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: Primary antibodies were: rat monoclonal anti-endomucin (V.7C7) (Santa Cruz, sc-65495, 1:100 dilution), goat polyclonal anti-Vegfr3 (R&D Systems, AF743, 1:50 dilution), rabbit polyclonal anti-Cav1 (Cell Signalling, 3238, 1:50 dilution), chicken polyclonal anti-GFP (Abcam, ab13970, 1:200 dilution), rabbit polyclonal anti-OSTERIX (Abcam, ab22552, 1:300 dilution), rabbit monoclonal anti-Runx2 (Abcam, ab192256, 1:200 dilution), rabbit monoclonal anti-Ki-67 (Cell Signalling, 12202, 1:200 dilution), rabbit monoclonal anti-vATPase (Abcam, ab200839, 1:200 dilution), rabbit polyclonal anti-collagen IV (AbD Serotec, 2150-1470, 1:100 dilution), rabbit polyclonal anti-collagen IIIA1 (Abcam, ab7778, 1:100 dilution), goat polyclonal anti-Hif1a (R&D Systems, AF1935, 1:100 dilution), rabbit polyclonal anti-Hif1a (Santa Cruz, sc-10790, 1:100 dilution), rabbit monoclonal anti-GPI (Cell Signalling, 94068, 1:200 dilution), rabbit monoclonal anti-CD73 (Cell Signalling, 13160, 1:200 dilution), rabbit monoclonal anti-Hmox1 (Cell Signalling, 86806, 1:200 dilution), rabbit polyclonal anti-Dach1 (Proteintech, 10914-1-AP, 1:200 dilution), rabbit polyclonal anti-NG2 (Millipore, AB5320, 1:200 dilution), goat polyclonal anti-Cxadr (R&D Systems, AF2654, 1:50 dilution), rabbit monoclonal anti-Sox11 (Abcam, ab134107, 1:800 dilution), rat monoclonal anti-Madcam1 (Abcam, ab80680, 1:100 dilution), rabbit polyclonal anti-Fabp4 (Abcam, ab13979, 1:100 dilution), mouse monoclonal anti-Mct4 (Santa Cruz, AF647, 1:100 dilution), rabbit monoclonal anti-Glut1 (Cell Signalling, 12939, 1:100 dilution), goat polyclonal anti-Dll4 (R&D Systems, AF1389, 1:50 dilution) and chicken polyclonal anti-Mct1 (Millipore, AB1286-I, 1:100 dilution).
Techniques: Control, Expressing, Quantitation Assay, Two Tailed Test, Cell Culture, Derivative Assay, Generated, Mutagenesis
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