Extended Data Figure 5. Inhibition of increased Notch signaling normalizes blood counts and rescues hematopoietic defects in βCat(ex3)_osb mice.

a, Microarray analysis of calvaria-derived osteoblasts from βCat(ex3)_osb mice. AML and Notch-related genes in βCat(ex3)_osb osteoblasts and with p<0.05 and fold change of ± 20% in one comparison. Genes which that are up- or down-regulated relative to WT are shown. b, Flow cytometry analysis of Jagged-1 expression in osteoblasts (MFI: mean fluorescent intensity). c, Luciferase activity in HEK293T cells co-transfected with β-Catenin, Lef1 and Jagged1-Luc reporter constructs (−4112/+130) and (−2100/+130). Results show fold induction over respective Jagged1-Luc reporter constructs. * p< 0.05 versus respective Jagged1-Luc. Results are mean ± SD. d, ChIP in primary osteoblasts using anti-β-catenin antibody. Primers spanned the putative TCF/LEF binding sites (indicated) on the Jagged-1 promoter. e, Expression of Notch1 and Notch2 in LSK+ cells. f-g, Expression of Notch targets in LSK+ subpopulations. i, Normal intestinal architecture and j, PAS staining showing lack of goblet cell (arrows) metaplasia in DBZ-treated mice. Images at 60x. k, Peripheral blood counts and bone marrow cellularity in wild type and βCat(ex3)_osb mice treated daily with vehicle or DBZ (2μmol/kg body weight) for 10 days. l-p, Percentage of k, LSK cells, l, LSK+ subpopulations m, myeloid progenitors, n, CD11b+/Gr1+ population, o, erythroid cells. m, Percentage of erythroid cells and p, LSK+/FLT3+ population in the bone marrow. q-s, Percentage of q, myeloid progenitor populations, r, CD11b+/Gr1+ cells and s, erythroid cells in the spleen. In a N=3 mice per group and in b N=4 mice per group. In c,d results represent two independent experiments. In (e-g) N=4 mice per group, and *p < 0.05 versus WT. In (h-s) N=8 mice per group and *p < 0.05 versus WT and # p < 0.05 βCat(ex3)_osb-vehicle versus DBZ-treated βCat(ex3)_osb group. Results are mean ± SD and show a representative of two independent experiments.