Supplemental Material

This article contains the following supporting material:

• Figure 4 - Movie S1. Time-lapse images of an MRLC-GFP expressing HeLa cell loaded with PBS (Mock). Images were acquired at 2 min intervals and displayed at 10 frames per second. Green, MRLC-GFP.
• Figure 4 - Movie S2. Time-lapse images of an MRLC-GFP expressing HeLa cell loaded with C3 exoenzyme. Images were acquired at 2 min intervals and displayed at 10 frames per second. Green, MRLC-GFP.
• Figure 7 - Movie S3. Time-lapse images of an MRLC-GFP expressing HeLa cell transfected with siRNA to Luciferase. Images were acquired at 2 min intervals and displayed at 10 frames per second.
• Figure 7 - Movie S4. Time-lapse images of an MRLC-GFP expressing HeLa cell transfected with siRNA to ECT2. Images were acquired at 2 min intervals and displayed at 10 frames per second.
• Figure 7 - Movie S5. Time-lapse images of an MRLC-GFP expressing HeLa cell transfected with siRNA to MgcRacGAP. Images were acquired at 2 min intervals and displayed at 10 frames per second.
• Figure 7 - Movie S6. Time-lapse images of an MRLC-GFP expressing HeLa cell transfected with siRNA to MKLP1. Images were acquired at 2 min intervals and displayed at 10 frames per second.
• Figure 10 - Movie S7. HeLa cells treated with DMSO after washout of nocodazole. Images were acquired at 1 min intervals and displayed at 10 frames per second.
• Figure 10 - Movie S8. HeLa cells treated with Roscovitine after washout of nocodazole. Images were acquired at 1 min intervals and displayed at 10 frames per second.
• Figure 10 - Movie S9. HeLa cells were pretreated with Roscovitine for 15 min in the presence of nocodazole and nocodazole was washout. Then cells were cultured with Roscovitine. Images were acquired at 1 min intervals and displayed at 10 frames per second.
• Figure 10 - Movie S10. Time-lapse images Luciferase RNAi cells treated with Roscovitine. Images were acquired at 1 min intervals and displayed at 10 frames per second.
• Figure 10 - Movie S11. Time-lapse images ECT2 RNAi cells treated with Roscovitine. Images were acquired at 1 min intervals and displayed at 10 frames per second.
• Figure S1 - ECT2, MgcRacGAP, MKLP1, or Citron-kinase RNAi leads to multinucleate cells.

  HeLa cells were treated with indicated siRNAs for 6 d. (A) Giant cells with multinuclei. (B) Cells with multipolar spindles. Control cells at the same scale are shown at right upper corner of the panels of the ECT2 RNAi cells.

• Figure S2 - C3 exoenzyme inhibits cytokinesis in HeLa cells.

  (A and B) Binucleate cell formation by loading with C3 exoenzyme. Cells were fixed 24 h after loading with PBS (Mock) or C3. (A) Morphology. (B) Mean percentage of binucleate cells of total observed cells (250 cells were examined in each of 3 independent experiments). Error bars indicate standard deviation. (C) Flow cytometry profiles of cells 48 h after loading with PBS (Mock) or C3. Horizontal axis, DNA content; vertical axis, cell counts. Positions of the 2N, 4N, 8N, and 16N are indicated. (D) Giant cells generated by C3-loading. Cells were fixed with 4% formaldehyde 5 d after loading with C3 and stained with anti--tubulin antibody (Green) and DAPI for DNA (Red). (a) a multinucleate giant cell; (b) a cell with multipolar sindles. Control cells at the same scale are shown in the upper right corner.

• Figure S3 - RNAi induced failure of cytokinesis in ECT2, MgcRacGAP, or MKLP1-depleted cells.

  (A) Time-lapse images of HeLa cells stably expressing Histone H2B-GFP. Time in min from onset of anaphase (time point 0) is indicated. Bar, 10 μm. (a) normal cell division in cells transfected with siRNA to Luciferase. (b-d) abnormal cytokinesis observed in cells transfected with siRNA to ECT2. Cytokinesis phenotypes are classified into the following 4 classes: “Normal”, cells form the cleavage furrow. The furrow ingresses and the midbody is formed. Then, two daughter cells separate (a); “Regression”, cells form the cleavage furrow. The furrow ingresses and the midbody is formed but the daughter cells merge (b); “Incomplete Furrowing”, cells form the cleavage furrow. Ingression starts but does not complete. The daughter cells merge without forming the midbody (c); “No Furrowing”, no cleavage furrow formation (d). (B) Cytokinesis phenotypes observed in cells transfected with siRNA to Luciferase, ECT2, MgcRacGAP, or MKLP1. Time-lapse images were recorded for 48 h and exhibited phenotypes were classified into “Normal”, “Regression”, “Incomplete Furrowing”, and “No Furrowing” (see panel A) in each 12 h period.

• Figure S4 - The depletion level of ECT2, MgcRacGAP, and MKLP1 correlates to accumulation of myosin to the contractile ring.

  HeLa cells stably expressing MRLC-GFP (Green) were treated with siRNA to Luciferase (A and B), ECT2, MgcRacGAP, and MKLP1 (C-F) for 36 h. Examples of cells with various depletion levels of these proteins are shown. The cells were fixed with 100% methanol and stained with indicated antibodies (Red). Bar, 10 μm.

• Table S1