SLFN11 KO AML cells present enhanced activation of the ATR/CHK1 pathway and decreased cell death in response to AraC treatment. (A-B) U937-Cas9 and U937-SLFN11 KO cells (single clones) were treated with VC (water) or AraC (500 ng/mL) for 6 hours and stained with propidium iodide (PI), followed by flow cytometry analysis. (A) Representative flow cytometry histograms showing U937-Cas9 and U937-SLFN11 KO cell populations in the sub-G1, G1, S, and G2-M phases of the cell cycle before (untreated [UT]) and after AraC treatment. (B) The bar graph of flow cytometry data shows the cell cycle distribution for each cell type and treatment condition. The mean ± standard deviation of 3 independent experiments are shown. For each phase of the cell cycle (sub-G1, G1, S, and G2/M), statistical analysis was performed using 2-way ANOVA followed by Sidak multiple comparisons test (∗∗∗P < .001; ∗∗∗∗P < .0001). (C) U937-Cas9 and U937-SLFN11 KO cells (pooled clones) were treated with either VC (water) or AraC (500 ng/mL) for the indicated lengths of time (hours). The cells were then lysed, and protein lysates were resolved by SDS-PAGE, followed by immunoblotting analyses using the indicated antibodies. GAPDH is shown as a loading control. Immunoblots are representative of 3 independent experiments. (D) U937-Cas9 and U937-SLFN11 KO cells (pooled clones) were seeded into 96-well plates and treated with either VC (DMSO) or increasing concentrations of the ATRi VE822 for 24 hours. Cell viability was assessed using the WST-1 assay. Data are expressed as a percentage of cell viability of VC-treated cells. Means ± SEM of 3 independent experiments are shown. (E) U937-Cas9 (left panel) and U937-SLFN11 KO cells (right panel; pooled clones) were seeded in 96-well plates and treated with either VC (DMSO), 0.3 μM VE822, or increasing concentrations of AraC alone or in combination with 0.3 μM VE822 for 24 hours. Cell viability was assessed by WST-1 assay. Data are expressed as a percentage of cell viability of VC-treated cells (for AraC alone) or 0.3 μM VE822-treated cells (for AraC + 0.3 μM VE822). Means ± SEM of 3 independent experiments are shown. Statistical analysis was performed using 2-way ANOVA followed by the Sidak multiple comparison test. ∗∗P < .01; ∗∗∗P < .001; ∗∗∗∗P < .0001. (F) U937-SLFN11 KO cells (pooled clones) were treated with VC (DMSO), AraC (500 ng/mL), and/or VE822 (0.3 μM) for 1, 4, or 8 hours, as indicated. Cells were lysed and protein lysates were resolved by SDS-PAGE, followed by immunoblotting analyses using the indicated antibodies. GAPDH is shown as a loading control. Immunoblots are representative of 3 independent experiments. ns, not significant.