Figure 3.

OSM stimulates activation of STAT3 and ERK by phosphorylating the adapter protein Shc1. (A) Mouse calvarial osteoblasts were cultured in the absence (Co) or presence of LIF or OSM (both at 100 ng/mL) for 24 h and RNA was extracted for RT-qPCR analysis of Shc1mRNA. (B) The cells were exposed to LIF or OSM at 100 ng/mL for 15 and 30 min followed by cell lysis and Western blot for phosphorylated Shc1; the bands were quantified and normalized by vinculin. (C) Calvarial osteoblasts had the Shc1 gene silenced by small interfering RNA and were exposed to LIF or OSM (both at 100 ng/mL) for 24 h before RNA extraction and analysis of Shc1 mRNA expression by RT-qPCR; a scrambled sequence (siSCR) served as control. (D) Osteoblasts that had the Shc1gene knocked-down by siRNA and control cells exposed to scrambled sequence (siSCR) were exposed to OSM at 100 ng/mL or vehicle for 15 min and proteins were extracted for analysis of the phosphorylation of Shc1, STAT3 and ERK. (E) The Stat3 gene was silenced in calvarial osteoblasts, which were subsequently exposed to OSM at 100 ng/mL or vehicle (Co) for 24 h before analysis of Stat3 gene expression by RT-qPCR. (F) The effect of Stat3 silencing on phosphorylation of STAT3 and Shc1 was analyzed by Western blot in osteoblasts treated with OSM for 15 min. (A,C,E) Values represent means for four wells and SEM is shown as vertical bars. (A) Significant differences compared to untreated cells (Co) at each time point are defined as ^***P < 0.001; analyzed by one-way ANOVA followed by Dunnet's post hoc-test. (C,E) Significant differences are indicated by horizontal lines where ^***P < 0.001 two-way ANOVA followed by Tukey post hoc-test. The difference in OSM-induced response with and without silencing analyzed by two-way ANOVA was statistically significant [interaction P-value in C (P < 0.005) and in E (P < 0.0001)]. OSM had no statistically significant effect (P > 0.05) on Shc1 or Stat3 mRNA expression in cells which had been silenced for Shc1 or Stat3, respectively (C,E).