Fig. 4: Targeting intrinsic dependency on ser/gly synthesis radio-sensitizes NSCLC cell lines and decreases their stemness.

a Quantification of clonogenic assays as survival fraction of sertraline treatment relative to DMSO controls in NSCLC cell lines. After pre-treatment with sertraline for 48 h, single cells were seeded in DMSO, or sertraline containing media and left for 7 days. Media was changed after 7 days allowing the cells to grow without treatment for 7–10 more days; colonies were counted, calculating the survival fraction. Data are represented as mean ± standard deviation. Individual dots represent independent observations. P-values were calculated using an unpaired 2-tailed t-test. b Survival curves for NSCLC cells upon increasing doses of irradiation 2, 4 and 6 Gy, with and without sertraline. NSCLC cell lines were pre-treated with sertraline for 48 h and, subsequently exposed to 2, 4 and 6 Gy irradiation doses. Cells were dissociated and single cells seeded in DMSO, or sertraline containing media and left for 7 days. Media was changed after 7 days allowing the cells to grow without treatment for 7–10 more days. Survival fraction was determined relative to the non-irradiated cells with and without sertraline. Average results of n ≥ 3 experiments are shown, and data are fitted to a quadratic linear model. c Timeline showing the experimental procedure. A clonogenic as described above was performed, followed by a clonogenic replating or a limiting dilution sphere formation assay in the absence of any treatment. d Representative images of Calu-6 clonogenic assay. e Graph showing the number of colonies (>50 cells) after replating 500 cells from end-stage clonogenic surviving colonies of the Calu-6 cell line. One-way ANOVA with Tukey’s multiple comparison test was used. Data are represented as mean ± standard deviation. Individual dots represent different technical replicates. f Graph showing the fold change in stem cell frequency of the different originating groups compared to the control cells. One-way ANOVA with Dunnett’s multiple comparisons test was used. Data are represented as mean ± standard deviation. Individual dots represent n = 3 independent calculations from limiting dilution spheroid experiments. g Representative pictures at 20× magnification of Calu-6 spheres at day 30 after an initial seeding density of 25 cells/well and condition. Statistical analysis *p-value < 0.05, **p-value < 0.01, ***p-value < 0.001.