Fig. 3: Respiratory chain composition and function is impaired in MADaM fibroblasts.
a Representative western blots of respiratory chain proteins of complex I (NDUFS1, NDUFA9), complex II (SDHA), complex III (CORE I), complex IV (COX1, COX2), and complex V (ATP5B) in whole lysates from healthy control (WT), MADM2, MADM3, and HGPS fibroblasts. Protein levels were quantified by ImageJ software and their expression levels were normalized to REVERT total protein. b Quantitative analysis of western blots of respiratory chain proteins from complex I (NDUFS1, NDUFA9), complex II (SDHA), complex III (CORE I), complex IV (COX1, COX2), and complex V (ATP5B) from a healthy control (WT), MADM2, MADM3, and HGPS fibroblasts. Protein expression levels were normalized to Revert total protein using ImageJ software. Results are expressed as the mean, for ATP5B the mean ± SD is shown; n = 3 independent experiments for ATP5B and n = 2 independent experiments for all other proteins. One-way anova was performed comparing patients’ values to control (exact p values: NDUFS1: pMADM2 = 0.0034, pMADM3 = 0.002, pHGPS = 0.008; NDUFA9: pMADM2 = 0.041, pMADM3 = 0.025, pHGPS = 0.140; SDHA: pMADM2 = 0.954, pMADM3 = 0.89, pHGPS = 0.689; COREI: pMADM2 = 0.0188, pMADM3 = 0.0154, pHGPS = 0.011; COX1: pMADM2 = 0.0014, pMADM3 = 0.0011, pHGPS = 0.0012; COX2: pMADM2 = 0.1129, pMADM3 = 0.074, pHGPS = 0.0257; ATP5B: pMADM2 = 0.0035, pMADM3 = 0.0034, pHGPS = 0.0016) (p values: *p < 0,05, **p < 0.01, ns not significant. c Basal (R), oligomycin (O), and FCCP (F) respirations were studied in healthy (WT) and MADM2 and MADM3 fibroblast cell lines at passage P8. Respiratory ratios, including RCR (O/F) and RCRp (R-O/F) are also expressed for the respiration measured at 48 h. Data are expressed as mean ± SD of n = 4 independent experiments; statistical significance was analyzed using two-tailed Mann–Whitney test, 95% CI; *p = 0.015 in all cases. d The graph (left panel) shows quantification of tetramethylrhodamine methyl ester (TMRM, 50 nM) mean fluorescence intensity (MFI) signal measured by FACS analysis (right panel) in control (WT) and MADM2 and MADM3 fibroblasts. The counts shown in the right panel indicate the number of mitochondria quantified in this study, obtained from n = 3 independent experiments. Data are expressed as mean ± SD; statistical significance was analyzed using two-tailed unpaired Student’s t test, 95% CI; *pMADM2 = 0.015. FACS sequential gating/sorting strategies are provided in Supplementary Fig. 12 and source data are provided as a Source Data file.
