Extended Data Fig. 4: Regulation of 2-HG production by ME2 and effect of pyruvate on mutp53 stability in SF188 cells.

a-e, The structure analysis of 2-HG. (a) The scheme of 2-HG in LC-MS/MS. (b) Relative levels of 2-HG in SF188 cells transfected with Ctrl or ME2 siRNA for 48 hours were determined by Thermo Fisher UHPLC Q-Exactive MS. (n = 3 biological independent samples) (c) 2-HG standard MS1 Spectra by Thermo Fisher UHPLC Q-Exactive MS. (d-e) 2-HG MS1 Spectra by Thermo Fisher UHPLC Q-Exactive MS in SF188 cells treated with Ctrl (d) or ME2 siRNA (e) for 48 hours. f, NADPH and position-specific labelled pyruvate (pyruvate ¹³C-labelled at the C1, C2, C3, C2 and C3, or all-carbon positions) were incubated with purified ME2 or vector control proteins (Ctrl) for 60 min at 37°C as indicated. 2-HG production was determined by LC-MS analysis and the incorporation of ¹³C atoms into 2-HG are denoted as m + n, where n is the number of ¹³C atoms. The insert shows Coomassie blue-stained SDS-PAGE gel of ME2 protein. n = 3 biological independent samples. g, Relative p53G266E protein expression in SF188 cells treated with CHX in the presence or absence of 8 mM pyruvate as indicated. h, SF188 cells treated with control Ctrl or ME2 siRNA for 48 hours in the presence or absence of 8 mM pyruvate were collected after 4 hours of 10 μM MG132 treatment and subjected to immunoprecipitation using anti-p53 antibody. Immunoprecipitates and input were analyzed by western blot. i, Relative 2-HG levels in SF188 cells treated with Ctrl or ME2 siRNA, and/or 8 mM pyruvate as indicated for 48 hours. (n = 3 biological independent samples). j, SF188 cells transfected with Ctrl or ME2 siRNA were cultured in medium containing 8 mM pyruvate, 2-HG or α-KG as indicated for 48 hours. p53G266E expression was measured by western blot analysis. Data in b, f, i are means ± SD, P values were determined by unpaired two-tailed Student’s t-tests. All immunoprecipitation and western blot experiments were repeated three times.

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