R723 inhibits JAK/STAT signaling and growth of JAK2V617F harboring hematopoietic cells. (A) BM cells from WT or V617F-TG mice were harvested and cultured without serum for 3 hours. Cells were incubated with vehicle or R723 for 1 hour, followed by the stimulation with IL-3, EPO, and G-CSF for 15 minutes. Mac-1/Gr-1⁺ myeloid cells were analyzed for levels of STAT5 phosphorylation by flow cytometry. One representative experiment of 3 is shown. (B) Effects of R723 on EPO-independent CFU-E colonies derived from V617F-TG. BM cells from V617F-TG were cultured in duplicate in methylcellulose culture medium in the absence of EPO with and without R723. The number of CFU-E colonies was counted on day 3. R723 treatment significantly suppressed CFU-E in V617F-TG. Three independent experiments were performed. Data are presented as means ± SE. (C) Effects of R723 on cytokine-dependent BM colonies derived from WT and V617F-TG mice. BM cells from WT and V617F-TG mice were cultured in cytokine-containing methylcellulose with and without R723. The number of CFU-E colonies was counted on day 3 (top left). The numbers of CFU-GEMM (top right), CFU-GM (bottom left), and BFU-E (bottom right) colonies were counted on day 7. R723 inhibited the cytokine-dependent colonies (CFU-E, CFU-GEMM, CFU-GM, and BFU-E) derived from both WT and V617F-TG cells to the same extent. Three independent experiments were performed, each using 1 different WT mouse and 1 different V617F-TG mouse. Data are presented as means ± SE.