Osteogenic differentiation of cultured 9p21s^+/− MDS/MPN–like mice BM stromal cells. (A) Alizarin Red staining of osteogenic differentiation of BM stromal cells at 7, 14, and 21 days. (B) Quantification of Alizarin Red staining. Results were carried out in triplicate wells from individual mice (WT mice, n = 5; 9p21^+/− tumor mice, n = 5). Symbols represent individual wells. (C) Expression of osteogenic marker genes in the differentiated BM stromal cells at different time points was confirmed via qRT-PCR. Symbols represent the average gene expression of 2 groups (WT mice group, n = 4; and 9p21^+/− mice group, n = 4). (D) Volcano plot showing the upregulated and downregulated genes expression of cultured 9p21^+/− tumor mice BM stromal cells compared with cultured WT mice BM stromal cells at 7 days after osteogenic differentiation. The significantly differentially expressed genes are labeled. (E) Heatmap showing increased levels of osteoblast signaling pathway–related genes in 9p21^+/− tumor mice (n = 2) and WT mice (n = 2) BM stromal cells at 7 days after osteogenic differentiation. (F) Signaling pathway enrichment analysis with Enrichr for significant upregulated genes in 9p21^+/− tumor mice BM stromal cells at 7 days after osteogenic differentiation. Gene count, the number of genes enriched in a Gene Ontology (GO) term; gene ratio, the ratio of genes counted in total of 199 upregulated genes. (G) Heatmap showing log2 Cxcl13 and Cxcr5 gene expression in 9p21^+/− tumor mice (n = 2) and WT mice (n = 2) BM stromal cells at 0 and 7 days after osteogenic differentiation. (H) Cxcl13 gene expression change in BM stromal cells at 0 and 7 days after osteogenic differentiation were confirmed via qRT-PCR. Symbols represent individual mice (WT mice, n = 4; 9p21^+/− tumor mice, n = 4). Data represent the mean ± SD. Statistical significance was defined using Mann-Whitney test and is shown as follows: ∗P < .05; ∗∗P < .01; ∗∗∗∗P <.0001 in panels B-C,H.