Figure 4: IL-6/c-Src interplay in in vivo pathological conditions. | Nature Communications

Figure 4: IL-6/c-Src interplay in in vivo pathological conditions.

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

Figure 4

(a) Eight-week-old mice were treated for 24 h with 100 mg kg−1 per day c-Src inhibitor and then for 6 h with vehicle (PBS), LPS (2.5 μg per mouse) or turpentine (100 μl per mouse). RT–PCR and relative quantification of Il-6 expression (mean±s.d.; n=3; **P<0.001 versus vehicle-treated mice. #P<0.001 versus anti-inflammatory agent-treated mice, Student's t-test), Il-1β expression (mean±s.d.; n=3; *P<0.05 and **P<0.01 versus vehicle-treated mice. #P<0.05 and ##P<0.005 versus anti-inflammatory agent-treated mice, Student's t-test) and tumour-necrosis factor-α expression (mean±s.d.; n=3; *P<0.05 and **P<0.01 versus vehicle-treated mice. #P<0.05 and ##P<0.005 versus anti-inflammatory agent-treated mice, Student's t-test) in tibias, normalized with Gapdh. (b) Human breast cancer MDA-MB-231 cells were treated with 2 μM PP1 for 24 h. RT–PCR of human Il-6 expression, normalized versus Gapdh expression (mean±s.d., n=3, *P<0.05, Student's t-test) and western blot analysis to control total c-Src protein downregulation and activating phosphorylation, normalized versus actin. (c) Four-week-old female BALB/c-nu/nu mice intracardiacally injected with PBS or with a suspension (100,000 cells per 100 μl of PBS) of MDA-MB-231 cells. X-ray images and incidence of bone metastasis in mice treated with vehicle or with the c-Src inhibitor CGP76030 (100 mg kg−1 per day) for 38 days; in X-ray imagine bone metastases are delineated by a white line. N=8 animals per group. ELISA assay for (d) human and (e) mouse IL-6 in the sera of mice carrying MDA-MB-231 cell-induced bone metastases treated with vehicle or with the c-Src inhibitor CGP76030 (100 mg kg−1 per day) for 38 days after intracardiac tumour cell injection. Mean±s.d.; n=3; **P<0.005, Student's t-test. Immunohistochemical staining for (f) human IL-6 (scale bar, 50 μm) and (g) mouse IL-6 (scale bar, 50 μm) in paraffin-embedded 5-μm-thick tibia sections of MDA-MB-231 cell-injected mice treated with the CGP76030 inhibitor. (h) Control of species-specificity of anti-mouse and anti-human IL-6 antibodies in the metastatic tissue. Scale bar, 50 μm. B, bone; BM, bone marrow; M, metastasis. (i) RT–PCR for Il-6 expression in the indicated cells treated with 2 μM PP1, normalized versus Gapdh (mean±s.d., n=3. *P<0.05 and **P<0.005, Student's t-test). Gapdh, glyceraldehyde-3-phosphate dehydrogenase; HUVEC, human umbilical vein endothelial cell.

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