Mutant JAK3s are sensitive to JAK3-specific tyrosine kinase inhibitor, tofacitinib. (A) BaF3 cells transduced with mutant JAK3s were treated with tofacitinib at concentrations of 0, 0.5, 2.0, and 4.0μM and analyzed by Western blot analysis at 4 and 24 hours. The Western blots and graphs that display their quantification are shown. Protein levels are expressed as fold over levels seen in cells treated with DMSO (solvent) alone. JAK3 (106 kDa) protein expression was comparable with DMSO alone but decreased in the mutant JAK3-transduced BaF3 cells. P-STAT5A (92 kDa) levels decreased markedly with increasing concentrations of inhibitor. P-JAK3 (106 kDa) levels were relatively stable. JAK3 is not phosphorylated in WT-transduced cells so it could not be quantified. Tubulin (55 kDa), a loading control, remained relatively stable. (B) Live cells were quantified by the MTT assay. Cell survival decreased with increasing concentrations of tofacitinib. BaF3 cells expressing mutant JAK3s were susceptible to tofacitinib (IC₅₀: A572V, 0.22nM; L156P, 0.27nM; E183G, 0.087nM; R172Q, 0.082nM). (C) BaF3 cells transduced with mutant JAK3 constructs treated with 0, 0.5, 1, and 2μM tofacitinib for 24 hours and analyzed for apoptosis by staining for annexin V and propidium iodide; percentages of cells staining positive for annexin V relative to DMSO only are shown. The percentage of cells stained for annexin V only as determined by flow cytometry when treated with tofacitinib. Graph shows increase in annexin V staining with inhibitor treatment in all JAK3-transduced BaF3 cells except L156P mutant JAK3. Data shown are representative of 4 independent experiments. (D) Cell-cycle profiles cells treated with DMSO; 0.5 or 1μM tofacitinib were stained with Vybrant dye cycle-violet and analyzed by flow cytometry. Graph shows the percentage of cells in G1 phase. BaF3 and WT-transduced cells have < 75% of cells in G1 after 24 hours on 1μM tofacitinib. All mutant-transduced BaF3 cells have > 90% G1 arrest with 1μM tofacitinib. Graph shows proportion of cells in G1 increases in a dose-dependent manner for all mutant JAK3s. BaF3 cells and those transduced with WT JAK3 were grown in IL-3. Data shown are representative of 2 independent experiments.