Figure 5

Pitstop 2 activates the SAC. A, Metaphase synchronized HeLa cells were treated as described in Figure legend 2C then fixed and stained for phospho-BubR1^S676(green), α-tubulin (red) and DNA (DAPI, blue). Representative immunofluorescence microscopy images show that the amount of phospho-BubR1^S676 on the chromosomes increases following treatment with pitstop 2, MLN8237, and following depletion of CHC or epsin. B-C, The dot blots show the fluorescence intensity ratio of BubR1^S676 on the chromosomes/whole cell in individual HeLa cells treated with the indicated drugs (B) or siRNA (C). D-E, Metaphase synchronized HeLa cells were treated and processed as described in A except that they were stained for MAD2 instead of BubR1^S676. The dot blots show the fluorescence intensity ratio of MAD2 on the chromosomes/whole cell in individual HeLa cells treated with the indicated drugs (B) or siRNA (C). The median fluorescence intensity ratio in all dots blots shown is indicated by the solid black line. n≥30 per sample from two independent experiments. Statistical significance was determined by a Student’s t-test (* p < 0.05, ** p < 0.01).