Fig. 5

PFK-158 triggers DNA damage due to limited ATM-driven DDR in erlotinib-treated cells. A NSCLCs were exposed to appropriate treatments for 24h. Visualization of a neutral comet assay showing therapy-induced DNA double-strand breaks compared to DMSO vehicle control. Scale bar 100 µm. B Quantification of the neutral comet assay was performed using the OpenComet plugin for ImageJ software, and DNA breaks presented as DNA olive tail moment. The violin plot represents the data for individual nuclei from 2 independent experiments with technical replicates (n=250). C ATM phosphorylation (S1981) and total protein levels in PC9 and HCC827 cells in response to treatments assessed in immunoblotting. β-actin was used as a loading control. Target/loading control ratios were quantified using densitometry and normalized to vehicle-treated samples. ATM phosphorylation ratio was calculated by dividing ATM (S1981) by the total ATM densitometry signals for each treatment and normalizing to vehicle-treated cells. D Representative images of nuclei showing phosphorylated γ-H2AX foci (S139, green) and nuclear DNA stained with DAPI (blue). Scale bar 50 µm. E Cells were analyzed using CellProfiler pipeline, and relative amounts of the cells with >10 γ-H2AX foci normalized to the total amount of cells per field and presented in %. Each data point represents one field analyzed (3 independent experiments, n=12). F, PC9 and HCC827 cells were exposed to appropriate treatments for 24h. Cytosolic (soluble) and chromatin fractions were separated and analyzed in immunoblotting. Histone H3 was used as a loading control for chromatin fraction, and β-tubulin – for soluble fraction. Target ratios were quantified using densitometry and normalized to vehicle-treated samples. B, E Statistical analysis by ONE-WAY ANOVA with Tukey’s post hoc tests (p-values are shown as follows: *, <0.05; **, <0.01; ***, <0.001; ****, <0.0001). ATM - Ataxia-telangiectasia mutated, XRCC3 - X-Ray Repair Cross Complementing 3, RAD51 - RAD51 Recombinase