Fig. 4: Deregulated purine biosynthesis is associated with NADH accumulation-induced cell death.
From: Identification of purine biosynthesis as an NADH-sensing pathway to mediate energy stress
a The rationale of screening for NADH accumulation-associated cell death-promoting genes. b Workflow for the CRISPR/Cas9 screening strategy. Two sgRNA pools (Human GeCKO v2 and Brunello libraries) were used. c Metabolic processes coupling glycolysis (black lines), pentose phosphate pathway (blue lines), and purine biosynthesis (purple lines). The green balloon shows the potential activation of PRPS by NADH. G6P, glucose 6-phosphate; F6P, fructose 6-phosphate; GAP, glyceraldehyde 3-phosphate; R5P, Ribose 5-phosphate; PRPP, phosphoribosyl pyrophosphate; IMP, inosine monophosphate; AMP, adenosine monophosphate; GMP, guanosine monophosphate; d Effects of TKT knockout on the proliferation of HeLaTet-on EcSTH or HeLaTet-on EcSTH-LbNOX cells (n = 3 biologically independent samples). Cells were counted after treatment with Dox (0.1 µg/mL) for 48 h. The immunoblot showed the levels of TKT. From left to right: **P = 0.00015, **P = 1.48e-05. e Principal-component analysis of metabolites in HeLaTet-on EcSTH cells with sgTKT versus sgControl cultured with Dox (0.1 µg/mL) for 24 h (n = 3 biologically independent samples). f Volcano plot of the differential metabolites (two-tailed Student’s t-tests) between sgTKT and sgControl in HeLaTet-on EcSTH cells cultured with Dox (0.1 µg/mL) for 24 h (n = 3 biologically independent samples). The red highlights represent metabolites involved in purine metabolism and the pentose phosphate pathway. g KEGG pathway analysis of the upregulated metabolites (fold change > 1.5, P value < 0.05) using MetaboAnalyst 5.0. h Effects of purine biosynthesis inhibitors on proliferation of TKT knockout HeLaTet-on EcSTH and MDA-MB-231Tet-on EcSTH cells (n = 3 biologically independent samples). Cells were pretreated with the indicated inhibitors for 2 h and counted after treatment with Dox (0.1 µg/mL) and inhibitors for 48 h. 6-Mercaptopurine (6-MP, 300 µM), pelitrexol (PTrexol, 10 µM), lometrexol (LTrexol, 10 µM). From left to right: **P = 1.53e-05, **P = 1.85e-05, **P = 3.40e-05, **P = 2.31e-05, **P = 7.37e-07, **P = 5.72e-06, **P = 2.24e-05, **P = 8.99e-05. d, h 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.
