FIG. 3.

Downregulation of c-Myc is critical for Aiolos and Ikaros to exert their growth-inhibitory effect on pre-B cells. (A) Reconstituting c-Myc expression antagonizes Aiolos-mediated growth inhibition. DKO pre-B cells were infected simultaneously with virus expressing MycER and Aiolos. At 12 h after infection, Tamoxifen at 1 μM was added to activate the MycER protein. After 48 h, the infected cells were incubated with Hoechst dye, and the cell cycle status of the infected cells was determined by FACS. The numbers indicate the percentages of cells in the cycle. The result is representative of at least three independent experiments. (B) Wild-type (Wt) and Eμ-Myc pre-B cells were cultivated in the presence of IL-7 until they were homogenous B220⁺ pre-BCR⁺ IgM⁻. The cells were infected with Aiolos and Ikaros, and the impact of Aiolos and Ikaros on cell cycle progression was analyzed 48 h later by FACS. The infected cells expressing high levels of GFP-positive (GFP+) and GFP-negative (GFP−) cells were analyzed separately. The result is a representative of at least three independent experiments. (C) The GFP+ and GFP− cells were also isolated by sorting, and real-time PCR analysis was performed to measure λ5 expression. The values are averages and standard deviations from three independent experiments.