CCDC51 likely promotes normal mitochondrial morphology independently of K ⁺ channel activity. (A) A schematic displaying the amino acid sequence of the predicted TM domains (TM1 and TM2) of CCDC51. NP mutants of CCDC51 were generated (TM1-NP and TM2-NP) with the indicated point mutations in amino acids indicated in red. Mutations in the GxxxG motif (TM1-G4A) are indicated in blue. (B) Representative single-plane confocal images are shown of CCDC51 CRISPRi cells expressing an empty vector or low levels of the indicated GFP-CCDC51 constructs that were stained with MitoTracker Red. Arrows mark sites of focal accumulation of GFP-CCDC51. (C) A graph is shown of mitochondrial morphology characterization from cells as in B. Data shown represent 25 cells per condition in each of three independent experiments, and bars indicate SEM. Asterisks (**P < 0.01) represent an unpaired two-tailed t test of tubular mitochondrial morphology. N.S. indicates not statistically significant. (D) Representative single-plane confocal images of U2OS cells treated with the indicated siRNA for 72 h and expressing an empty vector (top) or high levels of GFP-CCDC51 (bottom) and stained with MitoTracker Red. Arrows mark examples of GFP-CCDC51 foci. (E) A graph is shown of mitochondrial morphology characterization from cells as in D. Data shown represent 25 cells per condition in each of three independent experiments, and bars indicate SEM. N.S. indicates not statistically significant results from an unpaired two-tailed t test of tubular mitochondrial morphology. (F) A graph is shown of relative mRNA levels as assessed by qRT-PCR of cells treated for 72 h with control- or ABCB8-targeting siRNA in three independent experiments. Asterisks (***P < 0.001) represent an unpaired two-tailed t test. (G) A schematic displaying GFP-CCDC51 variants with in-frame deletions of the matrix coiled-coil (∆matrix CC) or the IMS coiled-coil (∆IMS CC). (H and I) As in B and C for cells expressing the indicated GFP-CCDC51 forms indicated in G. Scale bars: 5 µm.