FIGURE 2.

JAK3 FERM domain integrity is required for the activity of the V674A mutant, but not for the L857P mutant. A, HEK293 cells were transiently co-transfected either with JAK3^WT, or different JAK3 mutants (Y100A, V674A, and L857P) or double mutants (V674A/Y100A and L857P/Y100A) together with γc and IL-9Rα or not, 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 between the control condition without JAK3 and the WT or mutant forms of JAK3 for each condition (with or without IL-9R complex). *, p < 0.05; ***, p < 0.001. K, kinase domain; PK, pseudokinase domain. B, in parallel of the luciferase assay, transfected HEK293 cells were lysed 24 h post-transfection and subjected to Western blot analysis using an anti-JAK3 antibody and an anti-β-actin antibody as loading control. 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 γc 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 < 0.05.