Figure 5. DMSO-induced autophagy is controlled through ATF4-mediated downregulation of phosphorylated AKT1. Expression of ATF4 and LC3 were assessed by western blotting and RT-PCR in the presence of siRNA against ATF4. (A and B) ATF4 siRNA were transiently transfected into HepG2 cells with LipofectAMINE 2000. The level of protein and mRNA expression of ATF4 was suppressed a dose- and time-dependent manner in HepG2 cells treated with 0.01% DMSO treatment. siRNA-transfected HepG2 cells were treatment 0.5 mM palmitate in the absence or presence of 0.01% DMSO for 6 h. (C) Knockdown of ATF4 expression significantly increased mRNA and protein expression of LC3 (D, left panel). And right panel of (D) indicated autophagic flux assays in the absence or presence of 50 nM CQ. The expression of LC3-II was further increased than by siATF4 alone. The experiments were performed five times under identical conditions. Results are shown as mean ± SEM *p < 0.05, **p < 0.01, ***p < 0.001 vs. untreated control cells. (E) Knockdown of ATF4 expression using siRNA suppressed ATF4 expression and phosphorylation of AKT1, MTOR and RPS6KB2, but increased LC3-II conversion. The experiments were performed four times under identical conditions. Results are shown as mean ± SEM *p < 0.05, **p < 0.01, ***p < 0.001 vs. palmitate-treated cells, ^††p < 0.01, ^†††p < 0.001 vs. palmitate and si-ATF4-treated cells, ^‡‡p < 0.01, ^‡‡‡p < 0.001 vs. palmitate and DMSO-treated cells. To demonstrate the attenuation of the palmitate-induced lipid accumulation by the knockdown of ATF4, lipid accumulation was determined by fat quantization in the presence of siRNA against ATF4. Scramble siRNA and ATF4 siRNA were transiently transfected into HepG2 cells with LipofectAMINE 2000. (F) Transfected HepG2 cells were treated palmitae for 6 h. siRNA against ATF4 statistically decreased palmitate-induced lipid accumulation. The experiments were performed four times under identical conditions. *p < 0.05, ***p < 0.001 vs. untreated control cells, ^†††p < 0.001 vs. scramble RNA-treated cells, ^‡‡‡p < 0.001 vs. siATF4-treated cells, ^∫∫∫p < 0.001 vs. palmitate-treated cells.