Fig. 2

Metabolite profiling and effects on DNA methylation. (A) Schematic overview of TCA cycle highlighting the loss of SDH function (red “X”). (B-C) Relative metabolite levels (log scale) in SDH-loss cells compared to their respective controls (n = 3 samples of million cells per line, data shown as mean ± SEM) normalized to protein content (B) or per cell (C). Cell normalization data were accounted for mathematically by calculating the number of cells in each sample using a standard curve for protein content per 1 M cells. Lac: lactate; Asp: aspartate; Mal: malate; Fum: fumarate; Succ: succinate; Glu: glucose; 2-hg, 2-hydroxyglutarate; AKG, 2-oxoglutarate; c-Acon: cis-aconitate; Isoc: isocitrate; Cit: citrate. (D) Western blot of H3K9me3, detected at 17 kDa, where hypermethylation serves as a marker of succinate inhibition of JMJD demethylases, a subfamily of OG-dependent dioxygenases. Anti-histone H3 is used as loading control, also detected at 17 kDa. (E) Western Blot analysis for HIF1/2α levels in various indicated oxygen concentrations. Protein extracts from the indicated cell lines were probed with anti-HIF1α or HIF2α, both detected at 120 kDa (two upper panels). α-tubulin was used as a loading control and detected at 50 kDa (lower panel)