Fig. 4
SGK1 inhibition decreases long-term survival and sensitizes to ionizing radiation. Colony formation of oxic and anoxia-tolerant NCI-H460 cells upon treatment with SGK1 inhibitor GSK650394 (5 μM), ionizing radiation (2 or 5 Gy), or the combination (pretreatment with GSK650394 for 2 h) under normoxic (20 % O2), a) and severely hypoxic conditions (0.2 % O2), b). Treatment in severe hypoxia was performed 2 h after pre-incubation in hypoxia. Upper panels show representative pictures of colonies formed after 10 days in normoxia (a) or severe hypoxia (b) as indicated. Values were normalized to the plating efficiency of the respective untreated cells. Lower panels represent quantification of the surviving fraction normalized to the plating efficiency of non-irradiated cells. Anoxia-tolerant NCI-H460 cells are characterized by increased radiation resistance as demonstrated by increased surviving fractions of anoxia-tolerant NCI-H460 cells compared to oxic controls upon exposure to ionizing radiation in normoxia (a) or hypoxia (b). SGK1 inhibition alone significantly reduced the number of clonogenic tumor cells in both cell lines. The effect was more prominent under normoxic conditions (a). (Pre-)treatment with GSK650394 was highly effective in reducing the surviving fraction of anoxia-tolerant NCI-H460 cells and potently sensitized both cell lines to the cytotoxic action of ionizing radiation under normoxic conditions (a). Combined treatment with ionizing radiation and GSK650394 under severely hypoxic conditions only decreased the numbers of clonogenic anoxia-tolerant cells but was almost without effect on the oxic control cells. Mean values ± SD are shown, n = 3. (* p ≤ 0.05, ** p < 0.01, *** p ≤ 0.001; two- way ANOVA with Bonferroni post-test). IR, ionizing radiation