Cell-autonomous TLR2 signaling promotes apoptosis of preleukemic HSPCs. The bone marrow KSL cells of 6- to 8-week-old WT, Tlr2^−/−, NHD13;Tlr2^+/+, and NHD13;Tlr2^−/− mice were analyzed by flow cytometry. (A) Percent of KSL cells in the S/G₂/M phase of the cell cycle as determined by Ki-67 and 4′,6-diamidino-2-phenylindole staining (n = 4-8 mice/group). (B) Bone marrow KSL cells from the same groups of mice were analyzed for Annexin V staining by flow cytometry. Shown are the percentage of Annexin V⁺ KSL cells (n = 6-7 mice/group). (C) Caspase-1 and (D) Caspase-3/7 activities were assessed from sorted KSL cells as described in the supplemental Methods (n = 3-8 mice/group). (E) Chimeras (containing a mixture of NHD13;Tlr2^+/+ and NHD13;Tlr2^−/− bone marrow cells) were generated as described in supplemental Figure 6, and Annexin V flow cytometry staining was performed on bone marrow. Shown is a representative flow analysis of the KSL cells of 1 of the chimeric animals. These data are quantified in panel F, which shows the MFI of Annexin V staining on the KSL cells from each of the genotypes in 8 total chimeras analyzed. (G). γ-H2AX staining was performed on the bone marrow KSL cells. Left, a representative flow plot comparing γ-H2AX levels between young adult (6-8 weeks old) WT (NHD13^-;Tlr2^+/+), NHD13, and NHD13⁺;Tlr2^−/− animals. Right, each data point represents the MFI for KSL cells from individual mice from each of the WT, NHD13, and NHD13⁺;Tlr2^−/− cohorts. n = 8 mice/group performed over 4 independent experiments. *P < .05; **P < .01, ***P < .001, ****P < .0001 by an unpaired Student t test or 1-way analysis of variance. Error bars represent mean ± standard error of the mean.