Supplemental Material

This article contains the following supporting material:

• Figure 1 - RNAi for hRad21 and NIPBL efficiently depleted the target proteins and suppressed the increase of cell number.
  Cells transfected with buffer or with siRNA for hRad21 or NIPBL were collected at 24 hr intervals for immunoblot (A and B) and counting the cell number (C). A-B. Immunoblot of hRad21 and NIPBL in HeLa cell extracts. Cdc2, the loading control, was detected by PSTAIRE antibodies. In control extracts, the hRad21 and NIPBL levels remained constant. The first lane (0 day) in A and B was copied and inserted next to the RNAi lanes for purpose of comparison. A. RNAi for hRad21 was effective enough to deplete hRad21 to the undetectable level after 24 hr. B. RNAi for NIPBL was also effective to deplete it to the undetectable level after 48 hr. C. A graph for the cell number increase. The rates of cell number increase were strongly (1/15) and significantly (1/4) repressed in the cultures of hRad21 and NIPBL RNAi, respectively.

• Figure 3 - Localization of MCAK and CENP-E is diminished in hRad21 knockdown cells
  HeLa cells transfected with buffer or siRNA for hRad21 were fixed with paraformaldehyde after 48 hr and immunostained for CENP-A (red), MCAK (green in A), CENP-E (green in B) and DNA (blue). The bars, 10 μm. A. Immunolocalization of MCAK. Kinetochore localization of MCAK was visible in hRad21 RNAi cells showing EMCs (lower panels), although the intensity was much weaker than that of control (upper panels). B. Immunolocalization of CENP-E. In mitotic control cells, CENP-E brightly localized to the prometaphase kinetochores (top row), while weak kinetochore staining was observed in metaphase (second row). In contrast, CENP-E staining was greatly diminished in hRad21 knockdown cells (bottom two rows).

• Figure 2A - Movies of HeLa cells after RNAi of NIPBL and hRad21
  A-C. HeLa cells stably expressing histone H2B-GFP were transfected with buffer (A) or with siRNA for hRad21 (B) or NIPBL (C) and observed under a DeltaVision microscope after 24, 104 and 120 hr, respectively. The starting time was set to zero. A second in movies A, B and C is equal to 4, 4 and 8 min, respectively.
• Figure 2B
• Figure 2C -

• Figure 4A - Movies of HeLa cells stably expressing GFP-hMis12
  HeLa cells stably expressing GFP-hMis12 were transfected with buffer (A) or with siRNA for hRad21 and/or Mad2 (B-E). The starting time was set to zero. A second in each movie is equal to 10 min. Green, GFP-hMis12; Blue, DNA stained with Hoechst33342. A. Movie of non-RNAi control cell after 24 hr. B and C. After 48 hr, hRad21-knockdown cells showed severe mitotic delay with a fragile metaphase plate (B) or with EMCs (C). D. Movie of Mad2-knockdown cell after 24 hr. Chromosomes were prematurely missegregated. Lagging chromosomes were visible. E. Movie of hRad21 Mad2 RNAi cell after 48 hr. Chromosomes were prematurely segregated in the similar kinetics to Mad2 RNAi.
• Figure 4B
• Figure 4C
• Figure 4D
• Figure 4E -

• Figure 5A - Movies of GFP-hMis12-expressing HeLa cells in which hRad21 and/or topo II were either knocked down or inhibited
  Topo II and/or hRad21 were functionally disrupted by RNAi or ICRF-193 in HeLa cells stably expressing GFP-hMis12. One second of each movie is equal to 10 min. Green, GFP-hMis12; Blue, DNA stained with Hoechst 33342. A. Movie of topo II αβ-knockdown cell after 48 hr. Chromosomes were aligned and kinetochores were segregated after a considerable delay, although chromosome mass stained by Hoechst did not separate but cytokinesis took place. B. Movie of hRad21, topo IIαβ-knockdown cell after 48 hr. The phenotype was similar to that of topo II RNAi. Paired kinetochores were visible out of metaphase plate during the delay. C. Movie of topo IIαβ Mad2-knockdown cells after 48 hr. Kinetochores were prematurely segregated to some extent, but chromosome mass failed to separate, producing the ‘cut’ phenotype. D. Movie of quadruple hRad21 topo IIαβ Mad2-knockdown cell after 48 hr. The phenotype was similar to that of topo II Mad2 RNAi. E. Movie of a buffer transfected cell treated with ICRF-193 after 24 hr. The cell was delayed in a metaphase-like state. F. Movie of hRad21 RNAi cell treated with ICRF-193 after 48 hr. Sister centromeres showed some back-and-forth movement during the delay, which was compromised with the fast cytokinesis. G. Movie of Mad2 RNAi cell treated with ICRF-193 after 48 hr. H. Movie of hRad21 Mad2 RNAi cell treated with ICRF-193 after 48 hr. In G and H, mitosis was quickly exited without separation of the whole chromosome.
• Figure 5B
• Figure 5C
• Figure 5D
• Figure 5E
• Figure 5F
• Figure 5G
• Figure 5H