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.
Amato et al. BMC Cell Biology 2009 10:79 doi:10.1186/1471-2121-10-79