Figure 3.

CENP-H/Chl4R^CENP−N and Nnf1R have separate roles in chromosome congression. (A) Successive frames every 3 min from live-cell movies of H2B-GFP-expressing HeLa cells transfected with control, siCENP-H, siChl4R or siChl4R+siCENP-H, siNnf1R-3 or siCENP-H+siNnf1R-3 RNA. NBD was set as T=0 min. Scale bar=10 μm. (B, D) Immunoblots showing protein levels of CENP-H, Chl4R or Nnf1R after treatment with siRNA as indicated. (C, E) Protein levels of CENP-H, Chl4R and Nnf1R as measured by quantitative immunofluorescence as in Figure 2D–H (see Supplementary Figure S4 for representative images). (F) Percentage of cells, treated as described in panel A, with uncongressed chromosomes at T=24 min. (G) Percentage of cells with one (dark blue), two (blue), three (red) or more than three (dark red) uncongressed chromosomes following 30-min treatment with MG132 as determined from images such as shown in (I) Error bars indicate the s.d. for the total number percentage of cells with uncongressed chromosomes. (H) Chromosomes in metaphase cells were counted as unaligned if they were located outside of the central 30% of the mitotic spindle, or if their kinetochores were aligned perpendicular to the spindle axis. (I) Representative images of cells treated for 30 min with MG132 and stained with DAPI (DNA; blue), anti-α-tubulin (green) and CENP-E antisera (red). Scale bar=10 μm, scale bar in zoom=1μm.