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Figure 3


Fig. 3. NO survival signals are independent of the cGMP-PKG pathway and require dynamin. (A) BAEC transfected with AdGFP or AdK44A were incubated with SNP or TNF-{alpha} or both. Cells were collected 24 hours later and prepared for western blot analysis of cleaved caspase-3. Cells transfected with AdK44A showed enhanced TNF-{alpha}-induced BAEC apoptosis as demonstrated by a significant increase in the amount of cleaved caspase-3 from cell lysates. In the presence of K44A overexpression, SNP-dependent protection from TNF-{alpha}-induced apoptosis was attenuated [representative blot (left panel) and densitometric analysis (right panel); n=5]. Blots were reprobed with anti-V5 antibody or anti β-actin antibody for verification of the expression of V5-K44A fusion protein and for protein loading control, respectively. (B) Tube formation in response to VEGF was assessed by the tube formation assay on Matrigel in EC transfected with AdGFP or AdK44A. Twenty-four hours after transduction, 30,000 cells were seeded in Matrigel-coated wells and the length of endothelial tubes after 12 hours was measured. In GFP-transfected EC, VEGF significantly promoted tube formation (*P<0.05 versus respective basal medium). By contrast, K44A-transfected BAEC failed to form tubes (n=3). Representative phase-contrast images of experimental conditions are shown in the upper panels and compiled data are shown in the graph (lower panel). (C) Left, BAEC were treated with TNF-{alpha} and co-incubated with SNP, 8-Br-cGMP or vehicle for 24 hours. Apoptosis was assessed by Hoechst 33342 stain, and TNF-induced apoptosis was assigned a relative value of 100%. SNP reduced TNF-{alpha}-induced apoptosis whereas 8-Br-cGMP did not (*P<0.05; TNF-{alpha}+SNP versus TNF-{alpha} alone; n=3). Right, BAEC were transfected with adenoviruses encoding GFP (AdGFP) or flag-tagged wild-type PKG (AdPKG), and treated with TNF-{alpha} alone or in combination with 8-Br-cGMP. Cell lysates were prepared 24 hours later for western blot analysis. Neither overexpression of PKG nor combination of PKG with the agonist 8-Br-cGMP protected cells from TNF-{alpha}-induced apoptosis. PKG expression was verified by the presence of Flag epitope.