Acceleration of granulocyte differentiation ex vivo by the RARα-specific agonist AGN195183 requires Mad1 and p27^Kip1. (A) Immature (CD11b/Gr-1^dim) and mature (CD11b/Gr-1^bright) granulocytes were isolated as described in “Materials and methods.” Protein (36 μg) from each fraction was prepared in sample buffer and separated on a 10% SDS-PAGE gel. Proteins were then transferred to a PVDF membrane and probed with anti-Mad1, anti-p27^Kip1, and anti–α-tubulin. (B) Expression of RARα in granulocyte subsets was analyzed using quantitative real-time PCR. Data shown are the expressions of RARα in the CD11b/Gr1^dim population ± SEM, with a similar relative expression observed in the CD11b/Gr1^bright and whole bone marrow populations (data not shown). (C) Whole bone marrow cells of the indicated genotype were cultured as described with either ethanol (control), the RARα-specific agonist AGN195183 (10^–6 M), or ATRA (10^–6 M). Following 3 days in culture, cells were isolated and phenotypically identified by CD11b/Gr-1 staining. Representative FACS dot plots following culture with ethanol (control) or retinoid agonist are shown. (D) ATRA can accelerate granulocyte differentiation independent of Mad1, p27^Kip1, and RARα. (E) The RARα-specific agonist AGN195183 requires Mad1 and both Mad1 and p27^Kip1 to accelerate granulocyte differentiation ex vivo. The specificity of AGN195183 is demonstrated by the lack of effect on RARα^–/– cells; n > 6 for all treatments/genotypes. Data are graphed as ratio of Gr-1^bright/Gr-1^dim cells ± SEM (**P < .01, *P < .05).