Expression of KRASG13D increases the misrepair frequency and deletion size. (A) Misrepair frequency was determined by an in vitro LacZα plasmid reactivation assay. Nuclear extracts were incubated with linearized pUC19 plasmids harboring a DSB in the LacZα gene. Misrepair frequency was calculated by the number of white colonies (misrepaired) related to the total colony numbers. Data represent the mean of ≥3 independent experiments ± SD (*P < .05, ***P = .0002, Student t test). (B) Microhomologies were defined by ≥2 bp. Bar graphs (white, EV; black, KrasG13D) show the percentage of repaired plasmids containing microhomologies at the site of religation. Twenty plasmids randomly selected from 3 experiments were sequenced and analyzed. (C) Analysis of deletion size of randomly selected, misrepaired (white colonies) plasmids incubated with nuclear extracts derived from (upper) CCRF-HSB2 or (lower) U-937. Data represent the mean values ± SD of 20 sequenced plasmids. White, EV; black, KRASG13D. (D) (Upper) CCRF-HSB2 cells and (lower) U-937 cells transduced with either EV or pLenti6.2-KRASG13D were transfected with either HindIII- or I-SceI–digested linearized plasmid in combination with pDsRed2-N1 as transfection control. After an incubation period of 72 hours, end-joining efficacy was determined by flow cytometry. Data shown represent mean values ± SD of 3 independent experiments (*P < .05, Student t test). (E) Protein expression of Ku86, Ku70, XRCC1, PARP1, Lig3α, and ligase 4 in cell extracts of transduced (left) CCRF-HSB2 and (right) U-937 cells. Shown is 1 representative analysis of 2 independent experiments. Bar graph show protein expression levels relative to actin determined by the means of densitometry of 2 independent experiments.
