Semiconfluent A374 cells were treated with 250 μM, 375 μM, and 500 μM of hydroxytyrosol and HT-144 cells with 250 μM, 350 μM, and 450 μM of hydroxytyrosol for 24 h and 48 h; therefore, the cells were lysed in RIPA buffer (50 mM Tris-HCl pH 8, 150 mM NaCl, 1% NP40, 0.5% DOC, 0.1% SDS) containing proteases and phosphatase inhibitors (4 mM PMSF and protease inhibitors cocktail, phosphatase inhibitors cocktail 2 and 3, Sigma). The protein concentration of cellular lysates was determined by Bradford protein assay (Bio-Rad laboratories GmbH, München, Germany). Furthermore, 20 μg or 40 μg of total cell extracts were resolved on sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and absorbed to nitrocellulose membrane (Hybond ECL, GE Healthcare, Biosciences, Pittsburgh, PA, USA) to perform western blot experiments as previously reported [49 (link)]. The membranes were processed and the protein bands were scanned and quantified by densitometric analysis, using Odyssey infrared imaging system (LI-COR Biosciences, Lincoln, NE, USA). The results of quantitative analysis are reported in the histograms as mean values of several western blot experiments and are expressed as a percent of unstimulated cells, normalized for actin amount.
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