Erk activation is necessary for TPO-mediated DSB repair in HSPCs. (A-B) γH2AX foci resolution analysis at the indicated times after IR (2 Gy) of (A) LSK and (B) LSK-CD34⁻ cells cultured in complete medium in the presence or absence of the MEK inhibitor U0126 (10 µM), or in medium without TPO. Data are means + standard error of the mean (SEM) (n = 3). (C) DSB analysis using a neutral comet assay of LSK cells cultured as in panel A. Mean of tail moments (left) and percent of cells with tail moments >5 or <5 (right) are shown. Representative experiment out of 3 similar performed with cells pooled from 3 to 4 mice. Data are means + SEM of tail moment values analyzed in at least 100 cells. (D) γH2AX staining in LSK-CD34⁻ cells isolated 16 hours after TBI of wild type (WT) and Erk1^−/− mice. Data are means + SEM normalized to the mean of γH2AX positive cells from WT mice (n = 5). (E) Experimental design to test the effect of Erk inhibition on LSK reconstitution ability (top). LSK cells were cultured as in panel A for 1 hour before injection in CD45-2 lethally irradiated congenic mice. CD45.1-chimerism in peripheral blood 4 months posttransplantation (bottom). Each dot represents an individual mouse. Mice were injected with LSK cells from a pool of 9 mice. Comp, complete medium; NIR, no IR.