Figure 3. Normal spindle morphologies and reduced CENP-E staining at the kinetochores of aligned chromosomes in PIASγ-depleted cells.

(A,G) Mitotic spindles (stained with anti-alpha-tubulin antibody) were indistinguishable in control and PIASγ-depleted metaphase cells, except that a small % at late time-points possessed extra poles (arrow in A): (A,G) Red = alpha-tubulin, Blue = DAPI.

(B–F,H–K) CENP-E staining (Red) is detected strongly on kinetochores of chromosomes away from the metaphase plate in both control-treated and PIASγ-depleted cells (B,H,I), but was much reduced in chromosomes at the plate (C,J,K).

After prolonged arrest in metaphase some of PIASγ-depleted cells had one or two chromosomes that left the plate and regained strong CENP-E staining at their kinetochores (D–F and see Fig. S1N,O ).

The same cell is shown in D (merged image), E (CENP-E straining) and F (DAPI straining).