Reduced DLL4 in the bone marrow of Ocn⁺ cell depleted mice led to decreased Notch signaling in CLP. (A) Intracellular Notch signaling was assessed by flow cytometry in the indicated cell populations. Representative flow plots are shown, and data are summarized by dot plot (right). Three independent experiments; n = 16–17 mice/group. Data show mean ± SEM. (B) Expression of the indicated Notch target genes in Ly6D⁻ CLP from OcnCre^+/−;iDTR mutants and controls. Mutant and WT mice were treated with DT for 28 d before being subjected to flow cytometry for Ly6D⁻ CLP isolation and quantitative PCR. Two independent experiments; n = 12 mice/group. Data show mean ± SEM. (C) Expression of the Notch ligands in the bones of OcnCre^+/−;iDTR DT-treated and control animals was assessed by qPCR. Mice were examined after 28 d after DT treatment. Three independent experiments; n = 12–14 mice/group. Data show mean ± SEM. (D) DLL4 expression in bone sections from control or Ocn⁺ cell deleted animals was assessed by immunohistochemistry. Two independent experiments; n = 6 mice/group. (E) Osx-mCherry cells and Ocn-Topaz cells were isolated from the OsxCre-mCherry;OcnCre-Topaz double transgenic mice by flow cytometry and expression of the indicated Notch ligands was assessed by qPCR. Experiment performed twice; n = 3/experiment. Data show mean ± SEM. (F) Gene set enrichment analysis (GSEA) comparing Ly6D⁻ CLP versus MPP microarray data. Experiment performed twice; n = 3/experiment. (G) GSEA comparing Ly6D⁻ CLP microarray data obtained from OcnCre;iDTR control versus mutant mice. Experiment was performed twice independently; n = 3–4/experiment.