Activation of STAT3 and STAT5 is blocked by EXEL-0862. (A) HMC-1.1 and HMC-1.2 cells were treated with EXEL-0862 at concentrations ranging from 0.35 μM to 1.0 μM for 24 hours. Cell lysates were then analyzed by Western blot with phosphospecific antibodies, as indicated. (B) Time-course study of STAT3 and STAT5 phosphorylation inhibition. Cells were exposed to EXEL-0862 at 0.35 μM for 24 hours. Phosphorylation of STAT3 and STAT5 was analyzed by Western blot. (C-D) EXEL inhibited the phosphorylation of STAT3 and STAT5 and their nuclear translocation on SCF stimulation. HMC-1.2 and HMC-1.1 cells were starved in serum-free medium for 24 hours. SCF (100 ng/mL, final concentration) in 20% serum was added for 1 hour. Cells were harvested to prepare cell lysates for Western blot (C) or to be fixed in 3% paraformaldehyde for immunofluorescence staining with FITC and/or DAPI (D; arrows indicate cytoplasmic [CONTL and EXEL+SCF] or nuclear [SCF] STAT3 localization). EXEL-0862 (1.0 μM) was added to the culture 20 minutes before the addition of SCF. Cells were visualized with a 40 ×/0.9 objective lens mounted on an Olympus BX60 epifluorescence microscope (Olympus, Melville, NY), and the images were recorded with an Optronics CCD camera and Fluoview software version 4.3 (Olympus, Center Valley, PA).