Journal: Molecular Pharmacology

Article Title: Alterations of Histone Modifications Contribute to Pregnane X Receptor-Mediated Induction of CYP3A4 by Rifampicin

doi: 10.1124/mol.117.108225

Figure Lengend Snippet: Effect of rifampicin on the enrichment of NCOA6 and p300 around the PXRE in the CYP3A4 promoter. LS174T cells were treated with a solvent control (0.1% DMSO, v/v) or rifampicin (10 μM) for 48 or 96 hours, followed by ChIP analysis. (A–C) Enrichment of nuclear receptor PXR, (D–F) cofactor NCOA6, and (G–I) histone acetyltransferase p300 in the CYP3A4 promoter after LS174T cells were treated with the solvent control or rifampicin. Data are shown as mean ± S.D. of three independent experiments. *p < 0.05, **p < 0.01 versus control (one-way analysis of variance followed by Dunnett’s test). RIF, rifampicin.

Article Snippet: LS174T cells that were grown on coverslip-coated 24-well plates and treated with rifampicin or solvent for 48 hours were fixed with 4% paraformaldehyde and permeabilized with 0.1% Triton X-100, then blocked with 1% bovine serum albumin for 1.5 hours followed by overnight incubation of a primary antibody at 4°C and 1.5-hour incubation of a secondary antibody at room temperature: For PXR localization, an anti-PXR (1:50; Santa Cruz Biotechnology) and a Alexa Fluor 647-conjugated rabbit secondary antibody (1:200; Abcam) were used; for NCOA6 or p300 localization, an anti-NCOA6 (1:50; Proteintech, Wuhan, China) or anti-p300 (1:100; Abcam) with a Alexa Fluor 488-conjugated mouse secondary antibody (1:200; Abcam) were used.

Techniques:

Journal: Molecular Pharmacology

Article Title: Alterations of Histone Modifications Contribute to Pregnane X Receptor-Mediated Induction of CYP3A4 by Rifampicin

doi: 10.1124/mol.117.108225

Figure Lengend Snippet: Functions of PXR, NCOA6, and p300 in the induction of CYP3A4 by rifampicin. Knockdown of target genes by RNA interference (RNAi) in LS174T cells was performed by transfection of shRNA plasmids. Transiently or stably transfected cells were treated with a solvent DMSO control (0.1%, v/v) or rifampicin (10 μM) for 48 hours before RNA isolation. Knockdown of PXR by RNAi on mRNA (A) and protein levels (B). Effect of silencing PXR expression on CYP3A4 mRNA and the induction by rifampicin (C). Knockdown of NCOA6 by RNAi on mRNA (D) and protein levels (E). Effect of silencing NCOA6 expression on CYP3A4 mRNA and induction by rifampicin (F). Knockdown of p300 by RNAi on mRNA (G) and protein levels (H). Effect of silencing p300 expression on CYP3A4 mRNA and induction by rifampicin (I). Data are shown as mean ± S.D. of three independent experiments. *p < 0.05, **p < 0.01versus control (Student’s t test or one-way analysis of variance followed by Bonferroni’s post-hoc test). RIF, rifampicin.

Article Snippet: LS174T cells that were grown on coverslip-coated 24-well plates and treated with rifampicin or solvent for 48 hours were fixed with 4% paraformaldehyde and permeabilized with 0.1% Triton X-100, then blocked with 1% bovine serum albumin for 1.5 hours followed by overnight incubation of a primary antibody at 4°C and 1.5-hour incubation of a secondary antibody at room temperature: For PXR localization, an anti-PXR (1:50; Santa Cruz Biotechnology) and a Alexa Fluor 647-conjugated rabbit secondary antibody (1:200; Abcam) were used; for NCOA6 or p300 localization, an anti-NCOA6 (1:50; Proteintech, Wuhan, China) or anti-p300 (1:100; Abcam) with a Alexa Fluor 488-conjugated mouse secondary antibody (1:200; Abcam) were used.

Techniques: Transfection, shRNA, Stable Transfection, Isolation, Expressing

Journal: Molecular Pharmacology

Article Title: Alterations of Histone Modifications Contribute to Pregnane X Receptor-Mediated Induction of CYP3A4 by Rifampicin

doi: 10.1124/mol.117.108225

Figure Lengend Snippet: Effects of silencing NCOA6 or p300 expression on alteration of histone methylation or acetylation levels of the CYP3A4 promoter. Knockdown of NCOA6 or p300 was performed by transfecting shRNA plasmids into LS174T cells followed by treatment with a solvent DMSO control (0.1%, v/v) or rifampicin (10 μM) for 48 hours, and cells were harvested for ChIP analysis. The relative levels of H3K4me3 (A–C) or H3K27me3 (D–F) in the CYP3A4 promoter in the control and treated groups. The relative levels of H3 acetylation (G–I) in the CYP3A4 promoter in control and treated groups. Data are shown as mean ± S.D. of three independent experiments. *p < 0.05, **p < 0.01 (one-way analysis of variance followed by Bonferroni’s post-hoc test). RIF, rifampicin.

Article Snippet: LS174T cells that were grown on coverslip-coated 24-well plates and treated with rifampicin or solvent for 48 hours were fixed with 4% paraformaldehyde and permeabilized with 0.1% Triton X-100, then blocked with 1% bovine serum albumin for 1.5 hours followed by overnight incubation of a primary antibody at 4°C and 1.5-hour incubation of a secondary antibody at room temperature: For PXR localization, an anti-PXR (1:50; Santa Cruz Biotechnology) and a Alexa Fluor 647-conjugated rabbit secondary antibody (1:200; Abcam) were used; for NCOA6 or p300 localization, an anti-NCOA6 (1:50; Proteintech, Wuhan, China) or anti-p300 (1:100; Abcam) with a Alexa Fluor 488-conjugated mouse secondary antibody (1:200; Abcam) were used.

Techniques: Expressing, Methylation, shRNA

Journal: Molecular Pharmacology

Article Title: Alterations of Histone Modifications Contribute to Pregnane X Receptor-Mediated Induction of CYP3A4 by Rifampicin

doi: 10.1124/mol.117.108225

Figure Lengend Snippet: Effects of silencing PXR expression on alterations of histone methylation and acetylation levels as well as enrichment of NCOA6 and p300 in the CYP3A4 promoter. Knockdown of PXR was performed by transiently transfecting shRNA plasmids into LS174T cells followed by treatment with a solvent DMSO control (0.1%, v/v) or rifampicin (10 μM) for 48 hours, and cells were harvested for ChIP analysis. The relative levels of H3K4me3 (A–C), H3K27me3 (D–F), H3 acetylation (G–I) in the CYP3A4 promoter in control and treated groups. The relative enrichment of NCOA6 (J–L) and p300 (M–O) in the CYP3A4 promoter in control and treated groups. Data are shown as mean ± SD of three independent experiments. *p < 0.05, **p < 0.01 (one-way analysis of variance followed by Bonferroni’s post-hoc test). RIF, rifampicin.

Article Snippet: LS174T cells that were grown on coverslip-coated 24-well plates and treated with rifampicin or solvent for 48 hours were fixed with 4% paraformaldehyde and permeabilized with 0.1% Triton X-100, then blocked with 1% bovine serum albumin for 1.5 hours followed by overnight incubation of a primary antibody at 4°C and 1.5-hour incubation of a secondary antibody at room temperature: For PXR localization, an anti-PXR (1:50; Santa Cruz Biotechnology) and a Alexa Fluor 647-conjugated rabbit secondary antibody (1:200; Abcam) were used; for NCOA6 or p300 localization, an anti-NCOA6 (1:50; Proteintech, Wuhan, China) or anti-p300 (1:100; Abcam) with a Alexa Fluor 488-conjugated mouse secondary antibody (1:200; Abcam) were used.

Techniques: Expressing, Methylation, shRNA

Journal: Molecular Pharmacology

Article Title: Alterations of Histone Modifications Contribute to Pregnane X Receptor-Mediated Induction of CYP3A4 by Rifampicin

doi: 10.1124/mol.117.108225

Figure Lengend Snippet: Effects of rifampicin on the nuclear accumulation of PXR, NCOA6, and p300, as well as interactions between PXR and NCOA6/p300. (A) Representative immunofluorescence images of PXR, NCOA6, and p300 in the treated LS174T cells. Bar, 10 μm. All cells were stained with DAPI (blue, column 1, rows I, II, III, and IV) to visualize the nuclei. Cells were stained with an antibody of anti-NCOA6 (green, column 2, rows I and II), anti-p300 (green, column 2, rows III and IV), and anti-PXR (red, column 3, rows I, II, III, and IV) to visualize the nuclear and cytoplasmic distribution of NCOA6, p300, and PXR. (B) LS174T cells were cotransfected with PXR and NCOA6 or p300 expression plasmids, and further incubated with rifampicin (10 μM) or a solvent DMSO control (0.1%, v/v) for 48 hours after the transfection. Cells were harvested for coimmunoprecipitation and detected by Western blot. All experiments were performed in three independent replicates. RIF, rifampicin.

Article Snippet: LS174T cells that were grown on coverslip-coated 24-well plates and treated with rifampicin or solvent for 48 hours were fixed with 4% paraformaldehyde and permeabilized with 0.1% Triton X-100, then blocked with 1% bovine serum albumin for 1.5 hours followed by overnight incubation of a primary antibody at 4°C and 1.5-hour incubation of a secondary antibody at room temperature: For PXR localization, an anti-PXR (1:50; Santa Cruz Biotechnology) and a Alexa Fluor 647-conjugated rabbit secondary antibody (1:200; Abcam) were used; for NCOA6 or p300 localization, an anti-NCOA6 (1:50; Proteintech, Wuhan, China) or anti-p300 (1:100; Abcam) with a Alexa Fluor 488-conjugated mouse secondary antibody (1:200; Abcam) were used.

Techniques: Immunofluorescence, Staining, Expressing, Incubation, Transfection, Western Blot