Fig. 5: Lima1 is required for proper mitochondrial energetics.

a Proportion of apoptotic and necrotic Lima1 KO and WT ESC. The cells were individualized by trypsinization and cultured on cell-repellent plates for 2 h at 37 °C. After that, the cell death was determined by annexin V assay in combination with DAPI, three independent experiments, data represent mean ± SEM, unpaired Student’s t-test, 2-sided. b Quantification of WT and Lima1 KO cell proliferation after 96 h of culture. The cells were treated with or without Y-27632 upon dissociation. Untreated Lima1 KO (n = 6 samples) and WT (n = 6 samples), three independent experiments. Treated with Y-27632 Lima1 KO (n = 6 samples), WT (n = 6 samples), four independent experiments. Data represent mean ± SEM, unpaired Student’s t-tests, 2-sided. c Pyruvate kinase activity measured in WT and Lima1 KO ESC, n = 4 independent experiments. Data represent mean ± SEM, unpaired Student’s t-test, 2-sided. d FACS analysis of WT and Lima1 KO ESC TMRE assay. Plot shows the TMRE signal normalized to the MitoTracker signal. CCCP is a mitochondrial oxidative phosphorylation uncoupler used as a negative control. e Quantification of TMRE signal intensity, normalized to MitoTracker signal intensity, n = 3 independent experiments. Data represent mean ± SEM, unpaired Student’s t-test, 2-sided. f Quantification of relative intracellular ROS levels in Lima1 KO ESC, relative to WT ESC. Three independent experiments, data represent mean ± SEM, unpaired Student’s t-test, 2-sided. g OCR measurement using the Seahorse mitochondrial stress test assay. FCCP−Carbonyl cyanide-4 (trifluoromethoxy) phenylhydrazone; Rot Rotenone, AA−Antimycin. Three independent experiments, mean values ± SEM. h Quantification of the OCR analysis, n = 3 independent experiments. Data represent mean ± SEM, 2-way ANOVA with Tukey’s multiple comparisons. Related to Fig. S4.