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. 2006 Dec 20;1(1):e53. doi: 10.1371/journal.pone.0000053

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Diaz-Martinez et al.

This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.

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Synchronous time-course experiment after release from early S-phase arrest in PIASγ-depleted HeLa cells (RNA-interference).

(A,B) Protocol for depletion of PIASγ and Western blot analysis (mit = mitotic shake-off; int = remaining interphase cells; Tub = alpha-tubulin). (C) Cyclin B and Phospho-H3 transiently peaked as control-treated cells passed through mitosis, while they accumulated in PIASγ-B treated cells (these blots are from mit + int cells).

Apc2 and Smc3 are loading controls. (D–G) Most PIASγ-depleted cells appeared to arrest in metaphase until the end of the experiment (∼12 hours after reaching mitosis).

Accordingly, chromosomes became progressively over-condensed (from D to G); arrow indicates the largest metacentric chromosome in each spread.

Unlike cells arrested in c-mitosis with nocodazole, PIASγ-depleted cells did not open chromosome arms. Proper chromosome alignment is evident in the side views of metaphase plates (D,F) and in the polar views (E,G).

In some cells one or two chromosomes lay off the metaphase plate (H); since these cells usually displayed overcondensed chromosomes and because this stage followed formation of a complete metaphase plate (see time-lapse material), we describe these as de-congressed metaphases.

For a detailed cytological comparison of PIASγ and control-treated cells, see Figure S1.

(I–K) Cells progressively accumulated in metaphase (I) after PIASγ depletion (1000 cells scored per time-point).

Shaded areas in I,J show control mitotic wave. (J) Anaphase/telophase index – Most PIASγ-depleted cells failed to initiate anaphase. (K) Accumulation in c-mitosis in the presence of nocodazole after control treatment or PIASγ-depletion and cell cycle synchrony in early S phase.

Some PIASγ-depleted cells failed to reach mitosis as indicated by the accumulation of only ∼50% c-mitotics after 16 hours compared with over 90% in control-treated samples, indicating that PIASγ might also have roles in interphase (see Fig. S2).