Treatment with panobinostat inhibits JAK2 mRNA expression and JAK/STAT signaling and induces apoptosis of myeloproliferative disorder (MPD)cells and Ba/F3 cells with ectopic overexpression of JAK2V617F. (A) HEL, Ba/F3-JAK2V617F, and Ba/F3-hEpoR cells were treated with the indicated concentrations of panobinostat (PS) for 48 hours. After this, 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 concentrations of PS for 24 hours. Then, total cell lysates were prepared and immunoblot analyses were performed for pJAK2 (Tyr1007/1008), JAK2, pSTAT5 (Tyr694), STAT5, pSTAT3 (Tyr705), pSTAT3 (Ser727), 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. (C) Ba/F3-JAK2V617F cells were treated with the indicated concentrations of PS for 24 hours. After treatment, cell lysates were prepared and immunoblot analyses were performed for pSTAT5 (Tyr694), STAT5, pSTAT3 (Tyr705), and STAT3. The expression levels of β-actin in the lysates served as the loading control. (D) HEL cells were treated with the indicated concentrations of PS for 16 hours, then mRNA was isolated and reverse transcribed. Quantitative real-time PCR was performed on the cDNA using TaqMan probes for the exon 8 to 9 boundary and exon 23 to 24 boundary of JAK2. Relative expression of JAK2 mRNA was normalized to GAPDH expression.