Ptpn2 modulates the oncogenic transformation of JAK1(A634D)-expressing Ba/F3 cells in a dose-dependent manner. (A) Quantitative real-time PCR analysis of Ptpn2 transcript levels in JAK1(A634D) mutation–positive Ba/F3 cells carrying 6 different shRNA constructs. Hprt1 was used for normalization of expression values and the y-axis displays Ptpn2/Hprt1 ratios. (B) JAK1 mutation–positive Ba/F3 cells cotransduced with the indicated shRNA constructs were deprived of IL-3 and cell growth was monitored every 24 hours for 4 days. Independent of the knockdown efficiency of Ptpn2, cell lines with reduced Ptpn2 expression (shRNA-Ptpn2) transformed appreciably faster to cytokine independence compared with the 2 control cell lines (shRNA-control). Differences in latency among the 4 cell lines carrying Ptpn2-targeting shRNA constructs were correlated with knockdown efficiency. (C) Protein blot analysis of transformed cell lines showed enhanced JAK/Stat activation in cells with a reduced amount of Ptpn2 protein. β-actin was used as an independent loading control. (D) Quantitative real-time PCR analysis of Ptpn2 transcript levels in JAK1(A634D) mutation–positive Ba/F3 cells after 48 hours of electroporation with various siRNAs. Hprt1 was used for normalization of expression values. The x-axis displays Ptpn2/Hprt1 ratios. (E) Knock-down efficiency of applied siRNAs was confirmed by protein blot analysis. Erk is shown as loading control. (F) STAT5 phosphorylation was determined in JAK1(A634D) Ba/F3 cells electroporated with different Ptpn2-targeting siRNAs or a control siRNA. Absorption values were normalized for total Erk. Bar graph shows the relative changes compared with control cells (mean values ± SEM). *P < .05.