Mechanisms underlying the inhibitory effects of AZD1480 treatment on MF CD34⁺ cells. (A) pJAK2, pSTAT3, and pSTAT5 levels in 1 JAK2V617F-negative and CALR mutant-positive (SP 7), 1 JAK2V617F-negative and CALR negative (SP 8) and 2 JAK2V617F-positive (SP 11, 12) primary SP MF CD34⁺ cells were determined using western blotting. Both primary JAK2V617F-positive and -negative/CALR mutant-positive MF CD34⁺ cells showed similar degrees of enhanced JAK-STAT activity as compared with normal CB CD34⁺ cells. (B) pJAK2, pSTAT3, and pSTAT5 levels in SP MF CD34⁺ cells following the treatment with AZD1480 for 4 hours were determined using western blotting. AZD1480 treatment resulted in the inhibition of pJAK2 and/or pSTAT3/5 levels to varying degrees in both JAK2V617F-positive and -negative/CALR mutant-positive MF CD34⁺ cells, whereas, only limited inhibition of JAK-STAT activity was observed with normal CB CD34⁺ cells. (C) AZD1480 treatment induced apoptosis of SP MF CD34⁺ cells. Two days after the culture, the percentage of CD34⁺Annexin V⁺PI⁻ cells was greater in SP MF CD34⁺ cells treated with cytokines plus AZD1480 as compared with that observed in SP MF CD34⁺ cells treated with cytokines alone (n = 10) or CB CD34⁺ cells treated with cytokines plus AZD1480 (n = 5) . ***P < .001; **P < .01. (D) AZD1480 treatment induced G0/G1 cell-cycle arrest in MF CD34⁺ cells. A significant accumulation of CD34⁺ cells in the G0 phase and a significant decrease of CD34⁺ cells in the G1 phase was observed in SP MF CD34⁺ cell (n = 10) treated with 150 nM AZD1480 for 3 days. By contrast, AZD1480 treatment did not alter the cell-cycle status of CB CD34⁺ cells (n = 5). *P < .05; ^#P = .07.