Fig. 2: Suppression of IDH2 leads to accumulation of α-ketoglutarate in TNBC cells due to blockage of the reductive TCA cycle.

a Effect of IDH2 knockdown by shRNA (IDH2-#1 and IDH2-#2) on intracellular α-ketoglutarate level in MDA-MB-231 cells. Non-target control shRNA (NC) was used as control. Cellular α-ketoglutarate was measured using an enzyme-based method as described in the method (n = 3, technical replicates, representative of two independent experiments, original data included in the Source Data). b Effect of IDH2 knockdown by shRNA (IDH2-#1 and IDH2-#2) on the cellular α-ketoglutarate level in HCC38 cells (n = 3, technical replicates, representative of two independent experiments, original data included in the Source Data file). c Relative α-ketoglutarate level in tumor tissues from mice inoculated with MDA-MB-231 cells transfected with IDH2 shRNA (n = 5, independent biological replicates) or non-target control RNA (NC, n = 6, independent biological replicates). d Schema of metabolic flux analysis of glutamine metabolic pathway feeding to the TCA cycle via α-ketoglutarate. The dark close circles represent [13C]-labeled carbons, whereas the open circles represent unlabeled 12C-carbons. The green arrow indicates the direction of oxidative TCA flow, while the orange arrow shows the direction of reductive TCA flow where IDH2 catalyzes the conversion of α-ketoglutarate to isocitrate. Inhibition of IDH2 led to an accumulation of α-ketoglutarate. e, f Comparison of glutamine uptake (e) and cellular glutamate (f) in MDA-MB-231 cells transfected with IDH2 shRNA (#1 and #2) or with non-target control RNA (NC). g Expression of PSAT1 in MDA-MB231 cells transfected with IDH2 shRNA (#1 and #2) or with non-target control RNA (NC). PSAT1 expression was measured by qRT-PCR. h Mole percent enrichment ratio of serine/PEP in MDA-MB231 cells transfected with IDH2 shRNA (#1 and #2) or with non-target control RNA (NC). i–k GC-MS analysis of α-KG abundance (i), its mole percent enrichment (MPE) (j), and the MPE/α-KG ratio in MDA-MB231 cells transfected with IDH2 shRNA (#1 and #2) or with non-target control RNA (NC) (k). Error bars in all panels represent mean values ± S.D., n = 3 (technical replicates) for each group in (e–k), a two-sided Student’s t-test was used to test the statistical significance. *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001.