NR4A2 siRNA knockdown inhibits myeloid cell proliferation. Hoxb8 conditionally immortalized murine myeloid progenitor cells were subjected to estrogen withdrawal and differentiated to mature neutrophils (>90% maturity) in the presence of SCF and G-CSF for 4 days with daily media replenishment. (A-C) Mature neutrophils were incubated with or without N6/8-AHA (100 μM) for 6 hours in apoptosis medium. (A) Cell viability and apoptosis was visualized by oil immersion light microscopy (arrow denotes an apoptotic cell) and (B-C) quantified by flow cytometry. RNA interference transfections were conducted 1 day after estrogen withdrawal by using Amaxa Nucleofector technology. Cells were transfected with siRNA for NR4A2, NR4A3, or a nontargeting control (siScrbl) on day 1. Total cell numbers were determined by hemocytometer counts (D-E) at days 1, 2, 3, and 4. Neutrophil maturity was assessed by light microscopy (F-G). Apoptosis was measured on (H) day 4 and (I) day 5 by flow cytometry. Data are expressed as means ± SEMs; (B-C, G) n = 4), (D-E) n = 3, and (F, H-I) n = 1. Statistical analysis was performed by ANOVA with Bonferroni’s posttest. Significant differences to media controls or siScrbl transfected cultures were denoted by **P <.01 or ***P < .001, respectively.