Supporting information for Hofseth et al. (2002) Proc. Natl. Acad. Sci. USA, 10.1073/pnas.0237083100
Fig. 5.
(a and b) Dose–response (a) and time-course (b) increase in DNA damage (assessed by Comet analysis) after exposure to either S-nitrosoglutathione (GSNO) or spermine NONOate (SPER/NO). (c) Dose–response increase ("D"’ represents the decomposed form of the donor) in p53 and serine 15 phosphorylation on p53 (P-Ser-15) after exposure to either GSNO or SPER/NO for 4 hr, as indicated. (d) DNA damage (CometAssay, Trevigen, Gaithersburg, MD) and time course increase in p53 posttranslational modifications after exposure to 0.5 mM GSNO. MCF-7 cells were cultured and exposed as described in Methods to 0.5 mM GSNO for the indicated times (hr). Equal amounts of protein were immunoprecipitated, and Western blot assays were performed as described in Methods. (e and f) Nitrate and nitrite, the stable end products of NO, accumulate quickly and to maximal levels of 208 μM or 215 μM in the media after exposure of MCF-7 cells to the NO donors GSNO or SPER/NO (e), respectively. Biochemical analysis of NO release by SPER/NO has been carried out in vitro and has shown to release a NO at a constant rate, with a half-life of 37 min (data not shown). (f) In coculture experiments with MCF-7 and ANA-1 mouse macrophages, nitrate plus nitrite accumulate in media to maximal levels of 42 μM over 6 hr only in conditions where ANA-1 macrophages are stimulated with cytokines.