Fig. 3. Mis-splicing of DNA repair genes alters protein function and results in accumulation of DNA damage.

(A) average PSI scores of BRCA2 exon12 and BRCA1 exon9 are plotted for each genotype and drug condition. data points represent the mean of biological triplicates (wild type and STAG2-KO2) or duplicates (STAG2-KO1 and SMC3-heterozygous) for each treatment condition. (B) Western blot analysis of BCRA1 and BRCA2 protein expression in wild-type and STAG2-KO2 cells treated with 50 nM H3B-8800 or DMSO for 3 days. each lane represents an independent single-cell clone of U937 cells transduced with a nontargeting sgRNA (wild type) or sgRNA targeting STAG2. actin was used as a loading control. (C) Schematic of CHEK2 exon structure (top) with the Thr⁶⁸ phosphorylation residue highlighted in red and the annotated kinase domain shown in green. Percent spliced in scores of exon2 and exon10 are shown for each treatment condition. data points represent the mean of biological triplicates (wild type and STAG2-KO2) or duplicates (STAG2-KO1 and SMC3-heterozygous) for each treatment condition. (D) Western blot analysis of pCHK2 and total CHK2 protein in wild-type and STAG2-KO2 cells treated for 3 days with 50 nM H3B-8800 or DMSO. each lane represents an independent single-cell clone of U937 cells transduced with a nontargeting sgRNA (wild type) or sgRNA targeting STAG2. Vinculin and actin were used as loading controls. (E) Western blot analysis of γh2aX protein in wild-type and STAG2-KO2 cells treated for 3 days with 50 nM H3B-8800 or DMSO. each lane represents an independent single-cell clone of U937 cells transduced with a nontargeting sgRNA (wild type) or sgRNA targeting STAG2. actin was used as a loading control. (F) representative images of cells treated with 50 nM H3B-8800 or DMSO control for 24 hours and stained for DNA/nuclei (DAPI, blue) and RAD51 (green).