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Figure S1. Use of shRNA interference and live cell imaging to identify candidate proteins. (A) The plasmid pU6YH was generated by integrating the ApaL1–AflII fragment from pH2B-YFP (Platani, M., I. Goldberg, A.I. Lamond, and J.R. Swedlow. 2002. Nat. Cell Biol. 4:502–508) containing the histone H2B-YFP expression construct into pBU6 (Girdwood, D., D. Bumpass, O.A. Vaughan, A. Thain, L.A. Anderson, A.W. Snowden, E. Garcia-Wilson, N.D. Perkins, and R.T. Hay. 2003. Mol. Cell. 11:1043–1054), an shRNA-expressing construct (a gift from N. Perkins, University of Dundee, Dundee, Scotland, UK). Sense and antisense pSilencer 2.0–compatible oligonucleotides were designed for each target protein, annealed, and ligated into the HindIII and BglII sites upstream of the U6 promoter of pU6YH. (B) Proteins screened using pU6YH. (C) Sequences inserted into the hairpin to target each particular protein. (D–G) Selected maximum intensity projections from time-lapse images of HeLa cells showing gene-specific mitotic phenotypes. Cells were transfected with pU6YH plasmid expressing shRNA targeting PSP1 (D), ABCF (E), FLJ13263 (F), or Npl4 (G). Images were taken 60 h after transfection over a period of 60–120 min. Numbers indicate time (minutes) from the establishment of a metaphase plate. (H) The percentage of cells showing anaphase segregation defects (mean of two independent experiments) was recorded. The number (n) of cells imaged for each protein is indicated. Bar, 10 μm.