Sub-confluent A549 were seeded in 6 cm diameter Petri dishes. After adherence, cells were treated for the indicated times with Ni(SalPipNONO) (0.5 mM) and specific pathway inhibitors. Protein extraction and Western blot were performed as previously described [58 (link), 61 (link)]. Electrophoresis (50 μg of protein/sample) was carried out in 4–12% Bis-Tris Gels (Life Technologies, Carlsbad, CA, USA). Proteins were then blotted onto nitrocellulose membranes, incubated overnight with primary antibodies [Anti-cytochrome c, anti-phospho-ERK1/2, anti-caspase- 3, anti-ERK antibodies from Cell Signaling (Celbio, Milan, Italy); anti-p53 antibody from Santa Cruz Biotechnology Inc (Dallas TX, USA); anti-COX-2 from Cayman Chemical (Ann Arbor, MI, USA); anti-HIF-1α from BD Biosciences (San Jose, CA, USA); anti-VEGF from Merck KGaA (Darmstadt, Germany)] and then detected by enhanced chemiluminescence system (Biorad, Hercules, CA, USA). Results were normalized to those obtained by using an antibody against beta actin from Merck KGaA (Darmstadt, Germany) or total ERK1/2 (Cell Signaling, Celbio, Milan, Italy), when indicated.
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