FIGURE 2.

FoxO1 knockdown reduces IGFBP1 transcript and curtails FoxA1/A2 binding and RNA polymerase II recruitment to the IGFBP1 promoter. A, Western blot of FoxO1 protein in unstimulated/insulin stimulated scrambled or FoxO1 shRNA-infected HepG2 cells. B, quantitative RT-PCR analysis of IGFBP1 expression in unstimulated/insulin-stimulated HepG2 cells infected with lentivirus expressing scrambled or FoxO1 shRNA. Values are expressed as the -fold change over non-insulin-treated cells infected with the scrambled shRNA. Data represent the mean ± S.E. (error bars) for four independent biological experiments and two technical replicates per biological replicate. C–E, ChIP analysis was performed using antibodies directed against FoxO1 (C), RNA polymerase II (D), and FoxA1/A2 (E) on chromatin isolated from unstimulated/insulin-stimulated scrambled or FoxO1 shRNA-infected HepG2 cells. Results were normalized to total input as an internal control, and values are expressed as a ratio over non-insulin-treated cells infected with the scrambled shRNA. All data represent the mean ± S.E. for four independent biological experiments and two technical replicates per biological replicate. **, p < 0.01, Student's t test. F and G, Western blot of FoxA1 (F) and FoxA2 (G) proteins in unstimulated/insulin-stimulated scrambled or FoxO1 shRNA-infected HepG2 cells.