Figure 2.

TSA treatment activates FOXO1 and causes MCF10A-ras cell death. (A) MCF10A and MCF10A-ras cells were treated with 0.5 µM TSA for 12 or 24 h. The cells were harvested for mRNA extraction and qPCR was performed to determine FOXO1 level. GAPDH was used as an internal control. **P<0.01, ***P<0.001. (B) MCF10A and MCF10A-ras cells were treated with 0.5 µM TSA for 12 or 24 h. The cells were harvested and subjected to western blotting analysis to evaluate FOXO1, P21 and cleaved Caspase-3 expression. β-actin was used as a loading control. (C) Control siRNA and FOXO1 siRNA were transfected into MCF10A-ras cells according the protocol. MCF10A-ras cells then were treated with 0.5 µM TSA for 24 h. Cells were harvested and immunoblotted for FOXO1, PARP1 and cleaved Caspase-3 antibodies. β-actin was used as a loading control. (D) MCF10A-ras cells were transiently transfected with control siRNA and FOXO1 siRNA according to the protocol and then were treated with 0.5 µM TSA for 24 h. The cell viability was measured using PI live cell uptake assay coupled with flow cytometry. Data are illustrated as the mean ± SD. **P<0.01, ***P<0.001. (E) Flow cytometry of MCF10A-ras cells were transiently transfected with control siRNA and FOXO1 siRNA and then were treated with 0.5 µM TSA for 24 h. TSA, trichostatin A; FOXO1, forkhead box O1.