Fig. 5
FASN deficit leads to an impairment of IDH1-dependent reductive carboxylation. a Isotopolog abundance of α-ketoglutarate, b fumarate, and c and citrate/isocitrate from [U-13C5]glutamine in 2D cultures determined by LC/MS (n = 4 biological replicates). d Isotopolog abundance of α-ketoglutarate, e fumarate, and f citrate/isocitrate from [U-13C5]glutamine in 3D cultures determined by LC/MS (n = 4 biological replicates). Presented data are the mean values ± SD. *P < 0.05; Student’s t test. g Relative levels of NADPH in cellular and mitochondrial lysates of FASNlox/lox-PyMT versus FASN∆/∆-PyMT MEFs in 3D cultures (n = 3). Presented data are the mean values ± SD. *P < 0.05 and ***P < 0.001; Student’s t test. h Digitonin-solubilized isolated mitochondria from FASNlox/lox-PyMT and FASN∆/∆-PyMT MEFs in 2D and 3D culture conditions were subjected to blue native gel electrophoresis (BNGE) followed by in-gel complex I activity assay. Complex I occurs both free and bound with complex III (I + III2 and I2 + III2 supercomplexes). Relative abundance of assembled complex I into supercomplexes (SC) versus free complex I (CI) after the quantification of band intensity in direct immunobloting of BNGEC against complex I subunit NDUFS1 in FASNlox/lox-PyMT versus FASN∆/∆-PyMT in 2D and 3D culture conditions is shown. Presented data are the mean values ± SD (n = 2 independent culture preparation). *P < 0.05; Student’s t test. i NAC (1 mM) and GSH-MEE (1 mM) rescue experiments. Data are represented as mean values ± SEM. Three independent experiments. j Rescue of soft-agar colonies (above) and spheroids (below) in FASN∆/∆-PyMT MEFs treated with NAC (1 mM) or GSH-MEE (1 mM). Representative images for each condition are shown. Scale bars, 500 μM. k Representative flow cytometry analysis showing constitutive mitochondrial superoxide levels in FASN∆/∆-PyMT MEFs treated with NAC or GSH-MEE (n = 4). MitoSOX red fluorescence intensity quantitation is shown on the right. Presented data are the mean values ± SD. **P < 0.01; Student’s t test. l NAD+/NADH ratio is restored after the addition of NAC or GSH-MEE in FASN∆/∆-PyMT MEFs in 3D cultures (n = 4). Presented data are the mean values ± SD. *P < 0.05; Student’s t test
