FIG. 6.

Salt stress in G₁ phase activates p53 expression, inhibits Hbo1 HAT activity, and blocks chromatin loading of the MCM2-7 complex. (A) MCF7 cells were arrested at metaphase with nocodazole for 16 h (“Noc Arrest”). The synchronized cell cultures were then split into fresh medium without nocodazole and allowed to release from arrest. After 2 h of recovery, one set of cells was left untreated (“Released Untreated”), while the other was treated with 0.26 M NaCl (“Released +NaCl”). Cells were harvested from the cultures after 6.5 h, stained for DNA, and assayed for their cell cycle distributions by flow cytometry. (B) Effect of salt stress on the amounts of key proteins. Whole-cell extracts were prepared from untreated (lane 1) (“−”) and NaCl-shocked (lane 2) (“+”) cultures described above (A). The extracts were separated by PAGE and assayed by Western blotting with antibodies against p53, Hbo1, Mdm2, p21, and γ-tubulin. (C) Immunoprecipitates were prepared using anti-Hbo1 antibody and whole-cell extracts of untreated cells (lane 1) and NaCl-shocked cells (lane 2) as described above (A). Portions of the Hbo1 immunoprecipitates were then assayed by Western blotting for the presence of Hbo1 and p53 (“p53/Hbo1 in the complex”) and for HAT activity by [³H]acetate incorporation (“Fluorography”) into chicken core histones (“Stain”). (D) MCF7 cells, either untreated or NaCl shocked as described above (A), were fractionated into total cell extract (TCE), a soluble cytoplasmic fraction (S2), a soluble nuclear fraction (S3), and a chromatin-enriched fraction (P3) as described previously (43). Fractions were separated by PAGE and assayed by Western blotting with antibodies against p53, Mcm2, Mcm6, Hbo1, Cdt1, Orc2, and Mek2.