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. Author manuscript; available in PMC: 2016 Aug 4.
Published in final edited form as: Cell Metab. 2015 Jul 23;22(2):304–311. doi: 10.1016/j.cmet.2015.06.023

Figure 3. Hypoxia-Induced L-2HG Arises via Promiscuous Substrate Usage by LDHA.

Figure 3

(A, B, C) SF188 cells were transfected with siRNAs targeting (A) IDH1 (IDH1-a, -b) and/or IDH2 (IDH2-a, -b), (B) MDH1 (MDH1-a, -b) and/or MDH2 (MDH2-a, -b), or (C) LDHA (LDHA-a, -b, -c, -d). 48 hr after transfection, cells were transferred to 21% or 0.5% O2 for an additional 24 to 48 hr, followed by measurement of intracellular 2HG. Western blot confirms target knockdown efficiency and shows hypoxia-induced expression of HIF1α. Expression of either S6 protein or α-tubulin are shown as loading controls.

(D) SF-XL cells were transfected with siRNAs targeting LDHA (LDHA-b) or both LDHA and MDH2 (LDHA-b/MDH2-b). 48 hr after transfection, cells were transferred to 0.5% O2 and cultured for an additional 48hr in the presence of vehicle (DMSO) or the indicated concentrations of dimethyl-α-KG (DMαKG), followed by measurement of intracellular 2HG. Western blot confirms target knockdown efficiency with α-tubulin shown as loading control.

(E). Schematic summarizing enzymatic sources of hypoxia-induced L-2HG. For each panel, representative data from 1 of 3 independent experiments are shown. See also Figures S2 and S3.