Cotreatment with PS enhances TG-mediated inhibition of JAK2 phosphorylation and downstream signaling and induces synergistic apoptosis of MPD cells. (A) HEL cells were treated with the indicated concentrations of TG and/or PS for 48 hours. Then, the cells were washed with 1 × PBS and stained with annexin-V and propidium iodide, and the percentages of apoptotic cells were determined by flow cytometry. Columns represent mean of 3 independent experiments; bars represent SEM. (B) HEL cells were treated with TG (200-800 nM) and PS (5-20 nM) for 48 hours. Apoptosis was determined by annexin-V staining and flow cytometry. Median dose effect and combination indices were obtained using Calcusyn software. CI values less than 1.0 indicate synergism of the 2 agents. (C) HEL cells were treated with the indicated concentrations of TG and/or PS for 24 hours. After treatment, cell lysates were prepared and immunoblot analyses were performed for pJAK2 (Tyr1007/1008), JAK2, pSTAT5 (Tyr694), STAT5, pSTAT3 (Tyr705), STAT3, pGATA-1 (Ser310), and GATA-1 and pAKT (Ser473). The expression levels of β-actin in the lysates served as the loading control.