Fig. 1: Fluorizoline inhibits mitophagy in cancer cells overexpressing Parkin.

HeLa cells overexpressing Parkin (CT) were treated for 16 h with 10 μM CCCP or 1 μM oligomycin/1 μM antimycin A (OA) in the absence (UT) or presence of 5 or 10 μM fluorizoline (F) (A, B). HeLa Parkin cells treated with 20 μM pan-caspase inhibitor Q-VD-OPh and 40 nM bafilomycin (CT) were treated for 8 h with 1 μM oligomycin/1 μM antimycin (OA), in the absence (UT) or presence of 10 μM fluorizoline (F). Cells were co-immunostained for DAPI (blue), TOM20 (green), and LC3 (red) and co-localization was analyzed by confocal microscopy. White arrows indicate the mitochondrial marker within the autophagosome. These are representative images of three independent experiments (C). The co-localization between LC3 and TOM20 was measured and is represented as the Pearson’s coefficient (n = 3) (D). HeLa cells overexpressing Parkin (CT) were treated for 16 h with 10 μM CCCP or 1 μM oligomycin/1 μM antimycin A (OA), in the absence (UT) or presence of 500 nM rocaglamide A (RocA) (E, F). m-Keima was measured by flow cytometry and it is expressed as the mean ± SEM (n = 5 independent experiments) of the percentage of mitophagy positive cells (A, E). Viability was measured by flow cytometry and it is expressed as the mean ± SEM (n = 4 independent experiments) of the percentage of non-apoptotic cells (annexin V-negative) (B, F). ^###p < 0.001 CT versus CCCP and OA-treated cells and **p < 0.01 and ***p < 0.001 UT versus F or RocA-treated cells.