Fig 1: Estimated metabolic flux distributions and isotopomer distributions (simulated and experimental) of malate and citrate for BCC cells. (a) Metabolic model with atomic transitions and estimated flux distribution for the mock (black) and siRNAs against FASN (red), ECHS1 (cyan), and ME (green) transfected BCC cells. (b–e) Malate and (f–i) citrate isotopomer distribution in mock and siRNAs against FASN (red), ECHS1 (cyan), and ME (green) transfected BCC cells. The bar plots show the fractions of citrate and malate labeled in zero (M0) to six (M6) carbons. Error bars represent 95% confidence intervals (n = 4).
Fig 2: Experiments assessing the existence of simultaneous FAS and FAO. (a) Metabolic model with atomic transitions showing how degradation of fatty acids derived from glutamine or ME activity can result in the M6 citrate isotopomer, and scheme showing the shuttle of redox potential from cytosolic NADPH to the mitochondrion, formed by simultaneous FAS and FAO. Labeled carbon atoms entering the TCA cycle from FAO are indicated in yellow. (b) Percentages of fully labeled citrate in three different breast cancer cell lines which were fed with 13C5-L-Glutamine. BCC showed 5 and 10 times more fully labeled citrate than MCF7 and BT-474 cell lines, respectively, which makes it more likely to have simultaneous FAS and FAO. Bar graphs represent mean ± SEM (n = 4). (c) Experimental changes in the fraction of M6 citrate resulting from the silencing of FASN, ECHS1, and ME, respectively. Bar graphs represent mean ± SEM (n = 4). (d) Effects of silencing FASN, ECHS1, and ME on the mitochondrial membrane potential. Bar graphs represent mean ± SEM of aggregate/monomer (R/G) ratios of JC-1 dye (n = 3). * p < 0.05, ** p < 0.01, *** p < 0.001.
Fig 3: (a) SCEH enzyme studies on patient fibroblasts. SCEH activity is expressed as percentage of the mean activity in six control cell lines whereas results represent the mean of two independent experiments (b) SDS-PAGE and ImageJ quantification (bottom) of ECHS1 steady state protein levels of patient and control fibroblast lysates. Values are displayed as percentage of age-matched control and STD (n of Family 1 = 3 and n of Family 2 = 2). (c) Quantitative PCR of RNA extracted from patient and control fibroblast using TaqMan assay in triplicate. Values were calculated as ??Ct and SEM
Fig 4: Pedigree of Family 1 (F1) and Family 2 (F2) with mutations in ECHS1. Haplotype is indicated below each tested individual. The silent exon 4 mutation is highlighted by red font. Black shading indicates affected status. P indicates patient, M indicates mother and F indicates father, U indicates unaffected, n.d. indicates studies were not done, (a) and (b) indicates phase of the maternal ECHS1 allele in Family 1
Supplier Page from Abcam for Anti-ECHS1 antibody [EPR11785]