Figure 5.

IL-6 protein accelerated cell migration and increased BMSC numbers through activation of the IL-6/gp130/STAT3 pathway. (a) BMSCs were treated with recombinant mouse IL-6 protein (10 ng⋅mL⁻¹) for 30 minutes with or without a 1-hour pretreatment of sgp130 (1 μg⋅mL⁻¹). Western blot analysis of BMSC protein samples compared phosphorylation of STAT3 (pSTAT3). IL-6 protein stimulated the phosphorylation of STAT3 in BMSCs, which was blocked by spg130 pretreatment. (b) Quantification of p-STAT3 protein expression was determined relative to expression of STAT3. This experiment was performed in duplicate. Data are mean ± SEM; *P < 0.05. (c) Transwell assays compared the migration of BMSCs cultured for 8 hours after treatment with IL-6 protein (10 ng⋅mL⁻¹) with or without 1-hour pretreatment of spg130 (1 μg⋅mL⁻¹). IL-6 protein treatment increased migration of BMSCs, while this effect was abolished by sgp130 pretreatment. 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 number of migrated cells per FV was counted. All experiments were performed in duplicate. Data were mean ± SEM (n = 2 in each group); *P < 0.05. (d) Image of BMSCs after fixation and hematoxylin staining in the Transwell assays. Images were taken at ×200 magnification. (e) Cell scratch assays compared the remaining scratch distance of BMSC cultures after 8 hours of IL-6 protein (10 ng⋅mL⁻¹) treatment with or without pretreatment of spg130 (1 μg⋅mL⁻¹). IL-6 protein treatment significantly reduced the remaining distance of cell scratch, which was abolished by sgp130 pretreatment. All experiments were performed in duplicate. Data are mean ± SEM (n = 3 in each group); *P < 0.05; **P < 0.01. (f) IL-6 protein treatment (10 ng⋅mL⁻¹) increased BMSC numbers after 1, 2, and 3 days of culture, an effect which was abolished by spg130 pretreatment (1 μg⋅mL⁻¹ for 1 hours). Data are mean ± SEM (n = 3 in each group); **P < 0.01.