Figure 7. Effects of autophagy inhibitors on cell death, apoptosis and senescence.

(A) Analysis of LC3-II accumulation in MDA-MB-231 cells. Cells were pretreated with or without 3-MA (5 mM) or CQ (50 μM) for 1 h and then RCE was added at the indicated concentrations for 48 h. Proteins were extracted and LC3-II accumulation was determined by western blot. (B) Inhibition of autophagy reduces cell death induced by RCE. MDA-MB-231 cells were pretreated as described above and treated for 48 h with 400 or 600 μg/mL RCE. Cell viability was determined using cytotoxicity assay kit as described in material and methods. Data are representative of three independent experiments. Statistical analysis of cell viability on control or treated cellswas performed using one-way ANOVA followed by LSD Post-Hoc test to determine significance (*p < 0.05, **p < 0.005, ***p < 0.001). (C) Western blot quantification of cleaved PARP in cells pretreated with and without autophagy inhibitors. (D) Effect of caspase inhibition of cell viability of RCE-treated MDA-MB-231 cells. MDA-MB-231 cells were pretreated with the pan-caspase inhibitor Z-VAD-FMK (50 μM) for 1 h and then treated for 48 h with 400 or 600 μg/mL RCE. Cell viability was determined using cytotoxicity assay kit as described in material and methods. Data are representative of three independent experiments. Statistical analysis of cell viability on control or treated cellswas performed using one-way ANOVA followed by LSD Post-Hoc test to determine significance (*p < 0.05, **p < 0.005, ***p < 0.001). (E) Effect of autophagy blockade on RCE-induced senescence. Cells were treated as described in A and stained, as described in material and methods, for SA-β-Gal activity to detect senescence. Data are representative of three independent experiments. Statistical analysis of senescent cells count on control or treated cells was performed using one-way ANOVA and univariate test to determine significance (**p < 0.01).