FIGURE 3.

TGFβ activation of autophagy acts through E2F1. A, HuH7 and H1299 cells were transiently transfected with siRNA against E2F1 or a control nonsilencing siRNA and stimulated with TGFβ for 24 h. mRNA levels for the specified autophagy genes were measured by real time qPCR analysis. The results are normalized to GAPDH and shown relative to levels observed in untreated cells (set to 1). The data are represented as means ± S.D. *, p < 0.05; **, p < 0.01. B, HuH7 cells were transiently transfected with a control nonsilencing siRNA or siRNA against E2F1 and E2F1 mRNA levels (left panel), and protein levels (right panel) were determined by real time qPCR and Western blot analysis, respectively. The data are represented as means ± S.D. ***, p < 0.001. C and D, following siRNA transfection, HuH7 cells were treated or not with TGFβ and LC3 conversion (C) and GFP-LC3 puncta formation (D) were analyzed by immunoblotting and fluorescence microscopy, respectively. The effect of E2F1 knockdown on Beclin-1 and p62 expression was also evaluated by immunoblotting (C). E and F, HuH7 cells were transiently transfected with siRNA against E2F4 or a control nonsilencing siRNA and stimulated with TGFβ for 24 h. The mRNA levels for the indicated genes were measured as in A. **, p < 0.01; (***, p < 0.001. E, middle and right panels, E2F4 mRNA levels (middle panel) and protein levels (right panel) were determined by real time qPCR and Western blot analysis.