Figure 3.

The CaMKII antagonist, KN-93, controls the expression and activation of cell cycle regulatory proteins in osteosarcoma. (a) MG-63 cells were treated with 10 μM KN-93 for the indicated time (upper panel), or treated for 24 h with the indicated concentrations of KN-93 (lower panel). A representative immunoblot of p21 and actin from three separate experiments is shown. (b) Real-time PCR was performed using primers specific for p21 or 18S rRNA (upper panel). Values were obtained from three separate experiments and represent the mean±s.e. of p21 messenger RNA expression relative to 18 S rRNA expression. *P<0.01. (Lower panel) MG-63 cells were treated with DMSO (0.1%), KN-93 (10 μM) or KN-93 (10 μM) + actinomycin D (Act D) (0.8 μM) for 12 h. Cells were then lysed and immunoblotting was performed using p21- and actin-specific antibodies. Representative images from three separate experiments are shown. (c) MG-63 cells were treated with DMSO (control), KN-92 (10 μM) or KN-93 (10 μM) for the indicated time. Protein was extracted and immunoprecipitation was performed using anti-CDK2 or anti-CDK4 antibodies, followed by Western blotting for p21 or CDK4 (upper panel), and p21 or CDK2 (lower panel). Representative images from three separate experiments are shown. (d) MG-63 cells were treated with either DMSO (control), KN-92 (10 μM) or KN-93 (10 μM) for 24 or 48 h. (Upper panel) Western blots using antibodies directed against p-Rb or total Rb were performed. (Lower panel) MG-63 cells were transfected with luciferase reporter plasmids driven by the E2F sequences and a CMV-β-galactosidase reporter constructs using Lipofectamine for 24 h. Cells were then treated with DMSO (control), KN-92 (10 μM) or KN-93 (10 μM) for 24 h. Reporter activity was then measured. Data are expressed relative to an internal control (CMV-β-galactosidase) and are the means±s.e. of three separate experiments, each performed in triplicate; *P≤0.01.