Figure 4.
Inhibition of PKC ameliorates prosurvival benefit conferred to HSCs by N-RasG12D. A and B, c-kit–enriched cells were transfected with either empty vector control or PKCδ shRNA, and mRNA level by qRT-PCR (A) and protein level by Western blot analysis (B) was measured to verify knockdown (n = 6). Unpaired Student t test was used to calculate statistical significance. WT, wild-type. C, FACS-purified SLAM HSCs from control or Nras mutant mice were exposed to cytokine starvation following transfection with either empty vector or PKCδ shRNA, and apoptosis was measured after 18 hours of culture using caspase activation (n = 5). Two-way ANOVA was used for statistical significance. D, Experimental design using in vivo administration of the PKC inhibitor AEB (10 μg/g body mass) to sensitize HSCs to apoptosis with and without γ-radiation. E and F, Representative flow cytometry histogram (E) and quantitation (F) showing apoptosis of SLAM HSCs as measured by Annexin V staining following in vivo radiation alone or together with AEB treatment in control and Nras mutant mice (n = 4–6). Two-way ANOVA was used for statistical significance. Data represent 10 mean ± SD; n, number of mice.