Fig. 5. Survivin depletion decreases BubR1 abundance at kinetochores of maloriented chromosomes. (A,B) Immunofluorescence images of control (A) and Survivin-depleted cells 60 hours post-transfection (B). Cells are stained for BubR1 (red), Survivin (green) and DNA (blue). Panels A'-B' show the BubR1 status of the inset regions indicated in a thermal scale where blue indicates a low signal intensity, green intermediate intensity and yellow-red high. Panels A"-B" show enlargements of the lagging chromosomes shown in the insets stained with DAPI and ACA. Unattached chromosomes of control cells have a high BubR1 signal (A'), while both high (B', upper chromosome) and low signal intensities (B", lower chromosome) are found on unattached chromosomes of a Survivin-depleted cell. (C) Chromosome spreads of HeLa cells following transfection with control or Survivin siRNA. Cells were treated for 2 hours with 0.1 µg/ml colcemid to depolymerize microtubules. In all panels Survivin is shown in green and DNA in blue. Top panels show BubR1 in red and lower panels show centromeres in red. All kinetochores are potentially capable of expressing high levels of BubR1 following Survivin depression. (D) Control and Survivin-depleted cells were treated with 0.1 µg/ml colcemid for 12 hours from 60 hours post transfection. A Survivin-positive and a Survivin-depleted cell are shown. BubR1 (red, D"), Survivin (greyscale, D'), centromeres (green) and DNA (blue). Survivin-depleted cell shows no BubR1 staining at the kinetochores. Bars, 5 µm.