Fig. 4. Depletion of cellular UBPY and AMSH causes cytokinesis defects. (A) Immunoblot analysis of the lysates of HeLa cells transfected with the mock, two UBPY siRNA or two AMSH siRNA expression vectors with anti-UBPY (top), anti-AMSH (middle) and anti-tubulin (bottom) antibodies. (B) HeLa cells were transfected with the mock, UBPY siRNA-1 or AMSH siRNA-1 expression vector, and double-stained with anti-tubulin antibody (red) and SYTOX green (green). Scale bars: 50 µm. (C) Percentage of multi-nucleated cells in randomly chosen 400500 HeLa cells transfected with the mock, two UBPY siRNA or two AMSH siRNA expression vectors. Mean±s.d. of five independent experiments is shown (^*P<0.01, t-test). (D) HeLa cells transfected with AMSH siRNA-1 were double-stained with anti-tubulin antibody (red) and FK2 (green) at stage 4 of cytokinesis. Double-headed arrow indicates an atypically long central spindle between two daughter cells (asterisks). Arrowhead indicates the midbody ring. Inset shows a high-magnification image of the midbody region. Scale bar: 10 µm. (E) HeLa cells were transfected with the mock, two UBPY siRNA or two AMSH siRNA expression vectors, and stained with anti-tubulin antibody. Cells with the long central spindle (>20 µm; e.g. arrow in D) were counted in a 15 mm diameter cover glass, and the numbers are shown as mean±s.d. of three independent experiments (^*P<0.01, t-test).