Effects of simultaneous RB/AURORA-A and RB/PLK1 silencing in IMR90 cells. A) Gene expression levels of RB, AURORA-A and PLK1 (top panel) evaluated by Real Time RT-PCR in silenced IMR90 cells as indicated. Western blots detecting protein levels for RB, Aurora-A (right bottom panel) and for RB, Plk1 (left bottom panel) in silenced IMR90 cells as indicated; β-tubulin was used as a loading control.B) Immunofluorescence microscopy detecting centrosome numbers (top panel) in untreated and double knockdown IMR90 cells. The graph in the middle panel summarizes the percentage of IMR90 cells with 1, 2 or more than 2 centrosomes after RNAi of RB, RB/AURORA-A and RB/PLK1. In the bottom panel are shown histograms summarizing hypodiploid (<46 chromosomes), diploid (46 chromosomes) and hyperdiploid (>46 chromosomes) metaphases. Cytogenetics analysis was carried out after three days following transfection except in RB/AURORA-A transcriptionally silenced fibroblasts which were examined after four days. C) Examples of metaphase spreads fro the indicated cell types. Numbers refer to chromosomes.