Figure 4.

OPN^−/− bone marrow has unaltered cell cycle profiles associated with increased stromal Jagged1 and Angiopoietin-1 expression and reduced primitive cell apoptosis. (A) Bone marrow Sca1⁺c-kit⁺lin⁻ cells with a bright staining for Hoechst 33342, representing cells in the G₂/M phase of the cell cycle (n = 3 pairs). (B) BrdU incorporation in Sca1⁺c-kit⁺lin⁻ cells at the specified time points in OPN^+/+ and OPN^−/− bone marrow. Data are the result of two independent experiments with four mice per group in each experiment. A Student's t test comparison revealed no P < 0.05. (C) Bone marrow adherent stromal cells were evaluated for Jagged1, Angiopoietin (Ang)-1, and N-cadherin expression by RT-PCR (n = 6 for each). Data were normalized to an intrasample GAPDH standard, and the results of the OPN^−/− vs. OPN^+/+ cells were compared by ratio. (D) Bone marrow cells of OPN^+/+ and OPN^−/− mice were stained with antibodies to differentiation markers, the apoptosis marker Annexin V, and the DNA dye 7-AAD. Annexin V–positive/7-AAD–negative apoptotic Sca1⁺c-kit⁺lin⁻ cells are shown (n = 4 pairs). (E) Stroma-dependent apoptotic rate demonstrated by reduced apoptosis of wild type primitive hematopoietic cells when transplanted into OPN^−/− mice compared with OPN^+/+ recipient mice. Analyses were performed on the lin⁻ fraction 12 wk after transplantation (n = 4). Error bars represent SEM.