SAMM50 KD alters mitochondrial morphology and depletes a subset of mitochondrial proteins. (A) Expression of SAM complex proteins in lysates from WT and two clones of SAMM50 CRISPR KD HeLa cells. LE, long exposure. (B) Densitometric analysis of MTX1 and MTX2 levels from A. Values are mean ± SD from three independent experiments. **, P < 0.005; one-way ANOVA. (C–F) Whole-cell lysates from WT and SAMM50 KD cells analyzed for expression of indicated mitochondrial proteins (C and E). LE, long exposure. Relative expression levels quantified (D and F) with mean ± SD from three different experiments. **, P < 0.005; *, P < 0.01; †, NS; one-way ANOVA. (G) High-resolution confocal images of WT and SAMM50 KD cells stained with antibodies to endogenous SAMM50 and TIMM23. DNA was stained with DAPI. Scale bars, 10 µm. (H) Quantification of relative fluorescence from G. Fluorescence intensity from 60–80 cells was quantified per sample using ImageJ software. Values are mean ± SD. ***, P < 0.001; *, P < 0.01; one-way ANOVA. (I) Mitochondrial cristae structure in WT and SAMM50 KD cells visualized by TEM. Scale bars, 0.2 µm. (J) Percentage of mitochondria with abnormal cristae were scored in I, based on 200 mitochondria from 8–10 micrographs per sample (see Materials and methods). Values are mean ± SD. **, P < 0.005; one-way ANOVA. (K and L) WT and SAMM50 KD HeLa cells analyzed for mitochondrial shape by TEM. Arrows indicate fused mitochondria (K). Percentage of fused mitochondria was scored in L (see Materials and methods). Values are mean ± SD. **, P < 0.005; one-way ANOVA. Scale bars, 0.5 µm. (M) OXPHOS measured in WT and SAMM50 KD cells using a Seahorse XFp flux analyzer. Graphs show one representative example from three independent experiments. Values are mean ± SD from three replicates. **, P < 0.005; one-way ANOVA.