Primary murine KrasG12D-expressing T-ALL and preleukemic cells show increased levels of DNA misrepair. (A-C) Single-cell suspensions were prepared from the thymus of leukemic KrasG12D-expressing mice or control animals (KRAS wild type). (A) Immunoblot analysis using anti-Lig3α and PARP1 antibodies as indicated. To control equal loading, the blot was stripped and reprobed with an anti-actin antibody. Numbers indicate expression levels relative to actin determined by the means of densitometry. (B) Expression of intracellular Lig3α in primary thymocytes and fluorescence-activated cell sorter analysis (red, unstained; blue, immunoglobulin G-isotype control; yellow, anti-Lig3α-Alexa488). (C) Cells were treated with either (upper) daunorubicin (125 nM) or (lower) were irradiated (10 Gy). Cells were stained with an anti-γH2AX antibody and counterstained with DAPI. Shown is the percentage of cells with >5 foci relative to the total number of cells. Data shown represent mean values ± SD of 3 independent experiments. (**P = .0039, upper; **P = .0045, lower, Student t test). (D) RQ-PCR analysis of XRCC1, PARP1, and Lig3α in sorted preleukemic thymocyte subsets of double-negative (DN) populations (DN1, DN2, DN3, DN4) from Lck-Cre × KrasG12D mice (n = 6). (E) Nuclear extracts were prepared from preleukemic CD4/CD8 thymocytes (Lck-Cre × KrasG12D) or littermate control animals (Lck-Cre). Shown is the frequency of misrepaired colonies of 4 independent experiments.
