Figure 2.

Suppressing the STAT3 pathway by inhibiting JAK1 kinase activity. (A) SKOV3 and MDAH2774 cells were first grown in regular culture medium for two days, then the medium was replaced with fresh medium for 30 mins. Cells, both before and after replacing the medium, were harvested and analyzed for STAT3 phosphorylation. (B) Old medium was added back to the SKOV3 cells, which had been in the fresh medium for 30 mins, either in the presence or absence of antibodies against the gp130 protein, and incubated for an additional 30 mins. Cells were harvested and analyzed for the phosphorylation of STAT3. (C) Relative levels of p-STAT3 and STAT3 were determined by measuring the density of each band and normalized to that of GAPDH. Densitometry data were relative changes in protein expression and were mean ± SD of 2-3 preparations. *, P<0.05 vs. control without anti-gp130 in old medium. (D) Effect of JAKi on cell viability of human ovarian cancer cells. Cells were plated and treated with various concentration of JAKi, cell viability was determined 72 hrs later. (E) Dose-dependent inhibition of STAT3 phosphorylation in SKOV3, MDAH2774 and primary human ovarian cancer cells by the JAK inhibitor (JAKi), AZD1480. Cells were treated with JAKi at various concentrations for 24 hours and the phosphorylation of STAT3 and JAK2 were analyzed on Western blots. (F) Effect of other kinase inhibitors on phosphorylation of STAT3. SKOV3 cells were treated with EGFRi (gefitinib), SRC inhibitor (dasatinib), multiple kinase inhibitor (sunitinib) and mTOR inhibitor (RAD001) for 24hrs. Phosphophrylation of STAT3 was determined by Western Blot. (G) STAT3 phosphorylation is inhibited by treating with an siRNA against JAK1. SKOV3-M-Luc cells were transfected with siRNA against STAT3, JAK1 or JAK2. Cells were analyzed for the expression levels of JAK1, JAK2 or the phosphorylation of STAT3 by Western blot.