Fig 2: MDH1 has significantly higher activity than MDH2 in tumor cells. (A) Enzyme kinetic analysis of MDH1 and MDH2 activities measured in 8 tumor cell lines for 30 min. (B) Cumulative from the tumor cell line panel (black bar) and cumulative data from the 3 normal cells (gray bar). Activity was normalized per mg protein. Error bars, ±SEM. ***p<0.0001; two-tailed student's t-test. n=4-6.

Fig 3: Deletion of SLC1A3 Impedes the ETC and Phenocopies Depletion of the Mitochondrial Aspartate Transporters AGC1 and AGC2 under Glutamine Deprivation(A) Schematic representation of the malate-aspartate shuttle (MAS). In brief, the MAS is a system that allows the transfer of electrons from cytosolic NADH to produce mitochondrial NADH where it is oxidized in the ETC. In the cytoplasm MDH1 catalyzes the reduction of oxaloacetate (OAA), where it accepts an electron from NADH to produce malate and NAD+. Malate can then enter the mitochondria where it is oxidized by MDH2 to OAA, resulting in the formation of mitochondrial NADH. Mitochondrial OAA is transaminated into aspartate by GOT2 whereby aspartate exits the mitochondria in exchange for cytosolic glutamate through a carrier. OAA is recovered in the cytosol by GOT1. By coupling aspartate-glutamate exchange, the aspartate-glutamate carrier is essential for the shuttle and is thought to represent the rate-limiting step.(B) Respiratory profiles of HCT116 WT cells transiently depleted of SLC1A3 and grown for 24 hr in glutamine-free medium, in the presence of mitochondrial inhibitors (oligomycin, FCCP [carbonyl cyanide-4-(trifluoromethoxy)phenylhydrazone] antimycin A/rotenone). Arrows indicate incubation of cells with the indicated inhibitors. Data are presented as mean ± SEM of one representative experiment (n = 6 wells).(C) Oxygen consumption rates (OCR) of HCT116 WT and p53-null clones 2 days after glutamine deprivation as in (B). Data are presented as mean ± SEM of one representative experiment (n = 6 wells).(D) Proliferation of HCT116 WT cells transiently depleted of AGC1 or AGC2 using siRNA and cultured in glutamine-free condition (LHS) or complete medium (RHS). Data are presented as mean ± SEM of one representative experiment (averages of triplicate wells). The downregulation of these two proteins was confirmed by western blot (middle panel).(E) HCT116 WT cells transiently depleted of AGC1 or AGC2 using siRNA were grown for 2 days in glutamine-free medium and pulsed with [U-13C]aspartate for the final 24 hr. Extracellular levels of aspartate (m+4), alanine, and serine normalized to cell number were quantified over 24 hr. Data are presented as mean ± SEM of one representative experiment (averages of triplicate wells).(F and G) HCT116 WT cells transiently depleted of AGC1 or AGC2 using siRNA were grown for 2 days in glutamine-free medium and pulsed with [U-13C]aspartate for the final 24 hr. Intracellular TCA-cycle intermediates (F) and glutamate and glutamine levels (G) were measured. Data are presented as mean ± SEM of one representative experiment (averages of triplicate wells).See also Figure S7.

Fig 4: MDH1 has functional and prognostic value in NSCLCs. (A) Total cellular malate dehydrogenase activity measured in 11 cell lines. The blue bar is cumulative data from the 3 normal cells and the red bar is cumulative from 8 tumor cell lines. Activity was normalized per mg protein. (B) Kaplan-Meier Analysis of MDH1 expression and overall survival in 226 NSCLC patients. (C) Tissue-Microarray analysis (TMA) of the MDH1 and MDH2 protein expression level in 33 NSCLCs and 33 normal adjacent lung tissues. Representative images are shown. (D) Expression level of the malate dehydrogenases in a cell line panel consisting of NSCLCs and normal lung cells. (E) Effect of genetic depletion on cell viability in the cell line panel, by two independent siRNAs, targeting MDH1 and MDH2, respectively. Cell death is presented as fold over control siRNA. Error bars, ±SEM. *p<0.01, **p<0.001, ***p<0.0001; two-tailed student's t-test. ns= non-significant.

Fig 5: Growth of SDHB knockout cells dependent on glutaminolysis(A) Cells were lysed 64 h after seeding, and expression levels of SDHB, glutaminase 1, citrate synthetase, IDH1, OGDH, DLST, succinyl-CoA, fumarase, MDH1, MDH2, GOT2, and β-actin were determined by western blotting. (B) The schematic illustration shows 13C5, 15N2-glutamine-derived oxidative glutaminolysis. The blue and red symbols (●, ●) represent 13C atoms and the black symbols (●) represent 12C atoms. Incorporation of 13C5, 15N2-glutamine into glutamate, α-ketoglutarate, succinate, fumarate, malate, aspartate, and citrate by oxidative glutaminolysis in SDHB knockout cells and control cells is shown as isotope enrichment of total metabolites. Data are given as means ± SD. *P < 0.05, **P < 0.01, ***P < 0.001 by the Bonferroni's corrected t-test. (C) The diagram represents 13C5, 15N2-glutamine-derived reductive carboxylation. The blue and red symbols (●, ●) represent 13C atoms and the black symbols (●) represent 12C atoms. Incorporation of 13C5, 15N2-glutamine into citrate, malate, fumarate, and aspartate by reductive carboxylation in SDHB knockout cells and control cells is shown as isotope enrichment of total metabolites. Data are given as means ± SD. *P < 0.05, **P < 0.01, ***P < 0.001 by the Bonferroni's corrected t-test. (D) SDHB knockout cells and control cells were treated with the indicated concentrations of the glutaminase 1 inhibitor Compound 968. After 4 days, cell viability was assessed. Ordinate values were obtained by setting the vehicle control value as 100%. Data are given as means ± SD (n = 3). Growth inhibition of 50% (GI50) was calculated using GraphPad Prism version 6 software. N.S., not significant. *P < 0.025, **P < 0.005, ***P < 0.0005 by Williams’ test.