FIGURE 4.

Effect of knockdown of HNF4α on TGF-β-induced gene expression in HepG2 cells. A, confirmation of HNF4α-knocked down samples for microarray analysis. HepG2 cells were transfected with HNF4α siRNA and treated with 120 pm TGF-β for the indicated times and harvested. Expression of HNF4α was determined by RT-qPCR and normalized by glyceraldehyde-3-phosphate dehydrogenase (GAPDH). siNC, negative control siRNA. B, down-regulation of HNF4α protein expression by siRNA. C, phosphorylation levels of Smad2/3 by using HNF4α siRNA. The top two panels show phosphorylation of Smad2 and Smad3. The 3rd panel indicates the expression of total Smad2/3, and the bottom panel is a loading control. IB, immunoblot. D, heat map of the TGF-β-induced expression of target genes of Smad2/3 and HNF4α and effect of HNF4α siRNA. Target genes that have overlapping binding regions of Smad2/3 and HNF4α were sorted by their induction of probe signal values by TGF-β stimulation for 1.5 h and are represented by color bars in the 1st column, using the TM4 microarray software (60). Relative expression of these genes in HNF4α siRNA samples to the control siRNA is shown in the 2nd column. In addition, a list of genes whose expressions changed more than 1.5-fold is shown in the right panel with their expression changes. E, heat map of target genes of TGF-β with Smad2/3 binding regions common to HNF4α at 24 h after TGF-β stimulation are shown as in D. Genes whose expressions were changed more than 2-fold are shown in the right panel.