Fig. 5

Altered energy metabolism and mitochondrial respiration in CRT-deletion fibroblasts. (A) Intracellular creatine levels in CRT-WT fibroblasts (WT) and CRT-deletion fibroblasts (Del). Data are presented as mean ± SD (n = 6 independent biological samples). (B–D) Intracellular ATP (B) and ADP (C) levels and the ADP/ATP ratio (D) in CRT-WT and CRT-Del fibroblasts. Data are presented as mean ± SD (n = 10 independent biological samples). *p < 0.05, **p < 0.01, ****p < 0.0001, significantly different from CRT-WT fibroblasts. (E-H) Mitochondrial respiration in CRT-Del fibroblasts. (E) Oxygen consumption rate (OCR) profile of CRT-WT and CRT-Del fibroblasts. Arrows indicate sequential addition of oligomycin (1 µM), carbonyl cyanide-p-trifluoromethoxyphenylhydrazone (FCCP, 4 µM), rotenone (0.5 µM), and antimycin A (0.5 µM). (F) Mitochondrial respiratory parameters, including basal respiration, ATP production-linked respiration, proton leakage, maximal respiration, spare respiratory capacity, and nonmitochondrial respiration, of CRT-WT and CRT-Del fibroblasts. (G) Relative mitochondrial DNA (mtDNA) copy numbers in CRT-WT and CRT-Del fibroblasts. (H) Ratio of OCR in mitochondrial respiratory parameters to mtDNA copy number. (I) Extracellular acidification rate (ECAR) profiles of CRT-WT and CRT-Del fibroblasts. Arrows indicate sequential addition of oligomycin (1 µM), FCCP (4 µM), rotenone (0.5 µM), and antimycin A (0.5 µM). (J) Estimation of basal ECAR, maximal (oligomycin-stimulated) ECAR, and glycolytic reserve of CRT-WT and CRT-Del fibroblasts. (K) Amount of lactate released into 24-h conditioned medium of CRT-WT and CRT-Del fibroblasts. Culture supernatants containing 10% FBS were assayed by the lactate assay kit-WST. Data are presented as mean ± SD (CRT-WT, n = 6–9; CRT-Del, n = 3–6 independent biological samples). *p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, significantly different from CRT-WT fibroblasts.