FIG. 9.

PIAS-1 expression is regulated during the cell cycle. (A) Western blot of Saos-2 cells grown in serum-free medium for 48 h (0 h) and then grown in medium containing 10% fetal calf serum (FCS) for 3, 6, and 24 h, showing that PIAS-1 is down-regulated in growth-arrested cells and is up-regulated in cells reentering the cell cycle. The same blot was reprobed with an antiactin antibody to show equal loading. IB, immunoblot. (B) Graph showing the distribution of cells in the different cell cycle phases evaluated by flow cytometry on an aliquot of the cells used for the Western blot in panel A. An increase of PIAS-1 expression parallels the increased number of cells in the S phase. (C) RT-PCR of RNA extracted from cells treated as described above, showing that there are no changes in PIAS-1 mRNA levels. (D) Hek293 cells untreated (lane 1) or blocked in G₂ by treatmentwith 0.2 μg of nocodazole per ml for 16 h (lane 2) and then released by culture in nocodazole-free medium and analyzed after 2, 4, and 8 h (lanes 3, 4, and 5, respectively). The same blot was reprobed with an antitubulin antibody to show equal loading. (E) Graph showing the distribution of cells in the different cell cycle phases evaluated by flow cytometry on an aliquot of the cells used for the Western blot in panel B. Nocodazole-treated cells are completely blocked in G₂ and show no expression of PIAS-1; again, an increase of PIAS-1 expression parallels the increased number of cells in the S phase. (F) RT-PCR of RNA extracted from cells treated as described above, showing that there are no changes in PIAS-1 mRNA levels.