Fig. 3: HPRT1 converts TMZ-derived AICA to AICAR.
a Cells were treated with 0.2 mM of 15N-TMZ for 2 h. Intracellular 15N-AICA were measured by HPLC-MS. Data represent the mean ± SD from sextuplicate experiments. **P < 0.001. U87, Control vs. HPRT1 shRNA, P = 5.27e-10; WT vs. D138N, P = 1.63e-09; WT vs. K166A, P = 1.95e-09; MES28, Control vs. HPRT1 shRNA, P = 2.98e-11; WT vs. D138N, P = 2.61e-12; WT vs. K166A, P = 1.96e-12. b Cells were treated with 0.2 mM of 15N-TMZ for 2 h. Intracellular 15N-AICAR levels were measured by HPLC-MS. Data represent the mean ± SD from sextuplicate experiments. **P < 0.001. U87, Control vs. HPRT1 shRNA, P = 4.75e-08; WT vs. D138N, P = 1.18e-10; WT vs. K166A, P = 1.32e-10; MES28, Control vs. HPRT1 shRNA, P = 1.61e-09; WT vs. D138N, P = 1.67e-08; WT vs. K166A, P = 1.31e-08. c Representative chromatograms of products of the HPRT1 kinase assay. d Michaelis-Menten curve of HPRT1 for AICA. Reactions were performed by mixing purified active HPRT1 and AICA. Data represent the mean ± SD from sextuplicate experiments. e and f Cells with or without shRNA-mediated HPRT1 depletion were treated with or without 0.2 mM of TMZ (e) or 0.25 of mM AICA for 2 h (f), respectively. Immunoblot analyses were performed with the indicated antibodies. Three biological repeats were repeated independently with similar results. g HPRT1-depleted cells with reconstituted expression of WT Flag-HPRT1, Flag-HPRT1 D138N, or Flag-HPRT1 K166A were treated with or without 0.2 mM of TMZ for 2 h. Immunoblot analyses were performed with the indicated antibodies. Three biological repeats were repeated independently with similar results. Statistics: a, b unpaired Student’s t-test for two-group comparison. Source data are provided as a Source Data file.
