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. 2022 Dec 2;15:966209. doi: 10.3389/fnmol.2022.966209

Figure 2.

Figure 2

Miro1 R272Q alters mitochondrial morphology concomitant with changes in cristae organization. (A) Representative image of hDaNs stained with 100 nM MitoTracker green to label mitochondria for analysis of mitochondrial morphology. Scale bar: 50 μM. (B) Analysis of mitochondrial morphology of hDaNs stained with 100 nM MitoTracker green to assess mitochondrial area, length, fragmentation, and form factor. Values of one differentiation normalized to mean of isogenic control of the same differentiation. nDiff = 3 (20 images per differentiation), data displayed as mean ± SD; Mann–Whitney test. (C) Tom20 protein levels in hDaN lysates. Representative blot and quantification of intensity of Tom20 bands relative to a-tubulin. nDiff = 8, data displayed as mean ± SD; paired t test (two-tailed). (D) Representative electron microscopy of hDaNs. Red arrows indicate mitochondria with regions devoid of cristae, green arrows indicate mitochondria with maintained cristae. Scale bar: 500 nm. (E) Blinded quantification of mitochondria with disrupted cristae structure per EM image. nDiff = 3, data displayed as mean ± SD; Welch’s t test (two-tailed). (F) Assessment of mitochondrial renewal in hDaNs by transfection with pMitoTimer. Quantification of mean red:green fluorescence and normalization of values of one differentiation to mean of isogenic control of the same differentiation. nDiff = 3 (nimages = 42), data displayed as mean ± SD; Mann–Whitney test. (G) Flow cytometric assessment of susceptibility to apoptosis. hDaNs treated for 4 h with 1 μM Staurosporine were stained with apoptotic marker Annexin V and necrotic marker 7-AAD. Mean fluorescence of each channel normalized to unstained signal. nDiff = 3, data displayed as mean ± SD; Two-way ANOVA with Tukey’s multiple comparisons.