Fig 1: Depletion or pharmacological inhibition of CDK1 or CDK2 increased endogenous levels of NICD in HEK293 cells HEK293 cells were cultured for 48 h after transfection with plasmids encoding scrambled siRNA (−) or siRNA specific for CDK2 (+) followed by Western blot for NICD, CDK2, CDK1, CDK4 and CDK8. β‐Actin served as loading control.Quantification of the density of Western blot bands in (A) performed by ImageJ software. Data are expressed as fold changes compared to control (scrambled siRNA transfected) cell lysate. All data represent the mean ± SEM from three independent experiments. Student's t‐test analysis was performed, with **P ≤ 0.01.HEK293 cells were cultured for 48 h after transfection with plasmids encoding scrambled siRNA (−) or siRNA specific for CDK1 (+) followed by Western blot for NICD, CDK1, CDK2, CDK4 and CDK8. β‐Actin has been used as loading control.Quantification of the density of Western blot bands in (C) performed by ImageJ software. Data are expressed as fold changes compared to control (scrambled siRNA transfected) cell lysate. All data represent the mean ± SEM from three independent experiments. Student's t‐test analysis was performed, with *P ≤ 0.05.Analysis of the inhibitory activity of three highly selective CDK2 inhibitors against a panel of kinases—a selection of those tested is shown here. At both 1 and 10 μM, Roscovitine is able to inhibit more than 95% of CDK2 activity, but is far less effective against other kinases. Purvalanol B (0.1 and 1 μM) inhibits more than 94% of CDK2 activity. At both 1 and 10 μM, GSK650394A is able to inhibit more than 98% of CDK2 activity. Source: The Kinase Profiling Inhibitor Database (http://www.kinase-screen.mrc.ac.uk/kinase-inhibitors).HEK293 cells were treated with three highly selective CDK2 inhibitors (10 μM of Roscovitine, 0.1 μM of Purvalanol B and 10 μM of GSK650394A) for 3 h. Endogenous levels of NICD were detected by Western blot. β‐Actin served as loading control.Quantification of the density of Western blot bands in (F) using ImageJ software. Data are expressed as fold changes compared to DMSO. All data represent the mean ± SEM from three independent experiments. One‐way ANOVA analysis, followed by Dunnett's test, was performed, with *P ≤ 0.05, **P ≤ 0.01, and ns = not significant.HEK293 cells were treated with RO‐3306, a specific CDK1 inhibitor, for 3 h at 10 μM. Levels of NICD, FBXW7, phospho‐histone H3 and histone H3 were detected by Western blot. β‐Actin was used as loading control.Quantification of the density of Western blot bands in (H) using ImageJ software. Data are expressed as fold changes compared to DMSO. All data represent the mean ± SEM from three independent experiments. Student's t‐test analysis was performed, with *P ≤ 0.05.