pRb depletion causes centrosome amplification, aneuploidy and altered gene expression. A) RT-PCR analysis for RB expression in IMR90 cells transfected with siRNA-luciferase (siLuc), siRNA targeting RB (siRB) and after RB re-expression (release). B) The histogram (top panel) summarizes the percentage of cells with 1, 2 or >2 centrosomes in control and pRb-depleted IMR90 cells. Presence of supernumerary centrosomes in interphase cells (bottom panel: b) and abnormal mitotic spindles (bottom panel: c, arrowheads) in IMR90 cells transfected with siRNA targeting RB in comparison with control cells (bottom panel: a) as revealed by immunofluorescence for γ-tubulin (green) or β-tubulin (green) respectively, nuclei were stained with DAPI (blue). C) Bar graphs showing the percentage of cells with normal (46 chromosomes), hypodiploid (<46 chromosomes) and hyperdiploid (>46 chromosomes) metaphases after RB silencing and in control cells, untransfected or transfected with siRNA-luciferase (siLuc). D) Expression levels of genes involved in mitosis progression by Real time RT-PCR. The x-axis indicates genes and the y-axis the relative quantification in IMR90 cells untransfected (wt), transfected with siRNA-luciferase (siLuc) and siRNA targeting RB (siRB). E) Western blot analysis in pRb silenced IMR90 cells showing lack of pRb, increase of Aurora-A, Plk1 and decrease of Mad2 protein at 72 hours. β-actin is used a loading control.
Amato et al. BMC Cell Biology 2009 10:79 doi:10.1186/1471-2121-10-79