Effect of NU2058 or NU6102 on cyclin-dependent kinase-2 (CDK2) and CDK1 kinase activity, p53 and RNA polymerase II phosphorylation levels in MCF7 cells. (A) CDK2 and (B) CDK1 immunoprecipitated from control MCF7 cells or from those treated with NU6102 or NU2058 for 1 h. Immunoprecipitated CDK levels were analysed by western blotting and kinase activity was measured by CDK kinase assay. CDK protein was incubated with γ[32P]ATP and histone H1, and analysed by SDS–PAGE and autoradiography. (C) Western blot for p53, phospho-Serine 2 and 5 of RNA polymerase II and total RNA polymerase II protein levels measured in MCF7 cells. Lysates were collected 16 h after exposure of exponentially growing cells to NU2058 (25 and 75 μ M), NU6102 (5 and 15 μ M) and flavopiridol (0.5 μ M). (D) MCF7 cells were transfected with either scrambled or CDK1 and CDK2 siRNA; at 3 days post-transfection, cells were treated with either DMSO, NU2058 or NU6102 for 24 h followed by replating. Colony formation was assessed 2 weeks after replating. Graph shows mean and s.e. of colony formation relative to the scrambled DMSO-treated control plates.