Fig. 2

Long-term lipophilic cAMP preincubation decreased apoptotic parameters. RAW 264.7 macrophages were prestimulated for 15 hr with 1 mM 8-Br-cAMP, or 1 mM db-cAMP, with a combination of LPS (10 µg/ml), IFN-γ (100 U/ml), and NAME (1 mM) (LIN) prior to the addition of NO donors, or remained as controls. (A) DNA fragmentation was initiated with 1 mM GSNO or 0.25 mM Sp-NO and quantitated after 8 hr with the diphenylamine assay (*p ≤ 0.05 versus NO-treated controls). (B) p53 expression in response to 1 mM GSNO was determined after 4 hr by Western blot analysis. (C) Caspase-3-like activity was measured after 4 hr by the cleavage of Ac-DEVD-AMC. (D) Mitochondrial membrane potential (Δψ) was assessed after 8 h by the DiOC₆(3) method in response to 1 mM GSNO. Histograms show single-color labeling. Representative results are shown. For details and statistical evaluations see Materials and Methods.