Polysome Profiling of Insulin-Deprived H1 Cells

Polysome profiling was performed as previously described with some modifications 23 (link). Briefly, H1 cells at 50% confluency were subjected to insulin removal or other treatments as specified. Prior to harvesting, 100 μg/ml cycloheximide (Sigma-Aldrich, St. Louis, MO, USA) were added to cells and incubated for 10 min. Wash cells with ice-cold 1 × PBS + 100 μg/ml cycloheximide, scrap off, and centrifuge to get cell pellet. Each sample was lysed on ice for 30 min in 500 μl polysome lysis buffer containing 12.5 mM Tris pH 7, 7.5 mM Tris pH 8, 15 mM MgCl₂, 200 mM NaCl, 1 mM DTT, 100 μg/ml cycloheximide, 1% TritonX-100, and 20 U/ml Superase Inhibitor (Ambion, #AM2696). Cell lysates were centrifuged at 13,000 rpm, 4ºC for 10 min. 10 to 60% RNase-free sucrose gradients (15 mM Tris pH 8, 100 mM KCl, 3 mM MgCl₂, 1 mM DTT, 100 μg/ml cycloheximide and 20 U/ml Superase Inhibitor (Ambion, #AM2696)) were prepared by a BioComp Gradient maker (BioComp Instruments, Fredericton, NB, Canada) according to the user guide. 500 µl cell lysate was put on top of the sucrose gradient and centrifuged at 41,000 rpm, 4°C for 4 h. A BioComp Gradient Station with fractionator and optical monitor at a 260 nm wavelength was used to get the polysome profiles.