Fig. 1.

Combination treatment with 5-azacytidine and talazoparib produces synergistic cytotoxicity in NSCLC models. (A) Colony forming assay in H460 NSCLC cells in presence of vehicle (DMSO), TAL (2 nM), 5-AZA (250 nM), or combination (day 10, n = 9 from 3 experiments performed in triplicate). Data are represented as mean number of colonies ± SEM. P value is calculated using 1-way ANOVA. (B) Combination index (CI) plot for 5-AZA+TAL in H460 cells (n = 9 from 3 experiments performed in triplicate). (C) Proximity ligation assay for PARP-γH2AX foci formation in H460 following treatment with vehicle (DMSO), TAL (2 nM), 5-AZA (250 nM), or combination (day 4, n = 100 per condition; 25 cells counted per condition from 4 experimental replicates). Data are represented as mean number of cells with >20 PLA foci ± SEM. P value is calculated using 1-way ANOVA. (D) Proximity ligation assay for DNMT1-PARP1 foci formation in H460 following treatment with vehicle (DMSO), TAL (2 nM), 5-AZA (250 nM), or combination (day 4, n = 100 per condition; 25 cells counted per condition from 4 experimental replicates). Data are represented as mean number of cells with >20 PLA foci ± SEM. P value is calculated using 1-way ANOVA. (E) DNA fiber assay in H460 following treatment with vehicle (DMSO), TAL (2 nM), 5-AZA (250 nM), or combination (day 4, n = 50 per condition; 25 fibers measured per condition from 2 experimental replicates). Data are represented as ratio of CldU fiber length to IdU fiber length for individual fiber tracts, overlaid with group mean ± SEM. P value is calculated using 1-way ANOVA. (F) Detection of γH2AX foci in H460 following treatment with vehicle (DMSO), TAL (2 nM), 5-AZA (250 nM), or combination (day 4, n = 100 per condition; 25 cells counted per condition from 4 experimental replicates). Data are represented as mean number of cells with >20 foci ± SEM. P value is calculated using 1-way ANOVA. (G) Detection of RAD51 foci in H460 following treatment with vehicle (DMSO), TAL (2 nM), 5-AZA (250 nM), or combination (day 4, n = 100 per condition; 25 cells counted per condition from 4 experimental replicates). Data are represented as mean number of cells with >20 foci ± SEM. P value is calculated using 1-way ANOVA. (H) Tumor volume in in vivo H460 NSCLC model. H460 xenograft (10⁷ cells per mouse) was delivered via flank injection (n = 8 per group). Treatment with 5-AZA (0.5 mg/kg SC) and/or TAL (0.3 mg/kg PO) was initiated once tumor volume reached 100 mm³ and continued until endpoint. Data are represented as mean external tumor volume ± SEM. P value is calculated using 1-way ANOVA.