Fig. 3

ENPP1 blockade synergizes with DDR inhibitors and boosts DNA damage post-IR. a Left panels: Heatmaps displaying the percentage of cell viability after 5 days of treatment with modulators of DNA integrity and ENPP1i from a targeted pharmacological screen. The x-axis features 11 kinase inhibitors that target components of the DNA damage response. Right panel: Synergy score. ATMi in combination with ENPP1i showed the highest synergistic effect (Combination Index close to 0). b Viability assay was conducted 5 days after incubation with ATMi or PARPi post-IR at increasing concentrations of ENPP1i showing a decrease in the IC₅₀. c Left panels: Quantification of the Comet assay. Right panels: Representative images of the Comet assay showing the tail moment of the indicated cells treated with IR (2 Gy), ATMi (5 µM) and ENPP1i (5 µM). Brackets point to the tail length. Kruskal-Wallis was used for comparison and Dunn’s post-hoc multiple comparisons test against the Control group. ***P < 0.001. Scale bar = 50 µm. d Left panels: Quantitative assessment of the number of positively labeled cells with anti-γH2AX antibody performed by an in-house developed macro based on ImageJ®. Cells were incubated with ENPP1i (5 µM) and ATMi (5 µM) for 24 h. n > 100 cells were examined over three biologically independent experiments. Median and interquartile range are represented. One-way ANOVA was used for comparisons, and Dunnett´s multiple comparisons test against the Control group. Right panels: Representative immunofluorescence images of nuclear γH2AX (red) and nuclei (blue) in cells subjected to the indicated treatments. Scale bar = 10 µm. e Immunoblot analysis of protein expression levels of γHA2X, PARP1, cleaved PARP1 (c-PARP), ENPP1, RAD51, GAPDH, and Tubulin from cell lysates extracted from a time course after treatment with IR (2 Gy), IR/ENPP1i (5 µM), IR/ATMi (5 µM), or the triple combination in ANV5-OE and OE-4T1 cells. Normalization in each immunoblot was performed relative to the control cells (C) at time 0. Cross-comparison between immunoblots should take into account variation among control samples on each membrane. Dashed lines segregate different treatments within individual immunoblots to enhance visualization