Figure 5

PL induces autophagy by increasing intracellular ROS. (a) U2OS/GFP-LC3 cells were treated with 10 μM PL for 0–8 h. Cell lysates were collected for the determination of ATP levels with a Bioluminescence Assay Kit CLS II from Roche Scientific (Indianapolis, IN, USA). (b) U2OS/GFP-LC3 cells were pre-treated with 1 mM MP for 6 h, followed by treatment with PL (10 μM) for 16 h. GFP-LC3 fluorescence was captured with fluorescence microscopy. White arrows indicate punctate GFP-LC3 fluorescence (autolysosomes/autophagosomes). (c) Expression of autophagy regulative genes and AMPK signaling molecules in U2OS/GFP-LC3 cells treated with PL and/or MP by western blot. Thirty micrograms of WCE proteins were loaded. GAPDH served as a loading control. (d and e) U2OS/GFP-LC3 cells were pre-treated with 5 mM NAC for 6 h, followed by treatment with PL (10 μM) for 16 h. GFP-LC3 fluorescence was captured with fluorescence microscopy. White arrows indicate punctate GFP-LC3 fluorescence (autolysosomes/autophagosomes) in (d). Folds of punctate GFP-LC3 numbers are shown in (e). (f) WT and null-ATG5 MEFs were pre-treated with 5 mM NAC for 6 h, followed by treatment with 10 μM PL for 48 h. Cell viability was determined by MTT assay. Data are shown as mean±S.D. (n=4). ***P<0.001