Fig. 4: PHGDH gene suppression sensitises while PHGDH overexpression protects human GBM cells from hypoxia-induced cell death.

a G55 PHGDHsh and control cells (non-targeting sequence, NTsh) were analysed by qPCR and immunoblot. PHGDH gene suppression was confirmed. qPCR values are normalised to 18 S as well as SDHA housekeeping gene expression (n = 3, mean ± SD). b G55 PHGDHsh and control cells were incubated in DMEM with or without 10% FCS as indicated. Medium was depleted for glycine and serine and serine was replenished where indicated. Cell density was measured by crystal violet staining at the beginning of cultivation and at 4 days (n = 6, mean ± S.D., n.s. not significant, *p < 0.05, **p < 0.01). c G55 PHGDHsh and control cells were exposed to glucose restricted (2 mM glucose) serum-free DMEM under normoxic or hypoxic (0.1% oxygen (O₂)) conditions. Serine was replenished as indicated. Cell death was quantified by LDH release (n = 4, mean ± S.D., n.s. not significant, **p < 0.01). d LNT-229 pTetOne PHGDH cells were cultured with vehicle or 0.1 µg/mL doxycycline for 24 h. PHGDH gene induction was confirmed by qPCR and immunoblot. qPCR values are normalised to 18 S as well as SDHA housekeeping gene expression (n = 3, mean ± SD). E-F, LNT-229 pTetOne PHGDH cells were preincubated with or without 0.1 µg/ml doxycycline for 24 h and exposed to glucose restricted (2 mM glucose) serum- and serine-free DMEM with or without 0.1 µg/ml doxycycline under normoxic or hypoxic (0.1% oxygen (O₂)) conditions. e Cell death was quantified by LDH release (n = 4, mean ± S.D., **p < 0.01). (n = 4, mean ± S.D.) f Analysis of NADPH/NADP⁺ ratios were performed by a luminescence-based assay (n = 3, mean ± SD, *p < 0.05).