Endogenous mutated KRAS causes increased expression levels of alt-NHEJ proteins in hematopoietic cell lines and primary AML patient cells. (A) Protein expression and quantitative densitometry of Lig3α, PARP1, XRCC1, and KRAS in cell extracts derived from KRAS-mutated (CCRF-CEM, SKM-1, Nomo-1, NB-4) and KRAS wild-type leukemic cell lines (CCRF-HSB2, THP-1, Mono-Mac-6, U-937). To control equal loading, the blot was stripped and reprobed with an anti-GAPDH antibody. Bar graphs indicate expression levels relative to GAPDH determined by the means of densitometry of 3 independent experiments. (B) RQ-PCR analysis of Lig3α, XRCC1, and PARP1 in primary AML patient samples. Shown are the count values (Ct values) of each gene in relation to actin mRNA and HPRT mRNA. White, KRAS wild-type AML patients; black, KRAS mutant AML patients. Samples were read in duplicates. (**P < .01, ***P < .001, Student t test). Cell lines with (left) wild-type KRAS and (right) mutated KRAS were lentivirally transduced with either tet-on–inducible nonsilencing miR-shRNA or miR-shRNA targeting Lig3α. Knockdown of Lig3α was induced on treatment with doxycycline (black and dark gray bars). Cells were then treated with (C) daunorubicin (72 hours) or (D) VP-16 (48 hours), and the percentage of sub-G1 cells was determined by flow cytometry. Data shown represent mean values ± SD of 3 independent experiments (*P < .05, ***P < .001, Student t test).