Fig. 1.

RRM2 acetylation at K95 downregulates RNR enzymatic activity. a, b Extracts from various human cells treated with the combination of NAM (10 mM) and TSA (2 μM) were incubated with ¹⁴C-CDP. The conversion from ¹⁴C-CDP to ¹⁴C-dCDP was analyzed by thin-layer chromatography (TLC). RNR activity was calculated as ¹⁴C-dCDP/(¹⁴C-CDP + ¹⁴C-dCDP). The error bars indicate ± s.d. of three separate experiments. ***P < 0.001, by two-tailed t test. c Various human cell lines were treated with NAM/TSA for 18 h, followed by IP using an anti-RRM2 antibody. Acetylation of RRM2 (Ac-K RRM2) was analyzed by western blot using acetylated-lysine-specific antibody. d H1299 cells were treated with NAM/TSA for 18 h, followed by RRM2 immunoprecipitation and analysis of LC-MS/MS peptide spectra of RRM2 acetylation. e H1299 cells expressing Flag-tagged WT or mutant RRM2 were treated with NAM/TSA, followed by Flag IP. RRM2 acetylation was analyzed as above. f, g Flag-RRM2 variants were immunoprecipitated from H1299 cells treated with NAM/TSA, then mixed with 1 µg of purified GST-RRM1 protein, followed by TLC analysis for RNR activity as above. h, i The effects of various acetyl-mimetic mutant RRM2 proteins on RNR activity were analyzed as above. The error bars indicate ± s.d. of three separate experiments. ***P < 0.001, by two-tailed t test