Perfection 1260 scanner

Analysis of protein composition was performed using the Proteome Profiler Human XL Cytokine Array Kit (R&D Systems), following the manufacturer’s instructions. In brief, 15 μg of proteins of CM were incubated with a cocktail of antibodies conjugated to the surface of a membrane overnight at 4°C. After several washing steps, the membrane was incubated with a cocktail of biotin-conjugated antibodies for 1 h at room temperature, then with HRP-conjugated streptavidin for 30 min at room temperature. After treatment of the membrane with substrate necessary for the HRP reaction (luminol and hydrogen peroxide) (LiteAblot^® TURBO, Euroclone), chemiluminescence was impressed on autoradiographic film (Fujifilm). Membranes were scanned using the Epson Perfection 1260 scanner, and analysis was performed using ImageJ to detect the pixel intensity. Four independent experiments were performed. Intensity values were normalized to the control spots of the membrane. Gene Ontology (GO) was performed on proteins with a normalized pixel intensity ≥0.3 using DAVID, focusing on the “Biological process (BP)” term. Graphical representations of cytokine array analysis and GO were carried out using R libraries: pheatmap, ggplot2, and circlize (Gu et al., 2014 (link)), setting the adjusted p-value < 0.05 as significant.

The release of cytokines and chemokines in CPCs- and ACs-CM was analysed using the Human XL Proteome Profiler™ Array (R&D Systems, Minneapolis, MN, USA) according to the user’s manual. Briefly, membranes spotted with antibodies were incubated with the same amounts (50 µg/mL) of each CM overnight at 4 °C. The following day, detection antibody cocktail was added for 1 h at RT, before visualization using ECL. Quantitative analysis was performed on scanned (Epson perfection 1260 scanner, Seiko Epson Corporation, Nagano, Japan) X-ray films (Fujifilm GmbH, Düsseldorf, Germany) using the Protein Array Analyser plugin available for ImageJ software (US National Institutes of Health, Bethesda, MD, USA). For each membrane, average spot signal density was determined by densitometry, followed by background subtraction and normalization to the reference spots. For a correspondence between a specific molecule and its position in the membrane, a reference to the manufacturer’s datasheet is provided (Catalog # ARY022B).

Confluent dedifferentiated chondrocytes were supplemented with 5% v-PL either in the presence or in the absence of bovine serum. At different times from the addition of the supplement, cells were washed with PBS and collected for western blot analysis. Electrophoresis was performed in reducing conditions using 25 μg of protein loaded on a 4–12% NuPAGE Bis-Tris gel (Invitrogen, Italy) as described (Ulivi et al., 2006 (link)). Primary antibodies tested were α-Cyclin D1 (1:250) and α-Actin (1:200) (Santa Cruz Biotechnology, USA), α-phospho Erk1/2 (1:1,000), α-phospho Akt (1:1,000) (Cell Signaling Technology, USA). Secondary HRP conjugated anti-mouse and anti-rabbit antibodies were both diluted 1:5,000 (GE Healthcare, UK), peroxidase conjugated anti goat antibody was diluted 1:10,000 (Jackson Immunoresearch, USA). ECL was purchased from GE Healthcare, UK.
Images were scanned using the Epson perfection 1260 scanner (Epson, Italy) and band densities were quantified using the ImageJ software (http://rsbweb.nih.gov/ij/download.html). Western blots were performed on three different independent primary cultures.