Figure 3: FoxM1 inhibits expression of FoxA2 by recruiting Rb and DNMT3b onto the FoxA2 promoter:

(A) Huh7 cells or HepG2 cells were transfected with empty vector (control) or vector expressing T7-FoxM1b. Forty-eight hours after transfection, cells were harvested for RNA assays. Total RNA (1µg) was analyzed by quantitative real time PCR (qRT-PCR) for expression of FoxA2 and GAPDH. Huh7 cells (B) or SNU449 cells (C) were transfected with control-siRNA or FoxM1-siRNAs. Seventy-two hours after siRNA transfection, cells were harvested for RNA (1 µg) assays. (D) Huh7 cells were subjected to crosslinking for chromatin-IP (18). The chromatin preparations were immunoprecipitated with FoxM1-ab or IgG. Enrichments of the FoxA2 promoter fragments were assayed by quantitative RT-PCR, and the relative enrichments with FoxM1-ab over that with IgG after normalization against a non-specific site are shown. (E-F) Huh7 cells transfected with control-siRNA or FoxM1 siRNA for 72h, and then processed for ChIP using Rb antibody (E) or DNMT3b-antibody. (F) Relative enrichments of the FoxA2 promoter fragments from control-siRNA transfected cells over those from the FoxM1-siRNA transfected cells are shown. Statistical calculations were done using GraphPad Prism online tool for t-test and p values stated as *p≤0.05and **p≤0.001 and ***p≤0.0001.