Fig. 3

Functional assays in CRISPR-edited iPSC-derived endothelial cells with TOM7 variant. (a) Schematic presentation of CRISPR/Cas9-mediated homology-directed repair with a single-strand oligodeoxynucleotide (ssODN) template carrying TOMM7 p.P29L (red) and three silent mutations (cyan). Asterisks represent phosphorothioate modifications. eCas9, enhanced Cas9 protein containing K484A, K1003A, and R1060A mutations. (b) Workflow of deriving endothelial cells from iPSCs through mesodermal cells. (c) Immunoblotting with anti-TOM7 antibody in whole-cell lysate from iPSCs and iEndo cells. (d) Immunoblotting with anti-TOM7 and anti-TOM40 antibodies in whole-cell lysate and in mitochondria-enriched lysates from iPSCs. (e) Protein half-live was evaluated by immunoblotting with anti-TOM7 antibody in whole-cell lysates from iEndo cells treated with cycloheximide (CHX) for 0, 2, 4, 6, 8, and 10 h. (f) Quantitative analysis of the level of TOM7 from (f). Statistical analysis was done by nonlinear regression with one-phase exponential decay fit and two-way ANOVA followed by Dunnett post hoc test. (g) Representative pictures of tube formation of iEndo cells on Matrigel. (h) Quantitative analyses of the number of extremities and the total length of tubes from (h). Statistical analysis was performed using Kruskal–Wallis test with Dunn's post hoc test for multiple comparisons. (i) Senescence associated β-galactosidase staining in iEndo cells. Arrows indicate positively stained cells. (j) Representative confocal images of immunofluorescent staining with anti-TOM40 (Red; staining mitochondria) and DAPI in iEndo cells. White arrowheads indicate large, round mitochondria. Green arrows indicate elongated mitochondria. (k) Quantitative analyses of mitochondrial morphology in the images of immunofluorescent staining by Mitochondrial Network Analysis (MiNA) toolset showing mean branch length, mean network branches, individual mitochondrial counts, and mitochondrial networks per cell in iEndo cells from (k). Statistical analysis was performed using Kruskal–Wallis test with Dunn's multiple comparisons test. (l) Immunoblotting with anti-DRP1 and anti-MFN1 antibodies in whole-cell lysate from iEndo cells. WT, wild type; Het, heterozygous TOMM7 p.P29L; Homo, homozygous TOMM7 p.P29L; KO, TOMM7 knockout. Data were presented as mean ± S.E.M. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001.