*4.7. Co-Culture Assays*

Cartilage explants (10 per well), were plated in a 12 well plate and co-cultured with synoviocytes in a transwell system (6.5 mm insert diameter, 3.0 μm polyester membrane, Corning Incorporated Life Sciences), in 1.8 mL of complete Adv DMEM, at 37 ◦C, in a humidified atmosphere containing 5% CO2.

To evaluate the effect of YP, co-cultures were supplemented with 10 μM of YP or 2 μM DXM for 24 h, followed by treatment with IL-1β (10 ng/mL) during 24 h, or HAP (750 μg/mL) during 72 h. Cartilage explants were collected as described above for RNA extraction, and cell media collected for ELISA analysis.

### *4.8. RNA Extraction, cDNA Amplification and Quantitative Real-Time PCR (qPCR)*

Cartilage tissue was immediately snap-frozen and manually grounded to powder in liquid nitrogen. Cells and tissue lysis was performed in a proportion of 1 mL of 4 M guanidine thiocyanate solution per 10<sup>7</sup> cells or 100 mg cartilage tissue, thoroughly mixed and passed 10 times through a 22G needle. Total RNA was further extracted as described by Chomczynski and Sacchi [55]. Briefly, homogenates were sequentially mixed with 2 M sodium citrate pH 4, phenol pH 4.2 and chloroform/isoamyl alcohol. After centrifugation, total RNA present in the aqueous phase was precipitated with isopropanol, redissolved in 4 M guanidine thiocyanate solution, reprecipitated in isopropanol, washed with 75% ethanol and resuspended in Sigma water. RNA concentration was determined by spectrophotometry at 260 nm (Nanodrop 1000, Thermo Scientific, Waltham, MA, USA). RNA was then treated with RQ1 RNase-free DNase (Promega, Madison, WI, USA) and reverse-transcribed using the qScipt cDNA SuperMix (Quanta bio, Beverly, MA, USA) according to manufacturer's recommendations. Quantitative real-time PCR reactions were performed using the CFX connect, Real time System (Bio-Rad, Richmond, CA, USA), SoFast Eva Green Supermix (Bio-Rad, Richmond, CA, USA), 300 nM of forward and reverse gene-specific primers for genes of interest (Table S2), and a 1:5 cDNA dilution. The following PCR conditions were used: initial denaturation/enzyme activation step at 95 ◦C for 13 min, 50 cycles of amplification (one cycle is 15 s at 95 ◦C and 30 s at 68 ◦C). Fluorescence was measured at the end of each extension cycle in the FAM-490 channel. Levels of gene expression were calculated using the comparative ΔΔCt method, and normalized using gene expression levels of glyceraldehyde-3-phosphate dehydrogenase (GAPDH), with the iQ5 software (BioRad).
