Figure 3

Bad and Bim depletion in JAK2 ^V617F mutant SET-2 cells suppress NVP-BSK805-induced apoptosis. A: SET-2 cells were transfected with control siRNA (Ctrl), Bad siRNA or siRNAs to deplete both Bad and Bim simultaneously. After 72 hours, cells were treated with 500 nM NVP-BSK805 or the drug vehicle DMSO for 30 hours and extracted for Western blot analysis. Arrowhead depicts cleaved PARP running below the full-length PARP protein. β-tubulin was probed for as a loading control. B: SET-2 cells were transfected with control siRNA (Ctrl) or Bim siRNA and analyzed as described above. The Bim Western blot depicts an extended molecular weight range to show that Bim-EL is the predominant form of the protein expressed in SET-2 cells. C: SET-2 cells were transfected with control, Bad or Bim siRNAs, or siRNAs to deplete both Bad and Bim simultaneously, for 48 hours and then treated with 1 μM NVP-BSK805 or the drug vehicle DMSO for 40 hours. Controls also consisted of non-transfected cells. Then, DNA content was measured by FACS using propidium iodide (PI) staining and the percentage of cells with less than 2N DNA content was determined (data represent the mean ± SD of three independent experiments). *Significantly different from respective DMSO control using t-test (p < 0.05). ^#Significant difference between groups as assessed by t-test (p < 0.05). D: SET-2 cells were transfected with control, Bad or Bim siRNAs, or siRNAs to deplete both Bad and Bim simultaneously, for 24 hours and then treated with increasing concentrations of NVP-BSK805 in cell proliferation assays for 48 hours. The histogram depicts half-maximal growth-inhibitory (GI₅₀) concentrations of NVP-BSK805 for each condition (data represent the mean ± SD of two independent experiments carried out in triplicate).