Figure 3.

CDK4 shuttles between the nucleus and the cytoplasm and promotes the nuclear accumulation of cyclin D1. A, HeLa cells were co-transfected with GFP- or Myc-tagged cyclin D1 and CDK4 for 24 h. Myc-tagged protein was immunostained. 200 cells were randomly counted in each of three repeated experiments. Representative images are shown. B, HeLa cells were transfected with CDK4 siRNA for 72 h and fractionated into nuclear and cytoplasmic fractions, followed by Western blotting. C and D, asynchronous HeLa cells co-transfected with control, CDK4 or CDK6 siRNA, and RFP-H2B for 72 h were stained using an anti-cyclin D1 antibody and DAPI (C), and the cells with cyclin D1 in the nucleus were counted (D). *, p < 0.05; **, p < 0.01 (Student's t test). E and F, HeLa cells co-transfected with GFP-cyclin D1 (GFP-D1) and NLS-CDK4-Myc for 24 h were visualized by IFM (E; scale bars, 10 μm) or a co-immunoprecipitation assay (F).