Proteoglycan Synthesis

³⁵S-sulfate incorporation was performed, as previously described, to measure PG synthesis²¹. Briefly ³⁵S-sulfate (10 μCi/mL, American Radiolabeled Chemical ARS-105) was added to cell culture media in duplicate wells per condition in a 24 well plate format. Cells were radiolabeled for two days and extracted by addition of homogenization buffer containing 200 mM sodium chloride, 50 mM sodium acetate, 0.1% Triton X-100 (Sigma-Aldrich X-100), 10 mM EDTA, 50 μM DTT (Sigma-Aldrich D9779), and 1x Protease Inhibitor (Sigma-Aldrich P8340) and shaking at 4°C for 1 hr in a separate 1.5 mL microcentrifuge tube. Proteoglycans in cell lysate were extracted with shaking in a guanidine hydrochloride solution (8 M guanidine hydrochloride (Sigma-Aldrich G3272) containing 50 mM sodium acetate, 10 mM EDTA, and 1x protease inhibitor at 4°C for 4 hours²². Extracted samples were mixed with alcian blue solution containing 0.02% alcian blue (Sigma-Aldrich A9186), 50 mM sodium acetate, and 85 mM magnesium chloride for an hour at room temperature then loaded onto nitrocellulose membranes (Millipore HAWP 025 00). The membranes were washed with a buffer containing 100 mM sodium acetate (Sigma-Aldrich S2889), 50 mM magnesium chloride, and 50 mM sodium sulfate (Sigma-Aldrich 239313) to eliminate unincorporated ³⁵S-sulfate. The membranes were dissolved in scintillation fluid (National Diagnostics LS-201) and counted in a scintillation counter (Packard Tri-Carb 2100TR). Counts per minute (CPM) were converted to number of pmoles of sulfate, using the specific activity of ³⁵S-sulfate measured in the conditioned media, and then normalized to the amount of DNA per sample as determined by Picogreen assay (Life Technologies P7589).