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. 2015 Apr 28;11(4):629–642. doi: 10.1080/15548627.2015.1023981

Figure 6.

Figure 6.

FOXO1 inhibits MTOR activity by elevating SESN3/Sesn3 expression. (A) HCT116 cells were transiently transfected with the FOXO1-specific siRNA followed by TSA treatment (0.5 μM) for 12 h (left panel). HCT116 cells were treated with 0.25 μM TSA in the absence or presence of FOXO1 inhibitor AS1842856 (100 nM) for 24 h (right panel). (B) Tsc2+/+ and tsc2−/− MEFs were treated with 1 μM TSA for 12 h and total RNA was isolated from cells. Total RNA was isolated from HCT116 cells, Tsc2+/+ and tsc2−/− MEFs, and the SESN3/Sesn3 mRNA level was quantified using real-time PCR. The Foxo1 mRNA level was also measured in Tsc2+/+ and tsc2−/− MEFs. Fold change in mRNA levels was calculated by normalizing to respective Gapdh groups. (C) and (D) HCT116 cells were transiently transfected with a nonspecific siRNA or the SESN3-specific siRNA followed by TSA treatment (0.5 μM) for 12 h. Total protein was extracted and subjected to immunoblotting for FOXO1, phospho-RPS6 (Ser235/236), RPS6, phospho-EIF4EBP1 (T37/46), and EIF4EBP1 antibodies. Total RNA was also isolated from HCT116 cells and the mRNA level of SESN3 was quantified using real-time PCR. Fold change in mRNA levels was calculated by normalizing to GAPDH.