Fig. 6. Flubendazole induces mitochondrial dysfunction and DRP1-mediated mitophagy via targeting EVA1A in MDA-MB-231 and MCF-7 cells.

MDA-MB-231 and MCF-7 cells were transfected with EVA1A siRNA or negative-control for 24 h, respectively, followed by treatment with or without flubendazole (0.5 μM). A Immunoblotting of EVA1A, DRP1, p-DRP1^ser616, Parkin, p-Parkin^ser65 and PINK1 expression. β-actin was measured as the loading control. B, C The autophagosomes are labeled by LC3 (green fluorescence) protein, and the mitochondria are labeled by TOM20 (red fluorescence) protein. The number of co-localized LC3 and TOM20 was quantified. Scale bar, 10 µm. D, E Colocalization of Parkin (red fluorescence) protein and TOM20 (green fluorescence) protein in MDA-MB-231 and MCF-7 cells following flubendazole (0.5 μM, 24 h) treatment. The number of co-localized Parkin and TOM20 was quantified. Scale bar, 10 µm. F ATP content measurement. G, H Flow cytometric analysis and quantification of mitochondrial membrane potential changes. Data represent mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001. Statistical significance compared with respective control groups (all P-values were obtained by one-way ANOVA).