Fig. 2

Excessive accumulation of mitophagosomes contributes to apoptosis induced by CQ/IH combination. a MDA-MB-231 cells were treated with CQ (20 μM) in the presence or absence of IH (10 μM) for 48 h, after which the mitochondrial fractions were prepared and subjected to Western blot analysis using antibodies against p62, LC3-I/LC3-II. b Cells expressed with EGFP-LC3 were treated with CQ (20 μM) in the presence or absence of IH (10 μM) for 48 h. The colocalization of EGFP-LC3 and MitoTracker (Deep Red FM) was examined using confocal microscopy. Scale bars: 10 μm. c Cells were transfected with control shRNA (shControl) or shATG5, and Western blot analysis was used to determine the expression of ATG5. For d-g, cells stably expressing shControl or shATG5 were treated with CQ (20 μM) in the presence or absence of IH (10 μM) for 48 h. d The mitochondrial fractions were prepared and subjected to Western blot using antibodies against LC3-I/LC3-II. e The colocalization of EGFP-LC3 and MitoTracker (Deep Red FM) was examined by confocal microscopy. Scale bars: 10 μm. f The expressions of total PRAP, C-PARP, pro-Caspase 3, C-Caspase 3 and Cyto C (Cytosolic fraction) were determined by Western blot. g Apoptosis was detected by flow cytometry analysis. The values obtained from the Annexin V/PI assay represent the mean ± SD for three separate experiments. ^***Values for cells combination-treated with CQ/IH after transfection with shATG5 are significantly decreased compared with those in shControl cells combined treated with CQ/IH, ^***P < 0.001