Extended Data Fig. 3: 2-HG accumulation augments protein levels, not mRNA transcription, of mutant p53.
From: Malic enzyme 2 maintains protein stability of mutant p53 through 2-hydroxyglutarate
a, Lysates from SF188 cells were subjected to immunoprecipitation using indicated antibodies followed by immunoblotting analysis. b, Colon tissue lysates from three pairs of ME2-/- and ME2+/+ mice were analyzed for the expression of ME2 by western blot. c and d, Western blot of SF188 cells (c) and MDA-MB-231 cells (d) treated with 8 mM 2-HG or left untreated (0) for 24 hours. e, Western blot of SF188 cells treated with 5 mM 2-HG or 5 mM α-KG as indicated. f, p53 mRNA levels in SF188 cells and MDA-MB-231 cells cultured in medium containing 0 or 5 mM 2-HG for 24 hours. (n = 3 biological independent samples). g and h, SF188 cells (g) and H1299 cells (h) transfected with luciferase constructs containing p53 promoter, p53 3’UTR region or vector control were treated with 5 mM 2-HG for 36 hours. Renilla vector pGL3 Basic-CMV was used as a transfection internal control. The relative luciferase activity was normalized to the co-transfected Renila activity. (n = 3 biological independent samples). i and l, Western blot of MDA-MB-231 cells (i) and p53+/+ HCT116 cells (l) cultured in medium containing 0 or 8 mM 2-HG for 36 hours prior to 200 μg/mL CHX treatment for indicated time points. Relative p53/actin ratios are shown. j, Western blot of SF188 cells treated with 8 mM 2-HG, 8 mM α-KG or left untreated for 36 hours followed by 200 μg/mL CHX treatment for indicated time points. Relative p53/actin ratios are shown. k, Western blot of Flag-p53R248W-expressing HEK293 cells treated with 8 mM 2-HG, or left untreated for 36 hours followed by 100 μg/mL CHX treatment for indicated time points. Relative p53/actin ratios are shown. m, Western blot analysis of p53G266E expression in SF188 cells stably expressing Flag-IDH1, Flag-IDH1R132H or vector control. n, Western blot of SF188 cells cultured in standard medium or in acidic medium (pH6.0) for 24 hours followed by 200 μg/mL CHX treatment for indicated time points. Relative p53/actin ratios are shown. Data in f, g, h are means ± SD, P values were determined by unpaired two-tailed Student’s t-tests.
