Extended Data Fig. 9: 2-HG supplementation restores anchorage-independent growth of p53 mutant tumour cells depletion of ME2 in soft agar.
From: Malic enzyme 2 maintains protein stability of mutant p53 through 2-hydroxyglutarate
a-c, p53-/- HCT116 cells stably expressing p53R273H (a), p53R175H (b) or p53R282W(c) were transfected with Ctrl, ME2 or p53 siRNA for 24 hours. Equal number of the cells were then cultured in soft agar medium for 2 weeks in the presence or absence of 8 mM 2-HG. Numbers of colonies with a diameter greater than 20 µm were quantified. In a, n = 6 (for each vehicle treatment) or 3 (for each 2-HG treatment) biological independent wells. In b and c, n = 3 biological independent wells. Protein expression was measured by western blot. Representative images of colonies stained with crystal violet at day 14 are shown. d, Colony formation assay of MDA-MB-231 cells treated with Ctrl, ME2 or p53 siRNA in the presence or absence of 8 mM 2-HG. Numbers of colonies with a diameter greater than 20 µm were quantified. n = 3 biological independent wells. Representative images of colonies stained with crystal violet at day 14. p53 expression was measured by western blot. e, Colony formation assay of HCT116 cells treated with control, ME2 or p53 siRNA in the presence or absence of 8 mM 2-HG. Numbers of colonies with a diameter greater than 20 µm were quantified. n = 4 biological independent wells. Representative images of colonies stained with crystal violet at day 14. p53 expression was measured by western blot. Data in a-e are means ± SD, P values were determined by unpaired two-tailed Student’s t-tests.
