Figure 2.

Cyclin A depletion reduces Cdh1 transcript levels. (A) Cells were transfected and synchronised as in A. At 4 h post release cells were treated with or without 20 μM of MG132, Plk1 inhibitor (Plk1i) or Cdk2 inhibitor (Cdk2i) for 3 h. All samples were then collected 7 h post release. Samples were lysed and immunoblotted for the indicated proteins. This is representative of duplicate experiments. (B) HeLa cells were transfected and synchronised as in A, although using only nonsense (NS) and Cyclin A siRNA#1. At 7 h post release when cells were in G2 phase, 10 μg/ml cyclohexamide was added, and washout out 3 h later. Cells were sampled at the indicated times after cyclohexamide addition and immunoblotted for Cdh1, and PCNA as a loading control. (C) Asynchronously growing HeLa cells were transfected with a nonsense control (NS), Cyclin A siRNA #1 or #3, or Cdh1 siRNA. Cells were synchronised with thymidine arrest overnight and released, harvested at 7 h post release when they were in G2 phase and their Cdh1 mRNA content assessed by qRT-PCR. The data are the mean and SEM of four independent experiments and expressed relative to the nonsense control. The level of Cdh1 was quantitated relative to the nonsense control.