Figure 1

Inhibition of mTOR leads to decreased lipogenesis in human hepatoma cells. (a) HepG2 cells were incubated with 100 nM rapamycin (Rapa), 250 nM of torin1 (Tor) and vehicle (Con) for 24 h and whole-cell lysates were analyzed by immunoblotting with indicated antibodies. β-Actin serves as a loading control. (b) HepG2 cells were transfected with scrambled siRNA (sc) and siRNA against raptor (Rap), rictor (Ric) or both (Rap+Ric). The whole-cell lysates harvested after 48 h of transfection were analyzed by immunoblotting. (c) HepG2 cells were treated with Rapa and Tor for 24 h or transfected with siRNA against raptor, rictor or both as described in panels a and b. SREBP-1c mRNA levels were quantified in triplicate samples using quantitative PCR and normalized with 18S RNA expression. Data are expressed as mean±SD relative to the expression in control- (Con, left panel) or scrambled siRNA- (sc, right panel) transfected cells. (d) HepG2 cells were treated with Rapa or Tor (left panel) and transfected with siRNA against raptor, rictor or both (right panel), followed by incubation with ¹⁴C-labeled acetate for 2 h. The incorporation of ¹⁴C in the lipid phase was measured in triplicate samples in a β-scintillation counter and normalized with total cellular proteins. (e) Following treatment with Rapa and Tor (left panel) or transfected with indicated siRNA (right panel), HepG2 cells were incubated with ¹⁴C-labeled glucose for 2 h. The incorporation of ¹⁴C in the lipid phase was measured in as described in Materials and Methods. All panels: n≥3, *P<0.05, **P<0.01 compared with Con or sc cells.