Figure 5: IGFBP5 involvement in the IL-6-dependent c-Src activation. | Nature Communications

Figure 5: IGFBP5 involvement in the IL-6-dependent c-Src activation.

From: c-Src and IL-6 inhibit osteoblast differentiation and integrate IGFBP5 signalling

Figure 5

(a) Mouse calvarial osteoblasts were treated with vehicle (acetic acid) or with 5 ng ml−1 rhIL-6 in a time-course experiment. Western blot analysis of c-Src activating phosphorylation and total c-Src, normalized versus actin. (b) RT–PCR for IGF-1 expression in osteoblasts treated with 5 ng ml−1 rhIL-6, normalized versus Gapdh (mean±s.d., n=3). (c) RT–PCR for Igfbp5 expression in osteoblasts treated with 5 ng ml−1 rhIL-6, normalized versus Gapdh (mean±s.d., n=3, **P<0.005, Student's t-test). (d) RT–PCR for Igfbp5 expression in primary osteoblasts treated with 100 nM of control siRNA (CTL) or siRNA for STAT3, normalized to Gapdh (mean±s.d., n=3, **P<0.005, Student's t-test). (e) Mouse calvarial osteoblasts were treated with the indicated concentrations of soluble IGFBP5 for 30 min. Western blot analysis of c-Src and IGF-1 receptor (IGF1R) activating phosphorylation, normalized versus the respective total proteins and actin. (f) Mouse calvarial osteoblasts were treated with 100 nM of control siRNA (CTL) or siRNA–IGFBP5. Western blot analysis of IGFBP5 and of c-Src activating phosphorylation, normalized versus the respective total proteins and actin. (g) Immunostaining analysis for IGFBP5 in primary mouse osteoblasts treated for 24 h with vehicle (DMSO) or 2 μM PP1. Scale bar, 50 μm. (h) RT–PCR for Igfbp5, normalized versus Gapdh expression (mean±s.d., n=3, **P<0.005, Student's t-test). (i) RT–PCR for Igfbp5 in primary mouse osteoblasts transfected with an empty vector (as a control) and WT and DN c-Src variants, normalized versus Gapdh expression (mean±s.d., n=3, **P<0.005, Student's t-test). (j) Relative quantification of Igfbp5 mRNA expression, normalized with Gapdh, in RNA extracted from tibias of eight week-old WT and NSE/hIL-6 (TG) mice treated for 1 month with 100 mg kg−1 per day of the c-Src inhibitor CGP76030 (mean±s.d., n=3, **P<0.005, Student's t-test). (k) Immunohistochemistry for IGFBP5 in paraffin-embedded 5-μm-thick tibia sections of WT and TG mice treated with vehicle or with the CGP76030 inhibitor. B, bone; BM, bone marrow. Scale bar, 50 μm. (l) RT–PCR for Igfbp5 in primary mouse osteoblasts transfected with an empty vector (as a control), c-Src DN and Runx2 WT vectors, normalized to Gapdh expression (mean±s.d., n=3, **P<0.005, Student's t-test). Gapdh, glyceraldehyde-3-phosphate dehydrogenase.

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