Fig. 4. KL-50 (4a) activates DDR pathways and cycle arrest in MGMT− cells, independent of MMR, and cells deficient in ICL or HR repair are sensitized to KL-50 (4a).

(A) γH2AX, (B) 53BP1, and (C) pRPA foci formation quantified by percent of cells with ≥10 foci in LN229 MGMT+/−, MMR+/− cells treated with 0.1% DMSO control, 20 μM KL-50 (4a), or 20 μM TMZ (1a) for 48 hours. Columns indicate the mean; error bars indicate SD; n > 5 technical replicates. Additional time course data are presented in fig. S6, B to D. (D) Representative foci images of data in (A) to (C). (E) Percentage of cells in G₁, S, and G₂ cell cycle phases after treatment as in (A) to (C), measured by using integrated nuclear (Hoechst) staining intensity. Columns indicate the mean; error bars indicate SD; n = 3 independent analyses. Additional time course data, cell cycle controls, and representative histograms are presented in fig. S7. (F) Change in percent cells with ≥1 micronuclei from baseline (DMSO control) after treatment as in (A) to (C). Columns indicate the mean; error bars indicate SD; n ≥ 15 technical replicates; ****P < 0.0001; ns, not significant. Additional validation is presented in fig. S9, A and B. (G) Short-term viability assay curves for KL-50 (4a) in PD20 cells, deficient in FANCD2 (FANCD2−/−) or complemented with FANCD2 (+FANCD2). (H) Short-term viability assay curves for KL-50 (4a) in PEO4 (BRCA2+) and PEO1 (BRCA2−/−) cells pretreated with 0.01% DMSO control or 10 mM O⁶BG (+O⁶BG) for 1 hour before KL-50 (4a) addition. (I) Short-term viability assay curves for KL-50 (4a) in DLD1 BRCA2+/− and BRCA2−/− cells pretreated with 0.01% DMSO control or 10 mM O⁶BG (+O⁶BG) for 1 hour before KL-50 (4a) addition. For (G) to (I), points indicate the mean, and error bars indicate SD; n = 3 technical replicates.