Figure 3.

Clofoctol inhibits autophagic flux in PC3 cells. (A) PC3 cells were treated with the indicated concentrations of clofoctol for 24 h. The protein levels of phosphorylated c-Jun, LC3B-I/II, PARP and tubulin were assessed by western blot analysis. (B) PC3 cells were transiently transfected with pEGFP-LC3 for 24 h and were treated with DMSO or clofoctol (20 μM) for 6 h. The EGFP-LC3 fluorescence was observed under the confocal microscope. Intense green dots are indicative of LC3 puncta. (C) PC3 cells were treated with indicated concentrations of clofoctol (CFT) and a JNK inhibitor, SP600125 (SP) for 24 h. The protein levels of PARP, phosphorylated c-Jun, LC3B-I/II and tubulin were assessed by Western blot analysis. (D) PC3 cells were treated with either DMSO (Ctrl), 20 μg·mL⁻¹ tunicamycin (TM), 3 μM thapsigargin (TG) or 25 μM clofoctol (CFT) for 6 h and then were harvested for Western blot analysis of LC3B-I/II. Bafilomycin A1 (BAF-A1, 100 nM) was added 2 h before harvesting the cells. (E) PC3 cells were transiently transfected with pEGFP-LC3 for 24 h and were treated with either DMSO (Ctrl), 20 μg·mL⁻¹ tunicamycin (TM), 3 μM thapsigargin (TG) or 25 μM clofoctol (CFT) for 6 h. Either DMSO (–BAF-A1) or bafilomycin A1 (+BAF-A1) was added 2 h before processing for the confocal microscope analysis of EGFP-LC3.