Fig. 3

DDIT4 gene suppression sensitises human glioblastoma cells to temozolomide, irradiation and hypoxia-induced cell death. a LNT-229 or G55 NTsh or DDIT4sh cells were incubated for 24 h with vehicle or temozolomide as indicated. After 24 h, the medium was replaced with fresh DMEM without temozolomide. Experiments were stopped by CV staining and clones counted manually under the microscope. Clonogenicity is depicted relative to the vehicle condition (n = 3, mean ± S.D, NS = not significant, *p < 0.05, Student’s t test). b Cells were seeded as in a, irradiated with 2 or 6 Gy, and 24 h thereafter, the medium was replaced with fresh DMEM. After an incubation period of 8 days, cells were stained with CV and clones counted manually as in a. Clonogenicity is depicted relative to unirradiated cells (n = 3, mean ± SD, NS = not significant, *p < 0.05, Student’s t test). c LNT-229 or G55 NTsh and DDIT4sh cells were incubated in serum-free (left panel) or serum-containing (10% FCS, right panel) culture conditions without glucose restriction (25 mM glucose) for 4 days in normoxia. Cell density was measured by CV staining (n = 6). d LNT-229 or G55 NTsh and DDIT4sh cells were incubated for 32 h under normoxia or 0.1% oxygen (hypoxia) in serum-free DMEM with glucose restriction (2 mM glucose). Cell death was quantified by LDH release (n = 4, mean ± SD, NS = not significant, **p < 0.01, Student’s t test)