Fig. 4: Excessive accumulation of mitophagosomes contributes to apoptosis induced by cepharanthine/epirubicin combination.

a MDA-MB-231 cells were treated with cepharanthine in the presence or absence of epirubicin for 48 h, after which the mitochondrial fractions were prepared and subjected to Western blot analysis using antibodies against p62, LC3-I/LC3-II, PINK1 and Parkin. VDAC1 was used as loading control. b Cells were transfected with control siRNA (siControl) or siATG5, and Western blot analysis was used to determine the expression of ATG5. GAPDH was used as loading control. For c–h, cells stably expressing siControl or siATG5 were treated with cepharanthine in the presence or absence of epirubicin for 48 h. c The mitochondrial fractions were prepared and subjected to Western blot using antibodies against LC3-I/LC3-II. VDAC1 was used as loading control. d Mitochondrial morphology was determined by MitoTracker Red CMXRos staining and confocal microscopy. Scale bars, 10 μm. e Mitochondrial length was measured with ImageJ software. 50 cells of 5 independent experiments. f Western blot was performed to detect the expression of PARP, cleaved-PARP (CF), C-caspase 3 and cytochrome c. g, h Apoptosis was determined by Annexin V-FITC/PI staining and flow cytometry. Data represented as mean ± SD (n = 5, ***P < 0.001, Student’s two-tailed unpaired t-tests).