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. 2023 Sep 19;33(11):835–850. doi: 10.1038/s41422-023-00874-4

Fig. 4. PHGDH mediates glucose starvation-induced apoptosis.

Fig. 4

a–c 3-PGA-unoccupied PHGDH induces apoptosis in SK-Hep-1 cells starved for glucose. Cells were infected with lentiviruses carrying HA-tagged PHGDH-T57A or PHGDH-T78A (a, b), or PHGDH-R135W or PHGDH-V261M (a, c) expressed under a doxycycline-inducible promoter. Cells were then incubated in RPMI 1640 medium or glucose-free RPMI 1640 medium, both containing doxycycline (100 ng/mL), for 8 h, followed by determining the levels of apoptotic cells via flow cytometry (a, see gating strategy for quantifying the populations of apoptotic cells in Supplementary information, Fig. S3a, and representative density plots in Supplementary information, Fig. S3b), and the levels of apoptotic markers by immunoblotting (b, c). Data in a are means ± SD, n  =  3, with P values calculated by two-way ANOVA, followed by Tukey. L.E., long exposure; S.E., short exposure. d, e PHGDH does not induce necroptosis, pyroptosis or ferroptosis in low glucose. SK-Hep-1 cells with inducible expression (as in a) of PHGDH-T57A (d) or PHGDH-R135W (e) were treated with 15 μM Necrostatin-1 (Nec-1; d), 0.75 μM GSK-872 (d), 0.5 μM Ferrostatin-1 (Fer-1; d), 20 μM Z-VAD (d), 10 nM Nigericin (Nig; e), 10 μM Erastin (Era; e), a combination of 10 IU/mL TNFα, 2.5 μM SM-164, and 10 μM Z-VAD (TSZ; e), all for 8 h, followed by determining the numbers of dead (PI-positive) cells via flow cytometry. Data are means ± SD, n = 3, with P values calculated by one-way ANOVA, followed by Tukey. See representative density plots in Supplementary information, Fig. S3h, i. Experiments were performed three times, except four times in a.