Analysis of transforming capabilities of Ptpn2 knockdown. (A) Transformation assay of Ba/F3 cells with or without knockdown of Ptpn2 and stably transduced with empty control vector, JAK1 wild-type, or JAK2 wild-type. Only JAK1-positive cells with knockdown of Ptpn2 transformed to cytokine independence. Ba/F3 cells carrying the indicated constructs were deprived of IL-3 and cell numbers were recorded in triplicate every other day. The y-axis displays the number of viable cells. Data shown are representative of 3 independent experiments. *P < .005. (B) Western blot analysis of Ba/F3 cells with knockdown of Ptpn2, overexpression of JAK1 (wild-type), or both. Growth factor–dependent Ba/F3 cells were cytokine depleted for 6 hours before cell lysis. Shown is 1 representative result of 3 independent experiments. (C) Ba/F3 cells transformed by JAK1 wild-type with knockdown of Ptpn2 or JAK1(R724H) mutant (positive control) or ZNF98-FGFR1 (negative control) were treated with a JAK inhibitor or with JAK1 siRNA and cell numbers were recorded after 48 hours. Values are displayed as relative cell growth (%). Graph represents mean values ± SEM. Significant differences in cell growth compared with those of control siRNA are indicated (n = 3). *P < .05; **P < .005.