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. 2009 Aug 11;4(8):e6603. doi: 10.1371/journal.pone.0006603

Figure 2. Laminar SS increases iNOS expression and NO production in the presence of Bleomycin.

Figure 2

HUVEC were treated for 16 h with Bleo (10 ug/ml) in static condition ST or exposed to SS. Control cells (open bars); Bleo treated cells (striped bars). a) The graph shows the average NO production, in cells cultured in control conditions or exposed to Bleo, SS or both in combination, determined by DAF incorporation and FACS analysis. Data are represented in arbitrary units and are the results of three independent experiments performed in duplicate. * = p<0.05; ** = p<0.01. b) The picture on the left shows a representative western blotting analysis (WB) depicting iNOS protein level in cells kept in static culture (ST) or exposed to laminar SS (SS) in the presence of Bleo (B). Densitometric analysis of three independent experiments expressed in arbitrary units is shown on the right. * = p<0.05. c) The graph on the left shows the effect of the NO donor DETA/NO (500 µM), administered to endothelial cells cultured in static conditions in the presence or absence of Bleo, on cell survival determined by propidium-iodide incorporation and FACS analysis. Data, expressed in arbitrary units, are the results of three independent experiments. Ns = not significant; *** = p<0.001. d) The graph shows the effect of the iNOS inhibitor GW274150 on endothelial cell survival determined by propidium iodide incorporation and FACS analysis. Data are represented in arbitrary units and are the average of three independent experiments performed in static culture (ST) or in the presence of laminar flow (SS). * = p<0.05. ** = p<0.01. # = p<0.005. e) The graph shows the effect of the iNOS inhibitor GW274150 on NO production determined by DAF incorporation and FACS analysis in endothelial cells culture in the presence of Bleo (B). Data are represented in arbitrary units and are the average of three independent experiments performed in static culture (ST) or in the presence of laminar flow (SS). *** = p<0.001. f) The picture shows a representative western blotting analysis (WB) performed by using an anti-nitrotyrosine antibody on total lysates obtained from cells cultured in static condition (ST) or exposed to laminar flow (SS) and/or Bleo (B). The Red Ponceau staining is shown below as loading control (Ponceau). The right panels indicates the average densitometric values expressed as arbitrary units. Data were generated by three independent experiments. * = p<0.05.