Figure 6.

Juxtacrine interaction of BMSCs and macrophages accelerated BMSC migration and increased BMSC numbers through activation of the IL-6/gp130/STAT3 pathway. (a and b) Conditioned medium harvested from either WT or IL-6 KO cell cultures were used to treat BMSCs for 30 minutes. Western blot analysis of BMSC proteins compared p-STAT3 levels. Only conditioned medium from co-culture of WT macrophages and BMSCs stimulated the phosphorylation of STAT3 in BMSCs. IL-6 KO conditioned medium did not. Quantification of p-STAT3 protein expression was determined relative to the expression of total STAT3. Data are mean ± SEM (n = 2 in each group); P = 0.059. (c and d) Transwell assays compared the migration of mouse BMSCs cultured for 8 hours under the stimulation of conditioned medium from co-culture of WT BMSCs and macrophages with or without pretreatment of spg130 (1 μg⋅mL⁻¹). Pretreatment with sgp130 blocked the ability of the conditioned medium to stimulate BMSC migration. The migrated cells were counted after fixation and hematoxylin staining. Images were taken at ×200 magnification. Five fields of view (FV) were randomly picked for each sample, and numbers of migrated cells per FV were counted. All experiments were performed in duplicate. Data were mean ± SEM (n = 2 in each group); *P < 0.05. (e) Cell scratch assays compared the remaining scratch distance of BMSC cultures after 8 hours of conditioned medium from co-culture of WT BMSCs and macrophages with or without spg130 pretreatment (1 μg⋅mL⁻¹). Pretreatment with sgp130 blocked the stimulation of conditioned medium on the migration of BMSCs. All experiments were performed in duplicate. Data are mean ± SEM (n = 3 in each group); *P < 0.05. (f) Pretreatment with spg130 (1 μg⋅mL⁻¹ for 1 hour) abolished the stimulation of conditioned medium on BMSC numbers after 1, 2, and 3 days of culture. Data are mean ± SEM (n = 3 in each group); *P < 0.05; **P < 0.01.