(A) Proliferation rates of 143B cells treated with vehicle (DMSO), 50 nM rotenone, or 5 µM atpenin A5 (AA5) cultured in pyruvate free DMEM with no addition, 1 mM pyruvate (PYR), 1 mM …
Proliferation rates and relative metabolite levels by LCMS in Figure 1.
Uncropped western blot for Figure 1F.
(A) Schematic demonstrating how the aspartate transporter SLC1A3 allows for cells to uptake aspartate, which can be measured by the incorporation of isotopically labeled extracellular aspartate …
Proliferation rates and metabolite levels by LCMS in Figure 1—figure supplement 1.
Uncropped western blot for Figure 1—figure supplement 1B and F.
(A) Proliferation rates of 143B cells cultured in DMEM with 1 mM PYR treated with vehicle (DMSO), 50 nM rotenone, 5 µM AA5, or 5 µM AA5 and 50 nM rotenone (n=3). (B) Aspartate levels of 143B cells …
Proliferation rates, relative metabolite levels by LCMS, and other data in Figure 2.
Uncropped western blots for Figure 2C and E.
(A) Mitochondrial oxygen consumption rates in WT 143B cells pretreated with vehicle (DMSO) or 5 µM AA5 for 4 hr, then injected with vehicle (DMSO) or 5 µM AA5 as indicated, followed by treatment of …
Proliferation rates, metabolite levels by LCMS, and mitochondrial oxygen consumption values in Figure 2—figure supplement 1.
Uncropped western blot for Figure 2—figure supplement 1D and I.
(A) Whole cell NAD+/NADH measured by LCMS metabolomics of 143B cells cultured in DMEM with 1 mM PYR and treated with vehicle (DMSO), 50 nM rotenone, 5 µM AA5, or 5 µM AA5 and 50 nM rotenone for 6 hr …
Proliferation rates, relative metabolite levels by LCMS, and enzymatic assay values in Figure 3.
Uncropped western blot for Figure 3D.
(A) Schematic showing the metabolic roles of the electron transport chain and ATP synthase and depicting the sites of action for rotenone, AA5, antimycin A, hypoxia, and oligomycin. (B) …
Proliferation rates and metabolite levels by LCMS in Figure 3—figure supplement 1.
(A) Schematic showing the mechanism of action of AZD7545 to activate pyruvate dehydrogenase (PDH) by inhibition of the negative regulators of PDH, pyruvate dehydrogenase kinases (PDKs). (B) …
Proliferation rates, relative metabolite levels by LCMS, and mitochondrial oxygen consumption values in Figure 3—figure supplement 2.
(A) Schematic depicting metabolic pathway usage for isotopologue patterns of aspartate derived from U-13C glutamine. (B) Relative ion counts for all aspartate isotopologues derived from U-13C …
Proliferation rates and relative metabolite levels by LCMS in Figure 4.
Uncropped western blot for Figure 4G.
(A) Schematic showing how the ACLY inhibitor BMS-303141 blocks a convergent step necessary for glutamine-derived aspartate synthesis by either cytosolic or mitochondrial reductive carboxylation of …
Proliferation rates and relative metabolite levels by LCMS in Figure 4—figure supplement 1.
(A) Western blot for MPC1 and SDHB in WT 143B cells, two MPC1 KO 143B clones, and MPC1 KO 143B clone 1 with MPC1-V5 cDNA added back (AB). SDHB is used as a loading control. (B) Proliferation rates …
Proliferation rates and relative metabolite levels by LCMS in Figure 5.
Uncropped western blot for Figure 5A.
(A) Model depicting three enzymes capable of mitochondrial AKG production from glutamate (GLU) and necessary cofactors. GPT2; glutamic-pyruvic transaminase 2, GDH; glutamate dehydrogenase, GOT2; …
Relative metabolite levels by LCMS in Figure 5—figure supplement 1.
(A) Western blot for GOT2 and Vinculin from WT 143B cells, GOT2 KO 143B cells, and the same GOT2 KO clone expressing GOT2 cDNA (AB). Vinculin is the loading control. (B) Proliferation rates of GOT2 …
Proliferation rates and relative metabolite levels by LCMS in Figure 6.
Uncropped western blots for Figure 6A and E.
(A) Proliferation rates of GOT2 KO and GOT2 AB 143B cells in pyruvate-free DMEM (n=3). (B) Relative malate levels by LCMS metabolomics of GOT1 KO 143B cells compared to GOT1 AB 143B cells, treated …
Proliferation rates and relative metabolite levels by LCMS in Figure 6—figure supplement 1.
Uncropped western blot for Figure 6—figure supplement 1D.
(A) Schematic showing how SDHB KO 143B clones were used to generate SDHB addback cells (AB), early passage SDHB KO cells (EP), or late passage (LP) SDHB KO cells. (B) Proliferation rates of …
Proliferation rates, relative metabolite levels by LCMS, volcano plot data, and complex I activity values in Figure 7.
Uncropped western blot for Figure 7I.
(A) Western blot for SDHB and tubulin from WT and SDHB KO HEK293T cells. Tubulin is used as a loading control. (B) Proliferation rates of SDHB KO HEK293T cells cultured in DMEM with 1 mM pyruvate …
Proliferation rates in Figure 7—figure supplement 1.
Uncropped western blot for Figure 7—figure supplement 1A.
(A) Western blot for NDUFA8 and tubulin from WT 143B, WT UOK269, and late-passage (LP) SDHB UOK269 cells. Tubulin is used as a loading control. (B) Mitochondrial oxygen consumption rate of WT UOK269 …
Proliferation rates, mitochondrial oxygen consumption values, complex I activity values, relative metabolite levels by LCMS, and tumor volumes in Figure 8.
Uncropped western blot for Figure 8A.
(A) Complex I activity assay of extracts from WT 143Bs and WT UOK269 cells (n=3). (B) Fractional isotopologue distribution of aspartate measured by LCMS metabolomics of WT or SDHB UOK269 cells …
Proliferation rates, relative metabolite levels by LCMS, fractional isotopologue distribution values, and mitochondrial oxygen consumption values in Figure 8—figure supplement 1.
(A) Detection of isotopically labeled aspartate by LCMS across a standard curve of aspartate levels. (B) Measurement of aspartate from 143B cells treated with vehicle, 50 nM rotenone, or 5 µM AA5, …
NAD+/NADH measurements from the indicated figure panels in Gui et al. 2016 or this revised manuscript, with each made relative to vehicle treated cells cultured in pyruvate-free DMEM.
Ion counts from each metabolomics experiment conducted in this study.
Organized by tab for each figure; tables include cell line, treatment, and metabolite measured.
Mitochondrial proteomics dataset used to generate Figure 7E-G, Figure 7—figure supplement 1F.