Fig 6. Silencing p21 Accelerates the Emergence and Proliferation of FLT3-ITD⁺ Cells Refractory to AC220.

(A) The numbers of viable N51-FLT3-ITD-Ba/F3 cells transfected with the p21 shRNA or control shRNA were quantitated in the presence or absence of 2 nM AC220. The cells were plated at a density of 1x10⁵ cells/ml and incubated with 2 nM AC220 or control DMSO for 10 days. The viable cells were enumerated using the trypan blue exclusion assay. The data shown represent one of three experiments that were analyzed in triplicate with identical results (*: P < 0.05 compared to the control shRNA). The expression levels of the p21 protein in both cell populations are shown in the inset (the same blot is shown in Fig 3D). The right panel indicates the AC220 dose-dependent inhibition of the control FLT3-ITD⁺ cells containing empty vector and those with shRNA for p21. The cells were incubated with different concentrations of AC220 for 10 days, and the percent inhibition of viable cells was calculated compared to the control cells incubated with DMSO. (B) The panel indicates the numbers of viable FLT3-ITD⁺ cells containing the empty vector or p21 shRNA that were cultured in the presence of 2 nM AC220 for 60 days. The cells were plated at a density of 1x10⁵ cells/ml and were incubated with 2 nM AC220 or the DMSO control for 60 days. The medium was replaced every 5 days and contained 2 nM fresh AC220.