Figure 3. Effect of BMA097 on STAT3 phosphorylation and activation.

(A–B) BMA097 inhibition of constitutive STAT3 phosphorylation. MDA-MB-231 cells were treated with different concentrations of BMA097 for 24 hrs (A and D) or at 2 μM for different times (B) followed by Western blot analysis of total and phosphorylated STAT3. STAT3 downstream target proteins Bcl-xL and Cyclin D1 were also tested. Actin was used as a loading control for Western blot. (C) BMA097 inhibition of IL-6-induced STAT3 phosphorylation. Serum-starved MDA-MB-231 cells were pre-treated with BMA097 and then with IL-6 followed by Western blot analysis of total and Tyr⁷⁰⁵-phosphorylated STAT3. (D–E) BMA097 effect on pSTAT3 subcellular localization. MDA-MB-231 cells were treated without or with BMA097 for 24 hrs followed by immunofluorescence staining or fractionation and Western blot analysis of Tyr⁷⁰⁵-phosphorylated STAT3. DAPI was used to counterstain nuclei in panel D. Histone and actin were used as markers for nuclear and cytoplasmic fractions, respectively, in panel E. (F–G) Effect of BMA097 on STAT3 dimerization. MDA-MB-231 cells were treated with BMA097 as described in panel A followed by separation using non-denaturing PAGE and Western blot analysis of STAT3. (H) Effect of BMA097 on JAK2 activation. MDA-MB-231 cells were treated with BMA097 followed by Western blot analysis of total and Tyr^1007/1008-phosphorylated JAK2.