*4.10. Histological Evaluation*

Paraffin-embedded cartilage tissue sections were processed at the Histopathology Department of Centro Hospitalar e Universitário do Algarve (CHUA, Faro) and used for histological assessment. Cartilage grading of initial tissue samples was conducted based on modified criteria originally established by Mankin et al., and the specimens were analyzed for abnormalities in structure, cellularity and matrix staining, based on hematoxylin-eosin (HE, Bio-Optica, Milano, Italy), safranin-O (SO)/Fast Green (Sigma-Aldrich, Steinheim, Germany) and toluidine blue (Merck, Darmstadt, Germany) stainings [56,57]. Four tissue sections from each sample were analyzed.

### *4.11. Protein Extraction and Quantification*

Total protein from chondrocyte inflammatory assays and YP treatments was obtained by extraction with RIPA buffer (50 mM Tris HCl pH 8, 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS) for 1 h, at 4 ◦C, with agitation, followed by a centrifugation at 16× *g* for 15 min at 4 ◦C. Protein concentration was assessed using Micro BCA kit (Thermo Scientific), according to the manufacturer's instructions.

### *4.12. Electrophoresis and Western Blot*

Aliquots of 20 μg of total protein extracts were size separated in a 4–12% (*w*/*v*) gradient polyacrylamide precast gel containing 0.1% (*w*/*v*) SDS (NuPage, Invitrogen, Carlsbad, CA, USA) and transferred onto a nitrocellulose membrane (Biorad, Richmond, CA, USA). Detection of pIKBα and GAPDH was performed through overnight (O/N) incubation with the pIKBα pSer32 ABfinity Rabbit Monoclonal antibody (2.5 μg/μL, Thermo Fisher, Waltham, MA, USA) and anti-GAPDH polyclonal antibody (1:500, Santa Cruz Biotechnology). Detection was achieved using Goat anti-rabbit IgG horseradish peroxidase-conjugated secondary antibody andWestern Lightning Plus-ECL (PerkinElmer Inc., Waltham, MA, USA). Image acquisition was obtained using an IQ LAS 4000 mini biomolecular imager.

### *4.13. Determination of Total and Phosphorylated IkB*α

Total IkBα and phosphorylated IkBα (pIkBα) were determined in chondrocyte cell lysates, using the InstantOne ELISA assay kit (Invitrogen) according to the manufacturer's protocol.
