Induction of apoptosis and cell-cycle arrest by the JAK2^V617F-specific shRNA and the anti-JAK2 inhibitor AZ960. (A) Apoptosis: pRRL-mt4 virus (left) and AZ960 (right) treatment increased the percentage of annexin V–positive cells in the absence of cytokine. SET-2 (top) and UKE-1 (bottom) cells were either cultured for 48 hours in the presence of 105 nM or 50 nM AZ960 (IC₅₀), respectively, or transduced with pRRL-Scr (Scr) or pRRL-mt4 (mt4), in the absence or presence of cytokines. Cells were stained with annexin V–allophycocyanin and 7-aminoactinomycin D and analyzed by flow cytometry analysis. (B) Cell cycle: pRRL-mt4 virus (left) and AZ960 (right) treatment of UKE-1 cells decreased autonomous cell-cycle progression (S + G₂/M phases, P ≤ .05). UKE-1 cell cycle was analyzed by propidium iodide staining, 48 hours after AZ960 and pRRL-mt4 virus treatment, or DMSO and pRRL-Scr virus treatment as controls, in the absence or presence of cytokines. The percentages of cells in the G₀-G₁, S, and G₂-M phases are shown on each histogram. (A-B) The presence of cytokines blunted the increase in apoptotic or nondividing cells induced by AZ960 treatment or pRRL-mt4 transduction. Data are mean value (± SD) from 3 independent experiments. *P ≤ .05.