FIG. 5.

Ikaros arrests cells in the G₁ phase of the cell cycle, and phosphorylation alleviates this effect. (A) Flow cytometric cell cycle profiles of NIH 3T3 cells infected with pMX-GFP-IRES (GFP), pMX-Ik-1-GFP-IRES (Ik-1 GFP), or pMX-Ik-2-GFP-IRES (Ik-2 GFP). Cells were left untreated (asynchronous [ASYNCHRO.], right column) or treated with 1 mM hydroxyurea (HYDROX., middle column) or 0.5-μg/ml nocodazole (NOCOD., left column) to arrest cells in the S or M phase, respectively. The percentage of cells in the G₀-G₁ (G₁), S, or G₂/M (M) phases under the different conditions is depicted in the table at the bottom of the panel. Photomicrographs of representative asynchronously growing cells, infected under each condition, are shown. (B) NIH 3T3 cells were infected with Ik-2 retroviruses at 1:5 or 1:2 dilutions in fresh medium. Seventy-two hours after infection, GFP-positive cells were sorted and treated with nocodazole for an additional 16 h. The percentages of cells remaining in the G₁ or S + M phases are represented. The level of expressed Ik-2 protein was determined by immunoblotting with anti-Ikaros antibody (IB:Ik). The YY1 protein level is shown as a loading control. The GFP mean fluorescence is shown in parentheses. (C) NIH 3T3 cells were infected with the indicated retroviruses, and 72 h after infection, cells were treated as in panel B. Results from two representative experiments out of six performed with similar results are shown. (D) NIH 3T3 cells were infected with the indicated retroviruses. Seventy-two hours after infection, cells were treated as in panel B. Results from one representative experiment out of three performed with similar results are displayed.