Journal: The Journal of Experimental Medicine

Article Title: Lnk-dependent axis of SCF–cKit signal for osteogenesis in bone fracture healing

doi: 10.1084/jem.20100321

Figure Lengend Snippet: Enhanced SL cell mobilization by SCF stimulation in Lnk KO mice. (a) Serum mouse SCF levels were measured in Lnk KO and WT mice after fracture by ELISA. ††, P < 0.01; *, P < 0.05 versus Pre, respectively. (b) The number of circulating SL cells in Lnk KO and WT mice with injection of PBS (control; Lnk KO-PBS and WT-PBS), SCF (SCF, 20 µg/kg; Lnk KO-SCF and WT-SCF), or soluble SCF receptor (=c-kit ligand, sKit, 20 µg/kg; Lnk-sKit) were assessed by FACS in the indicated time course after the treatment. †† and †, P < 0.01 and P < 0.05 versus Pre, respectively. ** and *, P < 0.01 and P < 0.05 versus WT-PBS, WT-SCF, Lnk KO-PBS, and Lnk KO-sKit, respectively. (c) The number of circulating SL cells in WT mice with fracture and injection of PBS, SCF (20 µg/kg), or SCF (20 µg/kg) + sKit (WT-fx-PBS, WT-fx-SCF, or WT-fx-SCF-sKit, respectively), WT mice without fracture and SCF (20 µg/kg; WT-SCF), and Lnk KO mice with fracture and sKit (Lnk KO-fx-sKit) were assessed by FACS in the indicated time course after the treatment. †† and †, P < 0.01 and P < 0.05 versus Pre, respectively. * and **, P < 0.05 and P < 0.01 versus WT-fx-SCF-sKit, respectively. (d) Immunofluorescent staining for CD31 (green) and OC (OC, green) were performed in granulation tissue samples of peri-fracture sites in Lnk KO (left one panel) and WT (right two panels) mice 7 d after fracture. Alcian blue/orange G stained sections were also shown in parallel with immunostained sections (lower panels to each immunofluorescent staining images). Mouse phenotype with SCF (20 µg/kg) or sKit (20 µg/kg) administration (+) is indicated in the lower part of images. Dotted line indicates bone surface. Bar = 50 µm. Quantification of capillaries (e) and OBs (f). CD31 positive capillaries and OC positive OBs were counted in 3 randomly selected high power fields and averaged.**, P < 0.01 and *, P < 0.05. All data averaged with SEM from five independent experiments. All experiments were obtained from triplicated assays.

Article Snippet: Purified recombinant human SCF and sKit were purchased from R&D Systems and supplemented concentrations of cytokines used for culture were as follows: SCF 100 ng/ml and sKit 10 μg/ml (Nakamura, 2004).

Techniques: Enzyme-linked Immunosorbent Assay, Injection, Control, Staining

Journal: The Journal of Experimental Medicine

Article Title: Lnk-dependent axis of SCF–cKit signal for osteogenesis in bone fracture healing

doi: 10.1084/jem.20100321

Figure Lengend Snippet: Enhanced mineralization of OBs in Lnk-deficient mice. (a) OBs (OBs) isolated from Lnk KO and WT mice were cultured in osteogenic condition medium. Cells were assessed morphologically by ALP staining (blue) at day 14 and by alizarin red staining (red) at day 21. Upper panels show macroscopic images of whole culture dishes and lower panels show magnified images of nodule with mineralized matrix formation. (b) Real-time RT-PCR of cultured OBs for SCF mRNA expression at day 7 in Lnk KO and WT mice. Mouse heart and bone are used as positive controls for mouse endothelial and bone-related gene detection (not depicted) *, P < 0.05. Data averaged with SEM from three independent experiments. Experiments were obtained from triplicated assays. (c) The mineralized matrix formation was assessed by alizarin red staining 21 d after osteogenic culture in Lnk KO OBs with sKit (10 µg/ml) and in WT OBs in the presence of SCF (100ng/ml) with or without sKit (10 µg/ml). Upper panels show macroscopic images of whole culture dishes and lower panels show magnified images of nodule with mineralized matrix formation. Calcium content was measured by ELISA ( n = 5 each; d) in culture medium and BMP2 mRNA expression (e) was analyzed by real-time RT-PCR ( n = 3 each) in Lnk KO OBs with sKit (10 µg/ml) and WT OBs in the presence of SCF (100ng/ml) with or without sKit (10 µg/ml). *, P < 0.05 and **, P < 0.01. Experiments were obtained from triplicated assays.

Article Snippet: Purified recombinant human SCF and sKit were purchased from R&D Systems and supplemented concentrations of cytokines used for culture were as follows: SCF 100 ng/ml and sKit 10 μg/ml (Nakamura, 2004).

Techniques: Isolation, Cell Culture, Staining, Quantitative RT-PCR, Expressing, Enzyme-linked Immunosorbent Assay