between HindIII and XbaI sites by PCR using the following pair of oligonucleotides: forward primer, 59- ATTAAGCTTCTGGAGGTGAGACCCAAAGA-39, reverse primer: 59- ATTTCTAGATCAGCTACCTGTGATGCCGA-39. The pVP-PXR wasFigure 1. Rifampicin induced PXR nuclear translocation. HEK293T cells were transfected with pCMV-36flag-hPXR for 24 h, then treated with rifampicin (Rif, 20 mM) for 2 h. PXR was detected using an anti-PXR polyclone antibody and FITC-tagged second antibody. Nucleuses were stained by DAPI. doi:10.1371/journal.pone.0067959.gSCD1 Contributes to the Lipogenic Effect by PXRFigure 2. Rifampicin induced lipid accumulation in HepG2 cells. A. Oil red O staining of HepG2 cells. HepG2 cells were treated with rifampicin 5 mM (A-3), 10 mM (A-4), 20 mM(A-5) or TO901317 (10 mM, A-2) for 48 h. Cells treated with DMSO (A-1) were used as control. B. The stained lipid content was quantified by measuring Title Loaded From File absorbance at 510 nm. The triglyceride (TG, C), total cholesterol (TC, D) 1315463 and free cholesterol (FC, E) levels were measured. F. The ratio of TC/FC. All experiments were repeated at least three times. *, P,0.05. doi:10.1371/journal.pone.0067959.gused as the PCR template. The different lengths of the 59regulatory sequences of human SCD1 gene were cloned by PCR. The forward primers were: 59- GGAAGATCTATGGTAAGGCTCCTACAGACA-39 for SCD1-1039, 59- GGAAGATCTACGGTTTCCACAAAGAAGAT-39 for SCD1-653,59- AATAGATCTGGGCAGAGCCATTGTTCG-39 for SCD1436 and 59- AATAGATCTCGAGGGTTCACCACTGTTT-39 for SCD1-267. The Title Loaded From File common reverse primer is 59- CCCAAGCTTAAATGCTAATGAGGCTTCTG-39. Genomic DNA isolated from the HepG2 cells was used as the PCR template. The PCRSCD1 Contributes to the Lipogenic Effect by PXRFigure 3. Genes expression analysis in HepG2 cells. HepG2 cells were treated with rifampicin at indicated concentrations for 48 h. Total RNA was isolated and the selected lipid metabolism genes expression was determined by RT-PCR. A. Expression of CYP3A4, CD36 and ABCG1. B. Expression of several lipogenic genes. C, The relative gene level was analyzed using ImageJ from at least three independent experiments. *, P,0.05; **, P,0.01. D, Knockdown of PXR by shRNA abolished rifampicin-induced SCD1 gene expression in HepG2 cells. E. The SCD1 gene protein level in HepG2 cells after incubation with rifampicin. The intensity of the bands was measured using ImageJ. *, P,0.05. F. The expression of LCAT and ACAT1 gene. doi:10.1371/journal.pone.0067959.gproducts were cloned into the pGL3 vector between the BglII and HindIII sites. Site-directed mutagenesis was performed by the PCR overextension method [19]. All newly constructed plasmids, as well as the site-directed mutagenesis, were confirmed by DNA sequencing.Cell Culture, PXR Stable Cell Line and PXR Knockdown ExperimentsHEK293T and HepG2 cells were obtained from the Institute Hospital Chinese Academy of Medical Sciences. Both cell lines were maintained in DMEM supplemented with 10 FBS, 100 units/ml penicillin and 100 mg/m.Markers were obtained from Pharmacia (Saclay, France). Polyvinylidene Fluoride (PVDF) membrane for western blotting was obtained from Millipore (Bedford, MA). All other reagents and chemicals were of the highest purity grade available.PlasmidspVP-PXR, pCYP3A4-Luc, pRL-tk and short hairpin RNA (shRNA) construct against the hPXR were kindly provided by Dr. Wen Xie (University of Pittsburg). pCMV-3Xflag was kindly provided by Dr. Richard G. Pestell (Georgetown University). The cDNA of human PXR was subcloned into pCMV-3Xflag between HindIII and XbaI sites by PCR using the following pair of oligonucleotides: forward primer, 59- ATTAAGCTTCTGGAGGTGAGACCCAAAGA-39, reverse primer: 59- ATTTCTAGATCAGCTACCTGTGATGCCGA-39. The pVP-PXR wasFigure 1. Rifampicin induced PXR nuclear translocation. HEK293T cells were transfected with pCMV-36flag-hPXR for 24 h, then treated with rifampicin (Rif, 20 mM) for 2 h. PXR was detected using an anti-PXR polyclone antibody and FITC-tagged second antibody. Nucleuses were stained by DAPI. doi:10.1371/journal.pone.0067959.gSCD1 Contributes to the Lipogenic Effect by PXRFigure 2. Rifampicin induced lipid accumulation in HepG2 cells. A. Oil red O staining of HepG2 cells. HepG2 cells were treated with rifampicin 5 mM (A-3), 10 mM (A-4), 20 mM(A-5) or TO901317 (10 mM, A-2) for 48 h. Cells treated with DMSO (A-1) were used as control. B. The stained lipid content was quantified by measuring absorbance at 510 nm. The triglyceride (TG, C), total cholesterol (TC, D) 1315463 and free cholesterol (FC, E) levels were measured. F. The ratio of TC/FC. All experiments were repeated at least three times. *, P,0.05. doi:10.1371/journal.pone.0067959.gused as the PCR template. The different lengths of the 59regulatory sequences of human SCD1 gene were cloned by PCR. The forward primers were: 59- GGAAGATCTATGGTAAGGCTCCTACAGACA-39 for SCD1-1039, 59- GGAAGATCTACGGTTTCCACAAAGAAGAT-39 for SCD1-653,59- AATAGATCTGGGCAGAGCCATTGTTCG-39 for SCD1436 and 59- AATAGATCTCGAGGGTTCACCACTGTTT-39 for SCD1-267. The common reverse primer is 59- CCCAAGCTTAAATGCTAATGAGGCTTCTG-39. Genomic DNA isolated from the HepG2 cells was used as the PCR template. The PCRSCD1 Contributes to the Lipogenic Effect by PXRFigure 3. Genes expression analysis in HepG2 cells. HepG2 cells were treated with rifampicin at indicated concentrations for 48 h. Total RNA was isolated and the selected lipid metabolism genes expression was determined by RT-PCR. A. Expression of CYP3A4, CD36 and ABCG1. B. Expression of several lipogenic genes. C, The relative gene level was analyzed using ImageJ from at least three independent experiments. *, P,0.05; **, P,0.01. D, Knockdown of PXR by shRNA abolished rifampicin-induced SCD1 gene expression in HepG2 cells. E. The SCD1 gene protein level in HepG2 cells after incubation with rifampicin. The intensity of the bands was measured using ImageJ. *, P,0.05. F. The expression of LCAT and ACAT1 gene. doi:10.1371/journal.pone.0067959.gproducts were cloned into the pGL3 vector between the BglII and HindIII sites. Site-directed mutagenesis was performed by the PCR overextension method [19]. All newly constructed plasmids, as well as the site-directed mutagenesis, were confirmed by DNA sequencing.Cell Culture, PXR Stable Cell Line and PXR Knockdown ExperimentsHEK293T and HepG2 cells were obtained from the Institute Hospital Chinese Academy of Medical Sciences. Both cell lines were maintained in DMEM supplemented with 10 FBS, 100 units/ml penicillin and 100 mg/m.
