Figure 3

NSC114792 affects the viability of BaF3 cells expressing an activating allele of JAK3. BaF3-JAK3^V674A cells were transformed by introducing an activating allele of JAK3 (V674A) [23]. (A) Treatment of BaF3-JAK3^V674A cells with NSC114792 results in a decrease in phosphorylated JAK3 and STAT5 levels in a dose-dependent manner. (B) NSC114792 decreases the viability of BaF3-JAK3^V674A cells in a time- and dose-dependent manner. BaF3-JAK3^WT cells were incubated for the indicated time periods in the absence or presence of IL-3, and with or without 20 μmol/L NSC114792. BaF3-JAK3^V674A cells were also cultured with NSC114792 at various concentrations for the indicated time periods. BaF3-JAK3^WT cells showed IL-3-dependent survival, and the viability of these cells was barely affected by 20 μmol/L NSC114792. By contrast, this dose of compound significantly decreased the viability of BaF3-JAK3^V674A cells. These data suggest that the effect of NSC114792 on BaF3-JAK3^V674A cell viability is not caused by the non-specific cytotoxicity of the compound. (C and D) NSC114792 blocks both JAK3 and STAT5 phosphorylation, and causes a significant decrease in cell viability in BLNK^-/- mice-derived BKO84 cells, which express constitutively-active JAK3/STAT5 due to the lack of BLNK inhibition of JAK3 [18]. (B) *, p < 0.001, indicates statistical significance compared to DMSO-treated BaF3-JAK3^V674 cells. #, p < 0.01, indicates statistical significance compared to IL-3-treated BaF3-JAK^WT cells. (D) *, p < 0.001, indicates statistical significance compared to DMSO-treated BKO84 cells.