Fig. 4: TMZ-activated AMPK phosphorylates RRM1 at T52.

a–e, g–i Immunoprecipitation and immunoblot analyses were performed with the indicated antibodies. a–i Three biological repeats were repeated independently with similar results. a Cells pretreated with or without 0.25 mM of AICA were treated with or without 0.2 mM of TMZ for the indicated time points. b Cells pretreated with or without 0.25 mM of A769662 were treated with or without 0.2 mM of TMZ for the indicated time points. c Cells were treated with or without 0.2 mM of TMZ for 2 h. d In vitro phosphorylation and SDS-PAGE analysis and autoradiography were performed by mixing purified WT His-RRM1 or His-RRM1 T52A protein with active AMPK in the presence of [γ-³²P]ATP. e Bacterially purified WT His-RRM1 or His-RRM1 T52A was incubated with or without active AMPK in the presence or absence of ATP. f Stoichiometry of RRM1 phosphorylation by AMPK. Bacterially purified His-RRM1 was incubated with active AMPK in the presence of [γ-³²P]ATP. The radioactive intensity of incorporated ³²P was measured and the incorporation of ³²P into RRM1 was calculated. Data represent the mean ± SD of triplicate samples. g The indicated cells expressing WT Flag-RRM1 or Flag-RRM1 T52A were treated with the indicated dose of TMZ for 2 h. h AMPKα1/2 double knockout (DKO) U87 cells expressing WT Flag-RRM1 or Flag-RRM1 T52A were treated with or without 0.2 mM of TMZ for 2 h. C1 and C2, two clones of AMPKα1/2 DKO U87 cells. i The indicated cells with or without HPRT1 depletion were transfected with vectors expressing WT Flag-RRM1 or Flag-RRM1 T52A. The cells were further treated with 0.2 mM of TMZ for 2 h.