Figure 4.

Effects of GNMT on mTOR signaling, cell cycle progression and proliferation. (A) Stably expression of GNMT in HuH-7 cells causes an increase in the phosphorylation of 4E-BP, which can be counteracted by transiently transfecting DEPTOR into a GNMT stable cell line. (B, C) GNMT does not interact with mTOR but interferes with the interaction between DEPTOR and mTOR. HuH-7 cells were transfected with indicated plasmids and harvested for immunoprecipitation analysis. Endogenous mTOR is not present in the anti-FLAG immunoprecipitants (B), and FLAG-tagged GNMT is not present in the precipitant pulled down by antibody against endogenous mTOR (C). In GNMT-expressing cells, the amount of DEPTOR in the mTOR precipitants is decreased and vice versa (C). (D) GNMT overexpression in HuH-7 cells led to a decrease in proliferation. GFP and GNMT stable cells were seeded on 48-well plate; cell proliferation was evaluated as described in Figure 3B. *P < 0.05. (E) GNMT overexpression in HuH-7 cells led to G2/M arrest. GFP and GNMT stable cells were synchronized at the G0/G1 phase and allowed to reenter the cell cycle. The cells were fixed at the indicated time point, and cell cycle profiles were recorded by flow cytometry. Quantification of the percentage of cells in the G2/M phase of the cell cycle is shown. Each experiment was repeated at least two times. (F) Expression of SA-β-gal in GFP and GNMT stable cells. Quantification of β-gal expression in GFP and GNMT stable cell lines are shown. Data are presented as means ± SD of 15–20 random fields with 100× magnification from two independent experiments. **P < 0.01. IP, immunoprecipitation; Ig H, IgG heavy chain; —○—, HuH-7-GFP; ----●----, HuH-7-GNMT.