Fig. 4

Analysis of mitophagy in A2780 and A2780 CIS cell lines. A A2780 and A2780 CIS cells were incubated in full medium, with 10 µM CCCP for 18 h and/or with 400 nM Bafilomycin A1 (BAF) for 3 h. Western blot analysis was performed on cellular lysates using antibodies against LC3B and PINK1. HSP90 was used as a loading control. Densitometric analysis of immunoblot shown in (B) and (D) was performed using Image Lab software (Bio-Rad). C The autophagic flux was calculated as the ratio of normalized LC3BII between BAF and untreated cells of the same sample. E A2780 and A2780 CIS expressing GFP-LC3 (green) were treated with CCCP and subjected to immunofluorescence analysis using an antibody against TOM20 (red). Nuclei were stained with DAPI (blue). Scale bar = 10 μm. F The colocalization rates of mitochondria (TOM20, red) and autophagosomes (GFP-LC3, green) were determined with ZEN Black Edition 2011 acquisition software (Zeiss, Germany). Data represent the mean ± SEM of at least three independent experiments. * p < 0.05; ** p < 0.01; ***p < 0.001