FRDA fibroblasts display CoA and PDH lipoylation defects. (A) Immunoblots of PDH-E2 and lipoylated PDH-E2 (PDH-E2-LA) in mitochondria-enriched extracts from control (C1-C3) and FRDA (P1-P5) grown in regular medium for 72 hours, with or without 5 mM of dichloroacetate (DCA). Porin was used as a loading control. Immunoblotting quantifications are given in supplemental Figure 5A. (B) Total CoA content in control (C1-C3) and FRDA (P1-P5) cells grown in untreated regular medium, with 25 µM of CoA or 5 mM of DCA, for 72 hours. (C) Biotin immunoblots correlating with extent of TfR1 palmitoylation in control (C1-C3) and FRDA (P1-P5) fibroblasts grown in untreated regular medium or supplemented with either 25 µM of CoA or 5 mM of DCA for 72 hours. For each condition, the topmost panel shows palmitoylated TfR1 levels (immunoprecipitated [IP]: biotin), the panel below shows the IP amount of TfR1 (IP: TfR1), and IP TfR1 was used as a loading control. Immunoblotting quantifications of palmitoylated and steady-state levels of TfR1 are given in supplemental Figure 5B-C. (D) Immunoblot analysis of control (C1-C3) and FRDA (P1-P5) immortalized fibroblasts overexpressing wild-type FXN complementary DNA (cDNA). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a loading control. Immunoblotting quantifications are presented in supplemental Figure 6A-C. (E) Iron quantification using ferrozine-based colorimetric assay in control (C1-C3) and FRDA (P1-P5) immortalized fibroblasts overexpressing wild-type FXN cDNA. Plots show mean ± standard error (n = 3). Two-way analyses of variance were used to compare untreated with treated values. *P < .05, ***P < .001. ns, nonsignificant.