Figure 3.

TSA inhibits mTOR activity and enhances FOXO1 transcriptional activity. A – U2OS cells were treated with 0.5 μM TSA for different times (6, 12 or 24 h). Total protein was extracted and subjected to immunoblotting for phospho-AKT (Ser473), AKT, phospho-S6 (Ser235/236), S6, phospho-FOXO1 (Ser256), FOXO1 and P21. β-Actin was used as a loading control. B – U2OS cells were treated with TSA (0.5 μM) for different times (6, 12 or 24 h). To track the subcellular localization of FOXO1, cytosolic and nuclear proteins from control and TSA-treated cells were probed for FOXO1. The same membrane was then stripped and reprobed for α-tubulin or lamin AC to ensure equal protein loading. C – U2OS cells were treated with TSA (0.5 μM) for 12 h. Total mRNA was extracted and real-time PCR was performed to evaluate changes in ATG4B, ATG12, PI3Kb, LC3 and ULK2 mRNA levels. GAPDH was used as a loading control for real-time PCR. The data are representative from 3 independent experiments