Figure 3.

The effects of DEPTOR on mTOR signaling in HuH-7 cells. (A) Knockdown of DEPTOR resulted in activation of S6K and a decrease of Akt phosphorylation. HuH-7 cells were infected with lentiviruses expressing shRNAs targeting DEPTOR or luciferase (shLuc). Cell lysates were analyzed by Western blotting for indicated proteins and phosphorylation states. P and T indicate phosphorylated and total protein, respectively. (B) DEPTOR knockdown in HuH-7 cells led to a decrease in proliferation. HuH-7 cells that bore shRNA against luciferase or DEPTOR were seeded on 48-well plates, and cell number at an indicated time point was evaluated by crystal violet staining. Data were normalized against OD595 values on d 1 of each treatment. Each experiment was performed in triplicate; error bars represent standard deviation (SD). **P < 0.01. (C) DEPTOR overexpression in HuH-7 cells caused an increase in the phosphorylation of Akt, whereas there was no obvious effect on the phosphorylation of S6K. (D) Transiently expressed DEPTOR in HuH-7 cells caused an increase in the phosphorylation of Akt, which could be counteracted by coexpression of GNMT or the N140S mutant GNMT. (E) DEPTOR overexpression extends survival of HuH-7 cells grown in medium containing 0.1% serum. GFP and DEPTOR stable cells were seeded on 48-well plates for 24 h (d 0); then cells were cultured in DMEM containing 0.1% serum. Cell proliferation was evaluated as described in (B). *P < 0.05; **P < 0.01. (F) Effects of DEPTOR and GNMT on apoptosis and autophagy in HuH-7 cells. Different HuH-7 stable cells were cultured in normal medium (10% serum) or in medium with 0.1% serum for 3 d and were then harvested and analyzed by Western blotting for indicated proteins. Each experiment was repeated three times. Panel B: ---○---, HuH-7-shLuc; —■—, HuH-7-shDEPTOR-1; — ▾— HuH-7-shDEPTOR-2; panel E: —○—, HuH-7-GFP; —●—, HuH-7-DEPTOR.