Supporting information for Hofseth et al. (2002) Proc. Natl. Acad. Sci. USA, 10.1073/pnas.0237083100



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Fig. 7. (a)p53 downstream proteins are induced after exposure of MCF-7 cells to 0.5 mM GSNO or SPER/NO.MCF-7 cells were exposed to the indicated NO donor, then lysed. Thirty-microgram whole-cell lysate were loaded onto an SDS/PAGE gel and Western analysis performed as described in Methods. Both p21WAF1 and HDM-2 increased with time. p21WAF1 continued to increase up to 24- to 48-hr exposure; HDM-2 reached maximal levels between 4 and 8 hr. Bax levels did not change significantly. Numbers under the bands indicate densitometry values as a ratio relative to control values. (b)Exposure of MCF-7 cells to GSNO () or SPER/NO (▲) results in cell cycle changes. FACS analysis was performed as described (1).There was a progressive decrease in the percentage of cells in active S-phase. Data also indicate a progressive increase in the number of cells in G1 along with a transient increase in the number of cells in G2/M. (c)p53 is induced, and serine 15 is phosphorylated in both MCF-7 and HCT 116 cells. Both cell lines were exposed to the SPER/NO, then lysed. Thirty-microgram whole-cell lysate were loaded onto an SDS/PAGE gel and Western analysis performed as described in Methods. MCF-7 cells exposed to 0.5 mM SPER/NO for 4 hr resulted in equivalent amounts of P-Ser-15 modification, because HCT 116 cells exposed to 1 mM SPER/NO for 4 hr ALLN (20 μM) was used as a negative control for post-translational modifications. (d) There is a p53- and p21-independentG1 arrest and decrease in active S-phase cells exposed to NO. HCT 116 cells () , HCT 116 p53-/- (), or HCT 116 p21-/- (▲) cells were exposed to 1 mM SPER/NO for indicated time periods (hr). Cells were harvested at the same time, then FACS analysis was performed. "Inactive S-phase" cells are defined as cells that were in S-phase at the time of fixation, as evidenced by DNA content intermediate between G1 and G2/M cells, but were not actively engaged in DNA replication, as evidenced by the lack of incorporation of the thymidine analog BrdUrd during the labeling period just prior to fixation. Data are presented as a percentage relative to untreated cells. Gating was done according to supporting e. (e) Gating used to generate the graphs shown in Figs. 3b and 7 b and d.

1. Linke, S. P., Clarkin, K. C., Di Leonardo, A., Tsou, A. & Wahl, G. M. (1996) Genes Dev.10, 934–947.