Fig. 5
TGFβ1 induces the expression of HNF4α-P2 and binding by c-JUN to its promoter. a, b Immunoblots of HNF4α-P1 and HNF4α-P2 isoforms in Hep3B cells treated with TGFβ1 and or AREG (50 nM) for a 12 and b 48 h (n = 2). c Immunoblots of HNF4α-P1 and HNF4α-P2 from Hep3B cells transfected with an HNF4α-P1 specific siRNA for 48 h and treated with TGFβ1. d Hep3B cells were pre-treated with TGFβ-RI inhibitor SB431542 (5 nM) and treated with TGFβ1 (for 8 h (n = 3); qPCR of HNF4α-P1 and P2, PCK1 and Ornithine Carbamoyltransferase (OCT). e SMAD4-silenced Hep3B cells were treated overnight with TGFβ1; qPCR of SMAD4, HNF4α-P1 and P2 isoforms, PCK1 and OCT f Hep3B cells were pretreated with TAK1 inhibitor NG25 at 0.5 or 1 μM and then treated with TGFβ1 for 8 h (n = 3). qPCR of HNF4α-P1 and P2 isoforms. g Hep3B cells were treated with TGFβ1 overnight in the presence of cellular Src (c-Src) inhibitor PP2 (10 μM); g qPCR of HNF4α-P1 and P2 m Immunoblots of HNF4α-P1 and HNF4α-P2. k Chromatin immunoprecipitation of Hep3B cells treated with TGFβ1 overnight; RNA Polymerase II (orange), phospho-c-JUN (red) antibodies and normal mouse IgG (blue) were used. qPCR of GAPDH promoter, HNF4α-P2 promoter, and HNF4α-P2 proximal intron 1. Fold Enrichment of Pol II or c-JUN to control IgG is presented. l Hep3B cells were treated with TGFβ1 for 24 h and with the addition of proteasome inhibitor MG132 (10 μM) 2 h before collection when indicated (n = 3); immunoblot of HNF4α-P1. o–r HNF4α-P2-silenced HepG2 cells were collected 8 h (RNA) or 24 h (Nuclei) after TGFβ1 treatment (5 ng/ml) (n = 4–6); o qPCR of HNF4α-P2; p immunoblot of nuclear HNF4α-P1 and P2 isoforms q qPCR of HNF4α-P1 target genes PCK1, ALB, F7 and r CYP7A1 and CYP27A1. s Primary human hepatocytes were silenced with siRNA-HNF4α-P2 and supernatant was collected 48 h after transfection and 8 h after TGFβ1 treatment. Total bile acids in supernatant were quantified (n = 3). Significance was determined by two-tailed Mann–Whitney U test in d, e, g, k, m, n, o *P < 0.05. For box-and-whisker plots: perimeters, 25th–75th percentile; midline, median. The TGFβ1 dose used was 5 ng/ml
