CEBPB effects on proliferation and differentiation require its DNA-binding activity. (A) Schematic representation of MigRI GFP ΔuORF C/EBPβ (top) and MigRI GFP ΔP231-242) CEBPB (bottom) plasmids. (B) DNA-binding activity of WT and mutant CEBPB assessed by EMSA using a ³²P-labeled double-stranded oligonucleotide corresponding to the G-CSFR C/EBP-binding site and lysates from untransfected 293T cells (lane 9) or cells transfected with MigRI GFP (lanes 4 and 8), MigRI GFP ΔuORF C/EBPβ (lanes 2 and 6), or MigRI GFP Δ(231-242) CEBPB (lanes 3 and 7). Binding specificity was assessed using the G-CSFR C/EBP-binding site oligonucleotide as cold competitor (lanes 6-9). Arrow indicates the DNA-protein complex. (C) Luciferase assay (representative of 3 different experiments) on cell lysate from 293T transfected with a reporter gene in which luciferase activity is driven by 4 C/EBP-binding sites (G-CSFR pTK G-CSFR-luciferase) alone, or in the presence of the MigRI GFP empty vector, or ΔuORF or Δ(231-242) CEBPB. Levels of HA-C/EBPβ expression (bottom) were tested by Western blotting with an anti-HA antibody; equal loading was demonstrated by monitoring GRB2 levels. (D) Proliferation of 4-HT–treated (250 nM) cells carrying the MigRI GFP (□), the MigRI GFP ΔuORF C/EBPβ-ER^TAM (♦), or the MigRI GFP Δ(231-242) C/EBPβ-ER^TAM (▴) retrovirus; cell number (× 10³) is on the y-axis, day of treatment is on the x-axis. Values are the mean plus SD of 3 different experiments. (E) May-Grünwald-Giemsa staining of cytospin from MigRI GFP ΔuORF C/EBPβ-ER^TAM (left) or MigRI GFP Δ(231-242) C/EBPβ-ER^TAM (right) 32D-BCR/ABL cells 3 days after treatment with 4-HT (250 nM) and G-CSF (25 ng/mL); G-CSFR levels are shown at the bottom.