CHCHD10 physically interacts with SLP2 and is involved in the stability of the PHB complex. (A) Co-immunoprecipitation (IP) experiment in HeLa cells, transfected with an empty vector (EV), WT CHCHD10-Flag (WT) or mutant CHCHD10^S59L-Flag (S59L) constructs, with an antibody against SLP2 revealed with an anti-FLAG antibody. (B) Co-IP of endogenous CHCHD10 and SLP2 in control (C) and patient (P1) fibroblasts. P1 corresponds to patient V-10 harbouring the p.Ser59Leu variant (CHCHD10^S59L/+).¹ (C) Duolink PLA between SLP2 and CHCHD10 in control (C) and patient (P1, P2) fibroblasts observed by confocal microscopy. Mitochondria were stained with MitoTracker. P2 corresponds to patient IV-3 (CHCHD10^S59L/+).¹² Scale bar = 20 µm. (D) Duolink spots per cell were quantified for 30–40 randomly selected individual cells per each studied fibroblast cell line from three independent experiments. Values are mean ± SEM. (E) Western blot on control (C) and patient (P1, P2) fibroblast extracts using antibodies against SLP2, HSP60 or GAPDH. (F) BN-PAGE analysis of mitochondria from control and patient (P1, P2) fibroblasts incubated with an anti-PHB2 antibody. Loading control was performed with an antibody against UQCRC2 (Complex III, CIII). (G) Western blot on control (C) and patient (P1, P2) fibroblast extracts using antibodies against PHB1, PHB2, HSP60 or GAPDH. (H) Duolink PLA between SLP2 and PHB1 in control (C) and patient (P1, P2) fibroblasts observed by confocal microscopy. Mitochondria were stained with MitoTracker. Scale bar = 20 µm. (I) Duolink spots per cell were quantified for 30–35 randomly selected individual cells per each studied fibroblast cell line from three independent experiments. Values are mean ± SEM. (J) Mitochondrial membranes were extracted from control (C) and patient (P1, P2) fibroblasts at the indicated pHs and separated into pellet (P) and supernatant (S) fractions by centrifugation. Fractions were analysed by SDS-PAGE and immunoblotting with antibodies against SLP2, PHB2, UQCRC2 (Complex III, CIII) and HSP60. To verify the purity of isolated mitochondria, total lysates (Total) and mitochondrial isolates (Mito) were analysed by immunoblotting using antibodies against HSP60 (mitochondrial protein), PCNA (nuclear protein) or GAPDH (cytosolic protein).