Silencing of NuMA activates the spindle assembly checkpoint. (A) Cells treated with NuMA siRNA1 and control cells were processed for immunofluorescence of centromeres with human CREST autoimmune serum (top, green), or with specific antibodies against the checkpoint proteins BubR1 (middle, green), and Mad2 (bottom, green). Antibody against BubR1 was provided by Dr Tim Yen (Fox Chase Center, Philadelphia, PA); antibody against Mad2 was from Berkeley Antibody Company. DNA was stained with DAPI (blue). Bar, 10 μm. (B) The percentage of mitotic cells in different phases is indicated for controls (blue) and cultures treated with NuMA siRNA1 for 72 hours (red). Standard deviations were calculated from n = 706 mitotic control cells, and from n = 601 depleted mitotic cells (data obtained in three independent experiments). The percentages of those prometaphases that show unaligned chromosomes besides mostly aligned chromosomes (as seen in A) are indicated separately on the left. (C) Growth curve of HeLa cells subjected to control treatment or NuMA siRNA1.