( s MCM2-MCM7 (14) (Fig 5 row 7 columns ...)siRNA screening of the cell cycle with two dynamic GFP sensors (from Discovery Matters Issue 1, July 2005)

Similarly, analysis across all siRNA pools (Fig 6) showed a diverse range of cell cycle distributions following gene knockdown. For example, treatment of cells with cyclin A2 siRNA (CCNA2) resulted in a significant accumulation of cells in prophase and mitosis to a similar degree to that observed for cyclin B1 (CCNB1), corresponding with the requirement for cyclin A for G1/S and G2/M transitions. In confirmation of the specificity of siRNA knockdown, cell cycle perturbation was not observed for the germ line functional homologue cyclin A1 (CCNA1), which is not expressed in differentiated U2OS cells. These changes correlated with additional changes in cellular morphology described below.

Changes in cell cycle distribution around the G1-S boundary following knockdown of cyclin E were not resolvable in G2/M CCPM cells because the cyclin B1 fusion protein is not expressed during this part of the cell cycle. Analysis of cyclin E siRNA treated cells showed 76% of cells in G1 or S phases compared with 74% in control siRNA treated cells. Analysis of G1/S CCPM cells treated under the same conditions (Fig 7) allowed the effects of cyclin E knockdown on G1 to S phase transition to be quantitated. Control G1/S CCPM cells showed the same proportion (76%) of cells in G1 or S phase as control G2/M CCPM cells (74%), which was resolvable in G1/S CCPM cells to 9% G1 cells and 67% S phase cells. On knockdown of cyclin E the proportion of cells in G1 or S phase remained constant at 76%, as observed with G2/M CCPM cells. However the balance between the two phases shifted significantly to 27% G1 cells and 49% S phase cells, reflecting the critical role of cyclin E in G1 to S transition.

Knockdown of Polo-like kinase (PLK) with siRNA has been previously shown
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