Effects of Cy/G treatment in ATM^−/− mice. (A) Osteoblast cell numbers are not increased in Cy/G treated ATM-deficient mice. The total numbers of osteoblasts isolated after Cy/G treatment were calculated based on the frequency of OPN⁺ cells (as determined by FACS) among total numbers of cells isolated from collagenase treated bones from either ATM^+/+ or ATM^−/− mice that were untreated (ATM WT untreated, ; ATM KO untreated, ▬) or treated with Cy/G (ATM WT D2, gray hatched; ATM KO D2, black hatched). Data are plotted as means ± SD (*P < .05). (B) Decreased osteoblast survival after Cy/G treatment in ATM-deficient mice. ATM WT or ATM KO mice were treated with Cy/G and osteoblasts were isolated from collagenase-treated bones and stained with osteoblast specific markers as well as cell viability markers (annexin V⁺, early apoptotic marker; and PI⁺, late apoptotic marker). Representative data for 2 independent experiments are shown. (C-E) Lower frequency and engraftment efficiency of wild-type HSC exposed for 12 hours to D2 ATM KO osteoblasts compared with HSC exposed to wild-type D2 osteoblasts. A total of 100,000 Lin⁻ BM cells (HPC) from wild-type, untreated mice were exposed for 12 hours in vitro to 2000 OPN⁺ osteoblasts isolated from ATM KO or WT D2 mobilized mice (ATM WT untreated, ; ATM KO untreated, ▬; ATM KO D2, black hatched; ATM WT D2, gray hatched) and tested for HSC function by measuring in vitro maintenance of HSC frequency (C,D) compared with an HPC only control (gray dotted bar in panel D) and in vivo reconstitution potential (E) as described previously (Figures 4,5). Data are shown compared with an HPC only control (gray dotted) and an uncultured HPC control (■). Data are plotted as mean (± SD; *P < .05) and represent chimerism at 16 weeks after transplant. (F) Equivalent frequency of apoptotic (annexin V⁺, PI⁻ early apoptotic) osteoblasts after 12-hour HPC:osteoblast coculture assays. Coculture assays were performed as described previously, and cells were stained for osteoblast specific markers and the cell death and viability markers annexin V (early apoptotic marker) and PI (late apoptotic marker). Representative data are shown for 2 independent experiments performed.