Figure 4. PSMF1 deficiency is associated with defects in mitochondrial bioenergetics and dynamics. (a-c) Mitochondrial membrane potential status and maintenance:

(a) Representative images of tetramethylrhodamine methyl ester (TMRM)-loaded mitochondria in fibroblasts from healthy controls (HC1, HC2) and PSMF1 patients (A-II-2, E-II-3; see Fig. 1a). Scale bar = 20 μm. (b) Quantification bar graphs depict the mean mitochondrial membrane potential for healthy controls’ (gray bars; see also red dash line), probands’ (orange bars) and unaffected carriers’ (yellow boxes) mitochondria. (c) Inhibitor analysis of the mitochondrial membrane potential maintenance and electron transport chain function in healthy controls (upper quadrants) compared to probands’ (lower quadrants) mitochondria. (d-f) Mitochondrial dynamics: (d) Quantification box charts depicting average length of all mitochondrial rod and branches and (e) average number of branches analyzed in individual fibroblasts from healthy controls’ (gray boxes), probands’ (orange boxes) and unaffected relatives’ (yellow boxes) fibroblasts. (f) Representative images of mitochondrial shape and dynamics in one unaffected carrier (B-II-1), one proband (B-II-3) and one healthy control (HC2). Each dot represents a cell and total number of cells analyzed in 3–9 independent experiments is shown in brackets. Non-parametric Kruskal-Wallis ANOVA with post-hoc Dunn’s test for each group. *p < 0.05, **p < 0.01, ***p < 0.001 compared to control HC1; # p < 0.05, ## p < 0.01, ### p < 0.001 compared to control HC2. Scale bar = 20 μm. (g-h) Mitophagy: (g) Basal mitophagy rate assessed as colocalization of LysoTracker Red DND-99 with MitoTracker Green in healthy controls (gray bars), probands’ (orange bars) and unaffected carriers’ (yellow bars) fibroblasts. (h) Mitophagy rate after induction with either lactate or pyruvate. Data are presented as mean ± SEM. ***p < 0.0001.