Fig. 7

FBXL4–/– human fibroblasts had reduced CS activity and abnormal mitochondrial structure that was improved with DCA treatment. (A) CS activity decreased in patient fibroblasts from participants 1 (n = 3) and 2 (n = 5) as compared with control fibroblasts (n = 3). (B–D) RC enzyme activity was not significantly changed in patient fibroblasts compared with control fibroblasts. (E) Extracellular lactate levels were significantly decreased in patient fibroblasts compared with control cells in control media. DCA treatment did not affect extracellular lactate levels in patient cells. n = 12 each condition. (F) Intracellular lactate level evaluation in untreated and DCA-treated (20 mM) fibroblasts from participant 1 and controls. n = 12 each condition. (G) CS activity after treatment of fibroblasts with 20 mM DCA (participants 1 and 2). CS activity level in treated fibroblasts was normalized for percentage in control media. Data are shown as the mean ± SEM. Significance was determined using unpaired Student’s t test. *P < 0.05, ***P < 0.001. Bonferroni’s correction method was applied for A and E–G to account for multiple comparisons, and significant findings still held. (H and I) Low-magnification images (original magnification, ×20) of FBXL4–/– fibroblasts from participant 1 after exposure to metabolic stressor and cotreatment with 20 mM DCA for 48 hours. Mitochondria were stained with MitoTracker green (Thermofisher Scientific). Scale bar: 200 μm. (J and K) High-magnification images of mitochondrial morphology in FBXL4–/– fibroblasts from participant 1 after exposure to metabolic stressor and cotreatment with 20 mM DCA for 48 hours (see representative fibroblast image from participant 2 in Supplemental Figure 5). Mitochondria (in green) were disarranged and fragmented when incubated with stressor. More elongated mitochondria were visible after coincubation with 20 mM DCA. Scale bar: 25 μm. (L and M) EM images of mitochondria in FBXL4–/– fibroblasts from participant 1, showing loss of matrix electron density and loss of mitochondrial cristae after incubation in metabolic stressor for 48 hours and rescue of mitochondrial ultrastructure, with defined cristae and normal matrix electron density after cotreatment with 20 mM DCA. Scale bar: 500 nm.