HDACIs increase FOXO1 expression at the mRNA and protein levels. (A) HCT116 cells were treated with TSA (0.125, 0.25 or 0.5 μM for 24 h; or 0.5 μM for 6, 12, or 24 h); HepG2 cells were treated with TSA (0.25, 0.5 or 1 μM for 24 h; or 1 μM for 6, 12, or 24 h). The cells were then harvested and subjected to western blotting analysis to evaluate FOXO1, CDKN1A and acetylated FOXO1 (Ac-FOXO1) expression. (B) HCT116 cells were treated with TSA (0.25 μM) in the absence or presence of actinomycin D (1 μg/ml) for 16 h. Cell lysates were analyzed for protein levels of FOXO1 and CDKN1A. (C) HCT116 cells were treated with TSA (0.125, 0.25 or 0.5 μM) for 24 h; HepG2 cells were treated with TSA (0.25, 0.5 or 1 μM) for 24 h. Total mRNA was extracted and real-time PCR was performed to evaluate changes in FOXO1 mRNA level. GAPDH was used as a loading control for real-time PCR.