Enhanced basal and drug-induced autophagy in chemoresistant 5637 cells. Detection of the autophagic flux with the mRFP-GFP-LC3 tandem fluorescence construct by confocal microscopy (A and B). 5637 (A) and 5637rCDDP1000(B) cells were transiently transfected with mRFP-GFP-LC3 and treated with 15 μM of pan Bcl-2 inhibitor (−)-gossypol for 24 h. After fixation, nuclei were labeled with DAPI and digital images of representative cells were acquired. Bar, 10 μm. Quantification of lysosomal activity (C) Bladder cancer cells 5637, 5637rGEMCI20 and 5637rCDDP1000 were treated with 10 μM (−)-gossypol with or without Bafilomycin A1 (10 nM ) for 48 h and the net amounts of acidic vesicles in the cultures were measured by staining with Lysotracker Red DND-99 (25 nmol/L) and flow cytometry. *, P < 0.05, compared with the control. #, P < 0.05, compared with cultures not co-treated with inhibitor of autophagic flux Bafilomycin A1. §, P < 0.05 compared with the parenteral cell line. Western blot analysis of the expression of Mcl-1,LC-3 I, LC-3 II and p62 in 5637, 5637rGEMCI20 and 5637rCDDP1000 cells (D) Cells were treated with 10 μM of Mcl-1 sparing Bcl-2 inhibitor ABT-737 and 15 μM (−)-gossypol with or without Bafilomycin A1 (10 nM ) for 48 h. GAPDH served as a loading control.