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. Author manuscript; available in PMC: 2021 Mar 1.
Published in final edited form as: Cancer Res. 2020 Jul 10;80(17):3492–3506. doi: 10.1158/0008-5472.CAN-20-0246

Figure 4. Cell death mediated by dmaKG is caused by aspartate and adenine nucleotide depletion.

Figure 4.

(A) Cytotoxicity in UOK262-ev, UOK268-ev, and 143B-CytB following 24 hr treatment with dmaKG at the indicated concentrations with or without 10 mM aspartate.

(B) Cell proliferation as measured by crystal violet staining in UOK262-ev, UOK268-ev, and 143B-CytB following 72 hr of dmaKG treatment at the indicated concentrations with or without 10 mM aspartate.

(C) Cytotoxicity in UOK262 cells expressing SLC1A3 (UOK262-SLC1A3). Cells were treated with 6.6 mM dmaKG with or without 150 μM of aspartate.

(D) Measurement of adenine nucleotide pool (AMP+ADP+ATP) using a biochemical assay in UOK262 cell pair following dmaKG (6.6 mM) treatment at the indicated times. dmaKG caused a rapid and stable decline of adenylates in UOK262-ev. In UOK262-FH, after an initial decrease, adenylates reverts back to baseline levels. All treatment samples were compared to the mean of the respective control group.

(E) Adenine nucleotide pool levels in dmaKG-treated UOK262-ev (6.6 mM, 6 hr) and 143B-CytB (9.9 mM, 6 hr). Cells were treated in media with or without 10 mM aspartate supplementation.

(F) Cytotoxicity in UOK262-ev, UOK268-ev, and 143B-CytB following 24 hr treatment with dmaKG at the indicated concentrations with or without 110 μM adenine, 1.2 mM ATP, or a cell-permeable ATP analog AMP-PCP (100 μM).

All data are shown as mean and individual data points (two-way ANOVA with Tukey’s multiple comparisons, ns p>0.5, *p≤0.05, **p≤ 0.01, ***p≤0.001, ****p≤0.0001).