Figure 4
The EGF receptor is involved in thrombin-enhanced CXCL8 expression. (A) The mRNA expression of EGF receptor family members in patients with different MRI grades. (B) Representative immunohistochemical images of EGFR expression in paraffin-embedded sections at various MRI grades of human spinal disc specimens (three patients in each group). The extent of EGFR staining is slight in tissues with MRI grade III and V patients and strong in the tissue of grade IV patients. (C) NP cells were treated with AG1478 (3 or 10 μM) for 30 min following treatment with thrombin (10 U/mL) for 24 h; expression of CXCL8 was determined by ELISA. Each bar represents the mean ± S.E.M. from at least three independent experiments performed in duplicate. (D) NP cells were treated with thrombin for the indicated time periods, cell lysates were separated by SDS-PAGE and immunoblotted with either anti-phosphorylated EGFR (Tyr1068) antibody, anti EGFR antibody or anti β-actin antibody. (E) NP cells were treated with the STAT3 inhibitor for 8 or 24 h, and expression of the EGFR was determined by western blot. (F) NP cells were treated with thrombin for the indicated time periods and cell lysates were separated by SDS-PAGE and immunoblotted with anti-phosphorylated STAT3, anti-STAT3, anti-phosphorylated-JAK1, or anti-phosphorylated-JAK2 antibodies. Similar results were obtained from four independent experiments. (G) NP cells were pre-incubated with the JAK inhibitor 1 or AG490 for 30 min followed by treatment with thrombin (10 U/mL) for 24 h; CXCL8 protein levels were determined by ELISA. Each bar represents the mean ± S.E.M. from three independent experiments performed in duplicate. *p < 0.05 compared with the vehicle control group. # p < 0.05 compared with the thrombin-treated group.
