Fig 2: A schematic of AMPK-mediated NAMPT activation under ionizing radiation. Ionizing radiation causes rapid activation of AMPK, which in turn phosphorylates NAMPT S314 and enhances NAMPT enzymatic activity. NAMPT activation promotes NAD+ synthesis, thereby facilitating DNA repair and cell viability.

Fig 3: AMPK phosphorylates NAMPT S314 under ionizing radiation. (b–d, f–k) Immunoblotting analyses were performed using indicated antibodies. (a) HOK cells were pre-treated with 10 µM KU55933, 1 µM NU7441, 2 µM AZD6738, 20 µM PD98059, 20 µM SP600125 or 5 µM Compound C for 2 h, and cells were treated with 10 Gy ionizing radiation. Cellular NAMPT activity was measured 30 min after irradiation. **p < 0.01; ns, not significant. (b,c) HOK and HUVEC cells were treated with 10 Gy ionizing radiation, and cells were harvested 15 min after irradiation. Immunoblots (b) and immunoprecipitations (c) were performed using indicated antibodies. IR, ionizing radiation; WCL, whole cell lysate. (d) Bacterially purified His/Flag-NAMPT protein was incubated with purified active AMPK proteins (His-AMPKα1, untagged AMPKβ1 and untagged AMPKγ1) in the presence or absence of Compound C and [γ-32P]-ATP for an in vitro kinase assay. Immunoprecipitation was performed using anti-Flag antibody, and radioactivity in the precipitates was measured by autoradiography. (e) Alignment analyses of NAMPT S314 or T304 was performed among indicated species. S314 and T304 were shown in red, and the residues matches AMPK phosphorylation consensus was shown in blue. (f) Bacterially purified WT His/Flag-NAMPT protein or indicated mutants were incubated with purified active AMPK proteins in the presence [γ-32P]-ATP for an in vitro kinase assay. Immunoprecipitation was performed using anti-Flag antibody, and radioactivity in the precipitates was measured by autoradiography. (g) HOK cells were treated with 10 Gy ionizing radiation. Immunoblots were performed using indicated antibodies in the presence or absence or NAMPT pS314 blocking peptide. (h) HOK and HUVEC cells with expression of WT Flag-NAMPT or Flag-NAMPT S314A were treated with 10 Gy ionizing radiation, and immunoprecipitation was performed 30 min after irradiation. (i) HOK cells with expression of Flag-NAMPT were pre-treated with 5 µM Compound C for 2 h, and treated with 10 Gy ionizing radiation. Immunoprecipitation was performed 30 min after irradiation. (j) HOK cells were treated with 0.5 mM A769662 for 30 min. (k) HOK cells were incubated with glucose-free medium for 12 h. Glc, glucose.

Fig 4: G6PD regulates cellular metabolism independently of its dehydrogenase activity.A, HEK293T cells were transfected with vector, Flag-G6PD-WT, Flag-G6PD-R198P, or Flag-G6PD-H263A plasmid and then co-IP assays were performed 48 h after transfection using anti-FLAG affinity M2 beads, followed by Western blot. B, schematic diagram of the metabolism of glucose, GSH, glutamine, and fatty acids. C–E, glucose uptake (C), lactate excretion (D), and glycerol excretion (E) in HeLa cells with different states of G6PD, as indicated, under the normal condition. F, enzyme activity of GAPDH in HeLa cells lysates with different states of G6PD. G and H, relative labeled carbons in fatty acids in HeLa cells with different states of G6PD cultured with 13C5-glutamine for 48 h in the normal condition. I, the total GSH (GSH + GSSG) and the ratio of GSH/GSSG was determined in HeLa cells with different states of G6PD treated with or without PMS (1 μM) for 6 h. J, cell survival of HeLaKO cells treated with antimycin A (1 μM) or PMS (1 μM) for 24 h in the presence of different concentrations of GSH and NAC (pretreatment for 6 h). K, the working model of antistress roles of G6PD by binding to AMPK, NAMPT, PFKP, GAPDH, PGP, GCLC, GCLM, ACLY, IDH1, and other proteins. ∗p < 0.05; ∗∗p < 0.01 (t test). G6PD, glucose-6-phosphate dehydrogenase; NAMPT, nicotinamide phosphoribosyltransferase; PMS, phenazine methosulfate.

Fig 5: G6PD directly binds to AMPK independently of its dehydrogenase activity.A, HEK293T cells were transfected with vector, Flag-G6PD-WT, Flag-G6PD-R198P, or Flag-G6PD-H263A plasmid and then co-IP assays were performed 48 h after transfection using anti-FLAG affinity M2 beads, followed by Western blot. B, interaction of GST-AMPKα1, GST-AMPKβ1, or GST-AMPKγ1 with His-G6PD in the reaction mixtures was analyzed by a GST-pulldown assay. C, the interaction between endogenous G6PD and AMPKα or NAMPT was determined in HeLa cells. D, validation of the interaction between AMPKα, NAMPT, NADK1, and G6PD by bimolecular fluorescence complementation (BiFC) analysis. The scale bar represents 10 μm. Randomly selected representative cells were quantified for fluorescence intensity ratios of vennus/mCherry (n = 6). E, HEK293T cells were transfected with vector, Flag-AMPKα1, Flag-AMPKα1-N, or Flag-AMPKα1-C plasmid and then co-IP assays were performed 48 h after transfection using anti-FLAG affinity M2 beads, followed by Western blot. F, HEK293T cells were transfected with vector, Flag-G6PD-WT plasmid, and then treated with or without PMS (1 μM) for 8 h. Co-IP assays were performed using anti-FLAG affinity M2 beads followed by Western blot. ∗∗p < 0.01 (t test). Co-IP, coimmunoprecipitation; G6PD, glucose-6-phosphate dehydrogenase; NAMPT, nicotinamide phosphoribosyltransferase; PMS, phenazine methosulfate.