Activation of p53 by nutlin-3 recapitulates the effects of RPS14 or RPS19 shRNAs. (A) Primary human CD34⁺ cells were treated with nutlin-3 for 72 hours and cultured in cytokines supporting erythroid and myeloid differentiation. Levels of p53 in erythroid and myeloid lineage cells were assessed by flow cytometry. (B) Cell cycle status of the nutlin-3-treated cells was analyzed by flow cytometry after labeling with BrdU and 7-AAD. (C) Primary human CD34⁺ cells were treated with nutlin-3 and cultured in cytokines supporting erythroid and myeloid differentiation for 10 days. The ratio of erythroid to myeloid cells was assessed by flow cytometric analysis GlyA (erythroid) and CD11b (myeloid) expression. (D) A total of 50 000 total bone marrow cells from mice injected with vehicle (dimethyl sulfoxide [DMSO]) or 93 mg/kg of nutlin-3 at alternate day for 5 days were plated on methylcellulose, and colonies were counted after 7 to 10 days in culture (mean ± SEM; n = 5 mice in each group). (E) Bone marrow cells from mice injected with vehicle (DMSO) or 93 mg/kg of nutlin-3 at alternate day for 5 days were plated on methylcellulose, and colonies were counted after 2 and 5 days in culture for CFU-E and BFU-E, respectively (mean ± SEM; n = 5 mice in each group). Results of all in vitro nutlin-3 treatment experiments are representative of 3 independent experiments performed in triplicate (mean ± SEM). *P < .05. **P < .01.