Figure 2. OGG1 and XRCC1 modulate cellular responses to ß-lap.

(A) Relative 8-oxo-G levels were monitored in MiaPaca2 cells before or after ß-lap (4.0 μM), with or without co-administration of dicoumarol (50 μM) and compared to DMSO-treated control cells after 30 min treatments (DAPI, SF3A). (B) Relative 8-oxo-G levels (Intensity Quantification) of MiaPaca2 cells treated as in A using NIH ImageJ after exposure to various ß-lap doses, with or without dicoumarol, vehicle alone or H₂O₂ at indicated doses. (C) MiaPaca2 cells were transfected with pooled RNAis against specific BER proteins and treated 48 h later with DMSO or ß-lap (3 μM, 2 h) and survival was assessed using colony forming assays. XRCC1 and OGG1 were identified as genes altering sensitivity to ß-lap. (D,E) OGG1 protein levels were depleted in 48 h by specific siRNAs in MiaPaca2 or ASPC1 cells. Cells were then treated with ß-lap ± Rucaparib (25 μM) for 2 h and ATP levels monitored using CellTiter-Glo assays. (F) Stable shXRCC1 knockdown MiaPaca2 clones were generated as assessed by Western immunoblotting. (G) Clonogenic survival assays were used to determine LD₅₀ values for each ß-lap-treated MiaPaca2-non-targeting (NS) or shXRCC1 clones in separate dose-response studies. Plating efficiencies were not altered by shXRCC1 depletion. (H) ß-Lap dose-response of shXRCC1 MiaPaca2 clone #8 by clonogenic survival assays. (I) Relative intracellular NAD⁺ levels in stable shScr or shXRCC1 knockdown MiaPaca2 cells before and after exposure to various doses of ß-lap (μM, 2h). Results were compared using Student’s t-tests (+/− standard deviations). *p < 0.05; **p < 0.01; ***p < 0.001.