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. 2018 Nov 28;4(11):eaat3834. doi: 10.1126/sciadv.aat3834

Fig. 4. Type II JAK2 inhibitor CHZ868 blocks proliferation of cells expressing wild-type JAK2 and JAK2V617F.

Fig. 4

(A) TF1.8 cells were starved overnight in the presence of 0.5% FCS and stimulated for 5 or 15 min with IL-3 (25 ng/ml) in the presence of 280 nM ruxolitinib or different concentrations of CHZ868. Cells were lysed and immunoblotted with p-JAK2, p-JAK1, p-ERK, JAK2, and actin antibodies. (B) Recombinant JAK2 kinase domain was mixed with recombinant tyrosine phosphatase PTP1B and either DMSO, CHZ868, or ruxolitinib in phosphatase assay buffer. Phosphatase reactions were incubated at room temperature for 0, 0.5, 1, and 2 hours, fractionated by SDS-PAGE, and immunoblotted with p-JAK2 antibody. Coomassie blue staining was used as a loading control. (C) Starved TF1.8 cells and SET-2 cells were incubated in IL-3 (50 ng/ml) and 10% FCS with titrations of ruxolitinib or CHZ868 in triplicate. After 48 hours, cell proliferation was assessed using CellTiter 96 reagent. The ruxolitinib and CHZ868 IC50 values required to inhibit proliferation of TF1.8 and SET-2 cells, as shown in fig. S5 (B and C), were determined using GraphPad Prism. Data are presented as the means ± SEM of IC50 values determined from three independent biological replicates. Statistical significance was determined using an unpaired two-tailed t test (*P < 0.05). ns, not significant. (D and E) FCS-starved TF1.8 or SET-2 cells were treated with increasing concentrations of ruxolitinib or CHZ868 in triplicate for 48 hours. Apoptosis was determined by annexin V staining. Bars show means ± SEM of three independent biological replicates, ***P < 0.01 and ****P < 0.001 determined by one-way analysis of variance (ANOVA) with Bonferroni’s multiple comparisons post-test.