Constitutive activation of JAK3/STAT5 signaling inhibits p27^kip1 expression in BKO84 cells. (A) Cell-cycle profiles of BKO84 cells that were cultured in medium containing αIL-7R for the indicated time period. Cells were stained with propidium iodide and analyzed by flow cytometry. Percentages of cells in subdiploid (if present), G₁, and S/G₂/M phases are indicated by numbers. (B) Western blot analysis of p27^kip1 protein (top) and β-actin as a loading control (top middle), and semiquantitative RT-PCR analysis of p27^kip1 mRNA (bottom middle) and GAPDH as a cDNA quantity control (bottom) of the same cells as in panel A. (C,D) Cell-cycle analysis (C) and Western blot analysis for p27^kip1 protein (D) in BKO84 cells infected with retroviral vectors carrying either dnSTAT5a or STAT5a coupled via an IRES to rCD2. Cells expressing the rCD2 were magnetically sorted at the indicated times after infection and analyzed. (E) Semiquantitative RT-PCR analysis of p27^kip1 mRNA and GAPDH mRNA (cDNA quantity control) in BKO84 cells transduced with dnSTAT5a as in panel D. All data are representative of 3 independent experiments.