Roles of HNF4α on Smad2/3 binding and transcriptional activity of MIXL1 promoter. A, effects of HNF4α knockdown on transactivation of the MIXL1 promoter. HepG2 cells were transfected with siRNAs 1 day before transfection with the reporter constructs. siNC, negative control siRNA. B, effects of exogenous HNF4α on transactivation of the MIXL1 promoter. HNF4α was exogenously expressed in HaCaT cells, and transcriptional activity of MIXL1 reporter was determined. The lower panel shows the protein expression of HNF4α. IB, immunoblot. C, HNF4α (variant 2, RefSeq ID: NM_000457) or its C115R (CR) mutant, which does not bind to DNA, was overexpressed in HepG2 cells. The lower panel shows the protein expression of HNF4α and its mutant. D, effect of HNF4α siRNA on Smad2/3 binding to the MIXL1 locus. HepG2 cells were transfected with siRNAs 24 h before TGF-β stimulation. Cells were fixed 1.5 h after treatment, and ChIP-qPCR was performed as in Fig. 1B. Error bars, S.D.; *, p < 0.05; n.s., not significant.