C/EBP inhibits the G1 to S transition and induces p27^Kip1 levels and Rb hypophosphorylation in 32D cl3 cells. (A) 32D-Puro cells were cultured in IL-3 or in G-CSF for 1, 2, or 3 days. 32D-Puro, 32D-WT-ER-1, 32D-WT-ER-2, and 32D-bcr/abl, 32D-bcr/abl-WTER cells in IL-3 were exposed to estradiol for 0, 1, 2, or 3 days. The proportion of cells in the G1, S, and G2/M cell cycle phases was determined daily by BrdU/PI staining. Representative FACScan data from days 0 and 3 are shown for 32D-Puro cells in IL-3 or G-CSF and from 32D-WT-ER-1 and 32D-bcr/abl-WTER cells in IL-3 and estradiol. The location of G1, S, and G2/M cells is shown in the upper left panel. Signals to the left of the G1 population have less than 2n DNA content and represent cells undergoing apoptosis. (B) The proportion of cells in S, G1, or G2/M on each day for these six cultures is shown from a representative experiment. Cells with less than 2n DNA content were excluded from these calculations. (C) Total cellular protein extracts were prepared from 32D-Puro, 32D-WT-ER-1, and 32D-WT-ER-2 cells exposed in IL-3 to estradiol for 0, 1, 2, or 3 days. These extracts were then subjected to Western blotting for Rb, p21^WAF1/CIP1, and p27^Kip1. Retinoblastoma protein was detected as a doublet (Rb-P, Rb).