Journal: Nature Communications
Article Title: A Wnt-mediated transformation of the bone marrow stromal cell identity orchestrates skeletal regeneration
doi: 10.1038/s41467-019-14029-w
Figure Lengend Snippet: a Structure of Cxcl12-iCreER-bGHpA bacterial artificial chromosome (BAC) transgene. b – o Short-chase analysis of Cxcl12-creER + cells. b Cxcl12 GFP/+ ; Cxcl12-creER ; R26R tdTomato (pulsed at P21) distal femurs with growth plates on top. Gray: DIC. Scale bar: 500 µm. n = 3 mice. c , d Immunostaining for endomucin (Emcn, c ) and CD31 ( d ). Sn: sinusoids, At: arterioles. Arrows: sinusoid-attaching GFP high tdTomato + cells. Gray: DAPI. Scale bar: 10 µm. n = 5 mice. e Percentage of cells attaching Emcn + sinusoids among GFP high tdTomato neg (green) or GFP high tdTomato + (red) cells. n = 5 mice. f Flow cytometry analysis of CD45/Ter119/CD31 neg bone marrow cells. Blue lines: control cells. n = 5 mice. g Leptin receptor (LepR) expression based on flow cytometry analysis. Blue line: isotype control. n = 3 mice. h LepR staining. Gray: DAPI (Center), DIC (metaphysis and endosteal zone). Scale bar: 20 µm. i Quantification based on histology. Percentage of BMSCs expressing Cxcl12-GFP (green dots), Cxcl12 CE -tdTomato (red dots) and LepR (purple dots). n = 7 fields from four mice (central marrow and endosteal surface), n = 5 fields from 4 mice (metaphysis). j Percentage of CD45/Ter119/CD31 neg subpopulations in S/G2/M phase. n = 5 mice. k Intracellular CXCL12 protein levels of CD45/Ter119/CD31 neg subpopulations. MFI: median fluorescence intensity. n = 4 mice. l EdU serial pulse assay of Cxcl12-creER + cells. Cxcl12-creER ; R26R tdTomato mice were pulsed with tamoxifen at P21, then with EdU every 8 h for 3 days prior to analysis. Percentage of EdU + cells among GFP high tdTomato neg (green) or GFP high tdTomato + (red) cells. n = 5 mice. m Intracellular SCF protein levels of CD45/Ter119/CD31 neg subpopulations. MFI median fluorescence intensity. n = 5 mice. n , o Col1 (2.3 kb)-GFP; Cxcl12-creER ; R26R tdTomato . n Left panel: trabecular bone. Right panel: endosteal space. Gray: DIC. Scale bar: 20 µm. n = 4 mice. o Flow cytometry analysis of CD45/Ter119/CD31 neg cells harvested from Col1(2.3 kb)-GFP ; Cxcl12-creER ; R26R tdTomato at P28 (pulsed at P21) bone marrow. Blue lines: control cells. n = 4 mice. **** p < 0.0001, *** p < 0.001, ** p < 0.01, * p < 0.05, two-tailed, Mann–Whitney’s U test ( e , l ). Two-tailed, one-way ANOVA followed by Tukey’s post hoc test ( i – k , m ). All data are presented as mean ± s.d. Source data are provided as a Source Data file.
Article Snippet: For immunostaining, sections were permeabilized with 0.25% TritonX/TBS for 30 min, blocked with 3% bovine serum albumin/TBST for 30 min. and incubated with rat anti-endomucin (Emcn) monoclonal antibody (1:100, Santa Cruz Biotechnology, sc65495), rat anti-CD31 monoclonal antibody (1:100, Bio-Rad, MCA2388), goat anti-leptin receptor (LepR) polyclonal antibody (1:100, R&D, AF497), goat anti-osteopontin (OPN) polyclonal antibody (1:500, R&D, AF808), goat anti-ALPL polyclonal antibody (1:100, R&D, AF2910), rabbit anti-Sox9 polyclonal antibody (1:500, EMD-Millipore, AB5535), rabbit anti-perilipin A/B polyclonal antibody (1:500, Sigma, P1873) or rabbit anti-PPAR gamma (PPARγ) monoclonal antibody (1:200, Invitrogen, MA5-14889) overnight at 4 °C, and subsequently with Alexa Fluor 633-conjugated goat anti-rat IgG (A21049), Alexa Fluor 647-conjugated donkey anti-goat IgG (A21082), or Alexa Fluor 647-conjugated donkey anti-rabbit IgG (A31573) (1:400, Invitrogen) for 3 h at room temperature.
Techniques: Immunostaining, Flow Cytometry, Control, Expressing, Staining, Fluorescence, Two Tailed Test
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