Fig. 7. Upregulation of gp130 leads to a complex signalling cascade upon treatment of embryoid bodies with prooxidants or chemical hypoxia. (A) Embryoid bodies were treated at day 4 of cell culture with either H₂O₂ (10 µM), menadione (20 µM) or CoCl₂ (50 µM). After 24 hours, gp130 protein expression and phosphorylation was assessed by quantitative immunohistochemistry using an antibody directed against unphosphorylated gp130 or the phosphorylated form of gp130. (B) Activation of ERK1,2, JNK p38 and PI3-kinase upon incubation of embryoid bodies with either menadione (20 µM) or CoCl₂ (50 µM). Activation was assessed by semiquantitative immunohistochemistry using phospho-specific antibodies. Maximum activation of ERK1,2 was achieved after 15 minutes; maximum activation of JNK was at 15 minutes for menadione treatment and 30 minutes for CoCl₂ treatment; maximum activation of p38 was at 30 minutes for menadione treatment and 60 minutes for CoCl₂ treatment; maximum activation of PI3-kinase was at 60 minutes. (C) Inhibition of menadione- and CoCl₂-mediated upregulation of CT-1 by the ERK1,2 inhibitor UO126 (10 µM), the JNK inhibitor SP600125 (10 µM), the p38 inhibitor SKF86002 (10 µM), and the PI3-kinase inhibitor LY294002 (20 µM). Embryoid bodies were treated at day 4 of cell culture with either menadione (20 µM) or CoCl₂ in the presence of the respective inhibitor. CT-1 protein expression was assessed after 24 hours by semiquantitative immunohistochemistry. ^*P<0.05, significantly different from levels in the untreated control; ^#P<0.05, significantly different to levels before addition of inhibitors as indicated.