Fig. 5

Penfluridol induces reactive oxygen species (ROS)-mediated autophagy via triggering light chain 3 (LC3) turnover and acidic vesicular organelle (AVO) formation. a and b U937 and HL-60 cells were treated with the indicated concentrations of penfluridol for 24 h. Cell lysates were probed with antibodies against cleaved caspase-3, LC3 (a), and p62 (b). c and d Treatment of U937 and HL-60 cells with 7.5 μM penfluridol (c) or treatment of MV4–11 cells with 5 μM penfluridol (d) for the indicated time points. LC3 conversion (LC3-I to LC3-II), pro-poly (ADP ribose) polymerase (PARP) cleavage, and p62 degradation were detected by a Western blot analysis. β-actin served as a loading control. e U937 and HL-60 cells treated with 7.5 μM penfluridol for 12 h were stained with acridine orange (AO) and subjected to flow cytometry. Top of the grid was considered AVOs. Data are presented as the mean ± SD. *** p < 0.001 versus the vehicle control group. f U937 cells were treated with 35 μM cycloheximide or cycloheximide plus penfluridol for the indicated time points. The p62 degradation was detected by a Western blot analysis. Quantitative results of p62 protein levels are shown at the bottom. g U937 and HL-60 cells were exposed to N-acetylcysteine (NAC) (5 mM) for 1 h, followed by treatment with penfluridol (7.5 μM) for 24 h, and levels of LC3 conversion and cleaved PARP were analyzed by Western blotting. β-actin was the internal standard for protein loading