Figure 3. Induction of autophagy has no impact on MGMT expression. (A) HepG2 and Huh7 cells were transfected with plasmid expressing LC-3 or a control vector, and treated with 0 or 4 μM epirubicin for 6 h. LC3-II puncta was observed by fluorescence microscope (scale bar, 50μm). Representative images are shown. (B) Determination of LC3-II puncta per cell. About 100 cells were used in each experiment. Data are expressed as mean±SD. ** P≤0.01; ***P≤0.001. (C, D) HepG2 and Huh7 cells were treated with increasing epirubicin concentrations for 24 h, and the autophagic markers LC3-II and p62 were analyzed by western blot assay; GAPDH was used as loading control. (E, F) HepG2 and Huh7 cells were treated with epirubicin for 24 h or 48 h, and MGMT levels were analyzed by western blot. (G–J) Quantification of P62 and LC3-II/I were shown as mean±SD. * P≤0.05. (K, L) Quantification of MGMT were shown as mean±SD. (M, N) HepG2 and Huh7 cells were treated with 2 μM epirubicin for indicated times, and MGMT mRNA levels were analyzed by quantitative RT-PCR. The data were normalized to GAPDH levels, and to untreated cells (mean±SD).