JAK-STAT mutations lead to increased pSTAT5 signaling, cytokine-independent growth, and enhanced colony formation. (A-B) Mutated IL2RG (p.G628_M630del), JAK1 (p.S703I), JAK3 (p.Q507P), and STAT5B (p.T628S and p.N642H) leads to increased activation of STAT5 transcriptional activity (A, bar graph; n = 3 for each mutant protein in separate experiments; asterisk indicates P < .05; dagger indicates P < .001) and increased phosphorylation of STAT5B (B, western blot; arrowhead indicates exogenous STAT5B, arrow indicates endogenous STAT5B; normalized densitometric pSTAT5/STAT5 ratios are indicated) in HeLa cells. STAT5B p.P267A represents a germline polymorphism. (C) Cytokine-independent cell proliferation in the presence of mutant p.T628S STAT5B protein in the cytokine-dependent Ba/F3 cell line cultured in the absence of growth factors (n = 6; asterisk indicates P < .01). (D) Enhanced colony-forming capacity of STAT5B p.N642H mutant in Jurkat T cells (n = 3; asterisk indicates P < .01).