Fig. 4

Decrease of combination therapy induced cell death by inhibiting autophagy in GSC11 cells. a–d GSC11 cells with 3-methyladenine (3-MA) treatment (a, b and upper panel of d) or with transfection of siRNA targeting Atg5 (siAtg5) (c and lower panel of d) were treated with vehicle (control), 0.6 μM erlotinib, 4 μM sorafenib or the combination for 8 h for immunoblotting using anti-LC3B or anti-Atg5 antibodies (d), or 72 h for apoptosis assay using propidium iodide (PI), annexin V (AV), and flow cytometer (a-i–a-iii ) or trypan blue dye exclusion assay (b and c). (a-i) Early apoptosis; PI (−) with AV (+). (a-ii) Non-apoptotic cell death; PI (+) with AV (−). (a-iii) Total cell death; PI (+). a–c Data are expressed as the means of three independent experiments. *means p < 0.05 in ANOVA and Tukey’s post hoc analysis comparing combination treatment group with others. p values shown were calculated by student’s paired t test. Error bars are 95 % confidence intervals. d Inhibitory effects of 3-MA and siAtg5 on LC3B-II increase was confirmed by immunoblotting. β-actin was examined as loading control. Results shown are representative of two independent experiments