Polysome Fractionation

Polysome fractionation was performed as described previously (Panda et al., 2017). Three 15-cm plates with ATDC5 cells were used to generate a single sample. At the day of sample collection, cells were differentiated for 2 h, then pre-treated for 5 min with 100 μg/ml Cycloheximide (Sigma), washed twice in 0.9% NaCl with Cycloheximide, and collected by scraping with a rubber policeman in cold 0.9% NaCl. Pelleted cells were lysed for 10 min in 1.8 ml of polysome extraction buffer [20 mM Tris–HCl (pH 7.5), 100 mM KCl, 5 mM MgCl₂, 0.5% Non-idet P-40, 100 μg/ml Cycloheximide, complete protease inhibitor cocktail (Roche), and RNasin (Promega, 40 U/ml)] on ice. Nuclei and cellular debris were removed by centrifugation at 12,000 × g for 10 min at 4°C and 9/10th of the total volume was transferred to fresh tubes and measured spectrophotometrically. Total yield was the same for siCtrl- and siSox9-treated cells. Sucrose gradients (linear 10–50%) were made using the Gradient Master (BioComp) in ultracentrifuge tubes (Seton, SW41 tubes). Cytoplasmic extracts (250 μg/sample) were loaded to each gradient in a fixed volume (400 μl). Gradients were run on an ultra-centrifuge (Beckman L60, Brea, CA, United States) at 39,000 rpm for 1.5 h at 4°C with max acceleration and deceleration 9. Samples were fractionated into 24 × 0.5-ml fractions using a Piston Gradient fractionator (BioComp, Fredericton, Canada) and fraction collector (Gilson FC203B, Middleton, WI, United States) with continuous A260 monitoring (Triax FC-1).