Figure 2. Kinase-dead CDK11^p58D/N but not wild-type CDK11^p58 overexpression leads to a centriole duplication failure in S-phase arrested HeLa cells.

A) Expression of Myc-CDK11^p58 and Myc-CDK11^p58D/N (left) or Myc-Plk4 proteins following transfection of the appropriate constructs. A Myc and an actin (as a loading control) Western blotting are shown. B) HeLa cells stably expressing GFP-tagged centrin were transfected with either active (CDK11^p58) or inactive (CDK11^p58DN) CDK11^p58 expression constructs. A positive control was also performed using Myc-Plk4. After 24 hours (duration of one cell cycle), the cells were arrested in S phase by aphidicolin treatment for 24 hours and the centrioles were counted after 48 more hours. C) The transfected cells expressing Myc-tagged proteins were visualised using an anti-Myc antibody (red). DNA is blue. The centriole (green), indicated by arrows, (also displayed in monochrome in the lower panels) were counted in control or transfected cells. In cells expressing CDK11^p58 as well as in control cells, the vast majority of the cells (∼70%) displayed four centrioles. This percentage dropped to 38.2% when CDK11^p58DN was expressed. Note that more than 80% of the Plk4 over expressing cells showed centriole amplification. Scale bar is 10µm. D) Graph (±SD) showing the percentage of cells with <4 centrioles (blue), 4 (green) or >4 (orange).