FIGURE 3.

JAK1 expression is required for the activity of the V674A mutant, but not for the L857P mutant. A, JAK1-deficient U4C cells were transiently co-transfected with JAK3^WT or JAK3^V674A, γc, and IL-9Rα with or without JAK1^WT or JAK1^KD, in addition to the STAT5-responsive luciferase reporter pLHRE-luc (firefly luciferase) and the pRLTK plasmid (Renilla luciferase) as transfection control. 24 h post-transfection, the cells were subjected to a luciferase assay. The relative luciferase activity corresponds to the firefly luciferase light emission values divided by the Renilla luciferase light emission values. The results are means ± standard deviation of three different experiments, each performed in triplicate. A Kruskal-Wallis test with Dunn correction was performed to determine p values. *, p < 0.05. B, JAK1-deficient U4C cells were transiently co-transfected with JAK3^WT or JAK3^L857P, γc, and IL-9Rα with or without JAK1^WT or JAK1^KD, in addition to the STAT5-responsive luciferase reporter pLHRE-luc and the pRLTK plasmid as transfection control. 24 h post-transfection, the cells were subjected to a luciferase assay. The results are means ± standard deviation of three different experiments, each performed in triplicate. A Kruskal-Wallis test with Dunn correction was performed to determine p values. *, p < 0.05. C, relative proliferation of IL-3-dependent Ba/F3 cells or autonomous Ba/F3 cells obtained after transduction with ALL-associated JAK3 mutants (V674A and L857P) and double mutant (L857P/Y100A) after knockdown of endogenous JAK1 compared with the proliferation observed with an irrelevant control siRNA. After 72 h, tritiated thymidine incorporation was measured. The results are means ± standard deviation of three different experiments, each performed in triplicate. A Kruskal-Wallis test with Dunn correction was performed to determine p values between the IL-3-dependent control Ba/F3 cells and the transformed Ba/F3 cells. **, p < 0.01.