Treatment with TG inhibits JAK2 phosphorylation and downstream signaling and induces apoptosis in HEL and Ba/F3-JAK2V617F cells. (A) HEL, Ba/F3-JAK2V617F, and Ba/F3-hEpoR cells were treated with the indicated concentrations of TG for 48 hours. After treatment, the cells were 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 the indicated concentration of TG for 16 hours. Total RNA was harvested and reverse transcribed, then used for quantitative real-time polymerase chain reaction for JAK2 expression. Relative expression of JAK2 was normalized to GAPDH. (C) HEL cells were treated with the indicated concentrations of TG for 24 hours. Then, cell lysates were prepared and immunoblot analyses were performed for pJAK2 (Tyr1007/1008), JAK2, pSTAT5 (Tyr694), STAT5, pSTAT3 (Tyr705), STAT3, Bcl-x_L, pAKT (Ser473), AKT, pERK1/2, ERK1/2, pGATA-1 (Ser310), and GATA-1. The expression levels of β-actin in the lysates served as the loading control. (D) Ba/F3-JAK2V617F cells were treated with the indicated concentrations of TG for 24 hours. After treatment, cell lysates were prepared and immunoblot analyses were performed for pSTAT5 (Tyr694), STAT5, pSTAT3 (Tyr705), STAT3, Bcl-x_L, pAKT (Ser473), AKT, pERK1/2, and ERK1/2. The expression levels of β-actin in the lysates served as the loading control.