FIGURE 4

The effect of IkBm on p53-mediated cell-cycle arrest and gene expression. (a) EU-1/neo and EU-1/IkBm cells were treated with Dox (0.5 g/ml), and cell-cycle analysis performed 12 h post-treatment. (b) Western blot analysis for the effects of transfected IkBm on expression of p53, and its target genes p21, MDM2 and Bax following treatment with Dox and VCR. After treatment of EU-1/neo and EU-1/IkBm cells with Dox (0.5 g/ml) and VCR (0.1 g/ml) for 2 h, proteins were extracted and analysed for protein expression using antibodies as indicated. Actin served as control for equal protein loading and protein integrity. (c, d) Effect of stable IkBm expression on p53-mediated p21 promoter activity. EU-1/neo and EU-1/IkBm cells were transfected with either 5 g p21 promoter-luciferase (p21-L) alone, or in combination with 10 g MDM2 plasmid. Transfection was performed as described in Figure 1. At 46 h after transfection, cells were treated with increasing concentrations of Dox (0.05, 0.1 and 0.5 g/ml) or VCR (0.001, 0.01 and 0.1 g/ml) for another 2 h, and then cell extracts were prepared for luciferase activity assay as described in Materials and methods. Results were normalized to -galactosidase activity and plotted as the means of duplicates of a representative experiment out of at least three independent determinations