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. 2015 Oct 5;112(42):E5669–E5678. doi: 10.1073/pnas.1516219112

Fig. 5.

Fig. 5.

PGC-1α regulates HSF1-mediated transcription. (A) Analysis of the effects of PGC-1α on HSP mRNA half-life. Primary human hepatocytes were infected with an adenovirus encoding PGC-1α or a control adenovirus and then were mock treated or were treated with Actinomycin D for 18 h. HSP mRNA expression was determined by RT-PCR, and the half-life was calculated for the indicated transcripts. (B) Primary human hepatocytes were infected with an adenovirus encoding PGC-1α or with a control adenovirus, and intronic RNA expression of the indicated transcripts was determined by RT-PCR. (C) PGC-1α regulates the activity of HSP promoters. The indicated promoters were cloned in front of a luciferase reporter and were transfected into HepG2 cells together with a construct encoding Renilla under the control of a CMV promoter. After infection with either a PGC-1α–encoding adenovirus or a control adenovirus, cells were lysed, and luciferase activity was determined. All luciferase readings were normalized to Renilla readings. (D) PGC-1α regulates HSF1 transcriptional activity from an HSE-driven template. HepG2 cells were transfected with combinations of plasmids encoding HSF1, HSE-driven luciferase, and a template encoding luciferase but lacking HSEs, in addition to a template encoding CMV-driven Renilla. After infection with either a PGC-1α–encoding adenovirus or a control adenovirus, cells were lysed, and luciferase activity was determined. All luciferase readings were normalized to Renilla readings. (E) ChIP analysis for HSF1 in HepG2 cells infected with an adenovirus encoding PGC-1α or with a control adenovirus. Values represent enrichment of selected target gene promoters over an upstream region of the GAPDH promoter. (F) The transcriptional activity of HSF1 artificially tethered to DNA is repressed by PGC-1α. G4DBD or G4DBD fused to HSF1 was transfected into HepG2 cells together with a template containing G4DBD-binding sites and a template encoding CMV-driven Renilla. PGC-1α was coexpressed by adenoviral infection. Cells were lysed, and luciferase activity was determined. All luciferase readings were normalized to Renilla readings. (G) The L2/L3 motifs of PGC-1α are dispensable for HSP repression. HepG2 cells were infected with adenoviruses encoding PGC-1α or the L2/L3 mutant PGC-1α, as indicated, and mRNA expression of the shown genes was determined by RT-PCR analysis.