Fig. 6: NADH accumulation activates purine biosynthesis to induce energy crisis.

a Cellular ATP levels of HeLa (n = 3 biologically independent samples) and MDA-MB-231 cells (n = 5 biologically independent samples) expressing Tet-on EGFP, EcSTH, LbNOX, or EcSTH-LbNOX cultured with Dox as indicated for 12 h. From left to right: **P = 2.44e-05, **P = 2.34e-05, **P = 6.13e-08, **P = 0.00277, **P = 8.62e-05, **P = 2.55e-05. b Cellular ATP level of HeLa^{Tet-on EcSTH} (n = 6 biologically independent samples) and MDA-MB-231^{Tet-on EcSTH} cells (n = 3 biologically independent samples) cultured with Dox and α-KB (2 mM) as indicated for 12 h. From left to right: **P = 1.56e-11, **P = 7.57e-10, **P = 3.68e-07, **P = 0.00020, **P = 0.00030, **P = 2.73e-05. c, Effects of PRPS1/2 knockdown on cellular ATP level in HeLa^{Tet-on EcSTH} cells cultured with Dox (1 µg/mL) for 12 h (n = 3 biologically independent samples for siPRPS2 and n = 4 biologically independent samples for shPRPS2). From left to right: **P = 2.45e-07, **P = 0.00015, **P = 3.82e-07, **P = 6.59e-08, **P = 5.67e-09, **P = 2.06e-09, **P = 1.12e-08. d Cellular ATP level of HeLa^{Tet-on EcSTH} and MDA-MB-231^{Tet-on EcSTH} cells cultured with Dox and purine synthesis inhibitors as indicated for 12 h (n = 3 biologically independent samples). From left to right: **P = 6.90e-08, **P = 6.46e-08, **P = 2.86e-07, **P = 9.23e-07, **P = 2.67e-05, **P = 0.00028, **P = 0.00125, **P = 0.00150. e Effects of purine synthesis inhibitors on AMPK pathway in HeLa^{Tet-on EcSTH} and MDA-MB-231^{Tet-on EcSTH} cells cultured with Dox (1 µg/mL) and inhibitors for 48 h. f Cellular ATP level of HeLa^{Tet-on EcSTH} and MDA-MB-231^{Tet-on EcSTH} cells cultured with Dox and AMPK activators as indicated for 12 h (n = 3 biologically independent samples). From left to right: **P = 3.52e-05, **P = 2.63e-05, **P = 5.27e-06, **P = 1.90e-07, **P = 2.87e-05, **P = 0.00033. g Effects of AMPK activators on the proliferation of HeLa^{Tet-on EcSTH} (n = 4 biologically independent samples) and MDA-MB-231^{Tet-on EcSTH} (n = 3 biologically independent samples) cells. Cells were counted after treatment with Dox (1 µg/mL) and activators as indicated for 48 h. From left to right: **P = 2.02e-05, **P = 0.00126, **P = 0.00064, **P = 0.00025, **P = 0.00145, **P = 0.00129. d–g Cells were pretreated with the indicated inhibitors or activators for 2 h. 6-MP (300 µM), PTrexol (10 µM), LTrexol (10 µM), A769662 (100 µM), and AICAR (400 µM) were used. All experimental data were verified in at least three independent experiments. Error bars represent mean ± SD. **P < 0.01; Two-tailed Student’s t-tests. Source data are provided as a Source Data file.