Effect of CLCN3 antisense transfection on cisplatin cytotoxicity and lysosome dysfunction. U251 cells were treated with or without cisplatin (15 µmol/l) for 24 h following transfection of nonsense or CLCN3 antisense. (A) Analysis of apoptotic pathways triggered by cisplatin and CLCN3 antisense. Western blot analysis of TP53, BCL2, BAX, Cathepsin D expression and caspase 3 cleavage. β-actin was used as the loading control. Values represent means of three independent experiments. (B) Cathepsin D activity in cell lysates. Values represent means of three independent experiments. (C) Effects of cisplatin and CLCN3 antisense on AO emission spectra in red-stained organelles. U251 cells were treated with or without cisplatin (15 µmol/l) for 12 h following transfection of nonsense or CLCN3 antisense. Fluorescence emission of AO from red-stained organelles after 30 min of incubation with AO (5 µmol/l). The contribution of the red band within the whole spectrum (R%) is shown. A total of 25 cells were analyzed for each condition. magnification ×400 (*P<0.05 vs. control group, #P<0.05 vs. cisplatin group). CLCN3, chloride voltage-gated channel 3; Bcl-2, B-cell lymphoma 2; Bax, Bcl-2-associated protein X; AO, acridine orange.