Fig. 7: B-Myb is a previously unrecognized osteoclastogenic regulator specifically involved in TGFβ-priming/TNF-mediated osteoclastogenesis.

a RNA-seq analysis and comparison of mRNA expression induced by RANKL or TGFβ-priming/TNF in human CD14(+)-monocyte cultures. Bottom: pathway analysis of non-DEGs between RANKL and TGFβ-priming/TNF conditions. b Volcano plot of the DEGs from a. Blue dots: genes more highly expressed in RANKL-induced-condition; red dots: genes more highly expressed in TGFβ-priming/TNF-condition (adjusted p < 0.001 and FC > 4). c De novo-motif-enrichment analysis of ATAC-seq peaks associated with OC genes in TGFβ-priming/TNF condition. d IGV track displaying the indicated seq signals at MYBL2 locus. e, f qPCR analysis of MYBL2 expression (n = 5/group, e) and immunoblot analysis of B-Myb (f) in human CD14(+)-monocytes treated with/without TGFβ-priming for 3 days, followed by TNF stimulation. g Immunoblot analysis of B-Myb in human CD14(+)-monocyte-derived macrophages transfected with LNAs. h, i, l TRAP staining and relative area of TRAP-positive-MNCs/well (h), and qPCR analysis of the indicated gene expression (i, l) in human CD14(+)-monocyte cultures treated with/without TGFβ for 3 days, transfected with the indicated LNAs, and followed by TNF stimulation for 6 days (h, i) or 1 day (l). (n = 5/group). j Immunoblot analysis of B-Myb in human CD14(+)-monocyte-derived macrophages stimulated with RANKL. p38 was used as a loading control (f, g, j). k TRAP staining and relative area of TRAP-positive MNCs/well in human CD14(+)-monocyte-derived macrophages transfected with the indicated LNAs, followed by 3-day-RANKL stimulation. (n = 5/group) m TRAP (upper) and Immunohistochemical staining of B-Myb (middle, brown) on calvarial slices from TNF-induced osteolysis model in 12-week-old-male mice. Nuclei: blue. Arrows: osteoclasts. n = 3. n–p μCT images and the quantification of resorption area (n), TRAP staining of bone surface (o, left) and histological sections (o, right), and histomorphometric analysis (p) of the calvarial slices from 12-week-old-male mice after PBS or TNF injection to calvarial periosteum daily for 5 days (n = 5/group). e, h, i, l, n, p *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001; ns, not statistically significant by two-way ANOVA with Bonferroni’s multiple comparisons test. k ns, by two-sided Student’s t test. Error bars: e, h, i, k, l, n, p Data are mean ± SD. Scale bars: h, k 200 µm; m 10 µm; n 1.0 mm; o 100 µm. Source data are provided as a Source Data file.