Journal: Nature Cell Biology
Article Title: Specialized post-arterial capillaries facilitate adult bone remodelling
doi: 10.1038/s41556-024-01545-1
Figure Lengend Snippet: a . Violin plots showing selected genes related to reduction of bmEC markers and increased arterialization in Dach1 OE and control. b . Heatmap of differentially expressed genes related to Notch signalling in Dach1 OE and control bone ECs. c . Representative confocal images showing increased Delta-like 4 (DLL4) (green, white arrowheads) expression in vessels around Dach1 OE trabecular bone (TB). Scale bars, 100μm. d . Scheme of tamoxifen-induced (TMX) EC-specific Dll4 inactivation with Cdh5-CreERT2 line. e, f , Tile scan confocal images of EMCN + VEGFR3 − ECs (white arrowheads) (e) and CAV1 + aECs (green arrowheads) (f) in 12-week-old Dll4 iΔEC loss-of-function and control femur. Trabecular bone (TB) is indicated. Scale bars, 500μm. g , Quantitation of EMCN + VEGFR3 − vessel density (left), and number of CAV1 + arteries (right) in 12-week-old Dll4 iΔEC and control femur (n =3 female mice per group). Mean ± SEM. P values, unpaired two-tailed t-test. h . Heatmap showing differentially expressed transcripts for secreted factors in bone EC subpopulations. i, j , Representative images (i) and quantitation (j) of calcified nodules in human mesenchymal stem cells (HMSC) cultured in osteogenic differentiation medium (ODM) supplemented with 100ng/ml Complement C1q and Tumour Necrosis Factor-Related Protein 9 (CTRP9), Neurotrophin 3 (NTF3), Platelet-Derived Growth Factor D (PDGFD), or Semaphorin 7A (SEMA7A) (n=3 independent experiments for each group). Scale bars, 100μm. Graph shows quantitation of absorbance at 405nm. Mean ± SEM. P values, ordinary one-way ANOVA and plotted with Dunnett’s multiple comparisons test. k, l , Representative images (k) and quantitation (m) of multinucleated TRAP+ osteoclasts generated from bone marrow-derived monocytes/macrophages treated with RANKL in osteoclast medium (OCM) supplemented with 100ng/ml CTRP9, NTF3, PDGFD, or SEMA7A n=8 (4 independent experiments) for each group. Scale bars, 500μm. Graph shows quantitation of TRAP + multinucleated cells between OCM and treated conditions. Mean ± SEM. P values, ordinary one-way ANOVA and plotted with Dunnett’s multiple comparisons test. Panels a and b show separated conditions derived from the integrated mutant and control data, whereas panel h is based on the integrated scRNA-seq data.
Article Snippet: The culture medium was replenished with a StemPro Osteogenesis Differentiation kit (Thermo Fisher Scientific, A1007201) supplemented with human CTRP9 (R&D Systems, 6537-TN), NT-3 protein (R&D Systems, 267-N3), PDGFD protein (R&D Systems, 1159-SB) or semaphorin 7A (R&D Systems, 2068-S7) at a final concentration of 100 ng ml −1 .
Techniques: Control, Expressing, Quantitation Assay, Two Tailed Test, Cell Culture, Derivative Assay, Generated, Mutagenesis
