Rnase inhibitors

Polysomal profiling was complete as described (Tcherkezian et al., 2014 (link); Zahreddine et al., 2017 (link))). In brief, cells were treated with cyclohexamide (100 μg/ml, Sigma Aldrich cat# C7698) 10 minutes prior to harvesting. Lysates were prepared with 1mL of polysome lysis buffer (15 mM Tris pH 7.4, 250 mM NaCl, 15 mM MgCl2, 1% Triton X-100, 100 g/ml cyclohexamide, 1 mM DTT, 400 U/ml RNase inhibitors (Invitrogen cat# 10777019) and protease inhibitors (Sigma Aldrich cat # 111697498001). Equal amounts (5–10 mg) of protein lysates were layered on a 20%–50% linear sucrose gradient (20% and 50% sucrose solutions in 15 mM Tris pH 7.4, 15 mM MgCl2, 150 mM NaCl, 1 mM DTT, 100 μg/Ml cyclohexamide and 20 U/ml RNase inhibitors), mixed on Gradient Station IP Biocomp and centrifuged in a Beckman SW41Ti rotor at 92,000 g for 3 hr at 4°C. Polysome fractions were collected by continuously monitoring and recording the A254 on a Gradient Station IP (Biocomp) attached to a UV-MII (GE Healthcare, CA) spectrophotometer. RNAs were isolated from polysomal fractions using TRIzol reagent. RNA transcripts from each fraction (1–10) were examined via RTqPCR. Calculations were performed as outlined in Chassé et al. (2017) (link) and standard t test analyses were conducted to conclude statistical significance.