Fig. 2

Low Mn concentrations do not affect mitochondria and inhibition of Drp1 attenuates its blockade on autophagy. HeLa autophagy reporter cells were treated with vehicle (DMEM media), and varying concentrations of Mn for 24 h. (A) Representative confocal images of mitochondrial morphology after TOM20 immunostaining (upper panels, scale bar 20 μm), and then skeletonized (lower panels) for subsequent analysis of mitochondrial morphology. (B) The MiNA plugin of Fiji ImageJ was used to quantified mitochondrial morphology/network. Data represent mean ± SEM (n = 4 independent experiments with 30–40 cells per group quantified per experiment), analyzed by one-way ANOVA, followed by Tukey’s post hoc test. Mn did not induce a significant change in rod or branch length (P = 0.8500 for Mn 62.5 µM, P = 0.9951 for Mn 125 µM and P = 0.0739 for Mn 250 µM). At 250 µM, a modest reduction in the number of branches per network (P = 0.0470) was detectable, but not at the lower doses (P = 0.5492, P = 0.8068 for 62.5 and 125 µM respectively). (C), Mitochondrial respiration was assessed by measuring OCR using the XF^e96 Extracellular Flux Analyzer. Data represent mean ± SEM, n = 4 independent experiments with 8 replicates per experiment. (D) HeLa autophagy reporter cells were transfected with siRNA-Drp1 for 24 h to knockdown (KD) Drp1, and then treated with Mn (62.5 µM) for another 24 h, followed by immunostaining for Drp1. (E) Images were captured and the number of autophagosomes and autolysosomes were quantified as described in Fig. 1. Data represent mean ± SEM (n = 3 independent experiments with approximately 30 cells per experiment) and groups were compared by using Kruskal-Wallis ANOVA test with Dunn’s post-hoc test. Scale bar = 20 μm