Effects of G-CSFR activation on ATRA-induced morphologic maturation and neutrophil gene expression in NB4 cells. (A) NB4 cells (i) were induced with 5 μM ATRA alone (ii), G-CSF alone (iii), ATRA plus 100ng/mL G-CSF (iv), or were primed with ATRA for 24 hours, washed, and then incubated in ATRA-free media either in the absence (v) or the presence of G-CSF (vi). Following 4 days of incubation, cells then were cytocentrifuged and stained with Wright-Geimsa stain for morphologic analysis. Original magnification, × 100. (B) Aliquots of NB4 cells (u) primed with ATRA (5 μM) for 2, 4, and 12 hours were either extracted for total RNA (lanes 2, 5, and 8) or washed in 1 × PBS and incubated for 48 hours in ATRA-free maintenance media alone (lanes 3, 6, and 9) or in the presence of G-CSF (100 ng/mL) (lanes 4, 7, and 10). After the 48-hour incubation period, cells in media alone (–) or in media with G-CSF (+) were collected and subjected to Wright-Geimsa staining or extracted for RNA. Total RNA (10 μg) from each sample was then blotted and sequentially hybridized with 32P-labeled cDNA probes for human G-CSFR, C/EBPϵ, HNP-1,3 and gp91-phox, and rat C/EBPα. The blot was also hybridized with human γ-actin to demonstrate equal loading of RNA in each lane.
