Cycloheximide (chx)

RA was added to SH-SY5Y cells 3 d prior to harvesting. Cells were treated with cycloheximide (Sigma) at 100 µg/mL for 3 min at 37°C, washed (1× PBS, 100 µg/mL cycloheximide) and trypsinized for 5 min at 37°C. Subsequently, cells were pelleted, washed (1× PBS, 100 µg/mL cycloheximide), and resuspended in ice cold lysis buffer (Supplemental Methods); 50 mM Tris-HCl pH 8, 150 mM NaCl, 10 mM MgCl₂, 1 mM DTT, 1% IGEPAL, 100 µg/mL cycloheximide, Turbo DNase 24 U/µL (Invitrogen), RNasin Plus RNase Inhibitor 90U (Promega), cOmplete Protease Inhibitor (Roche), for 45 min. Cells were then subjected to centrifugation at 17,000g for 5 min, to pellet nuclei. Cytoplasmic lysates were loaded onto 18%–60% sucrose gradients (∼70 × 10⁶ cells per gradient) at 4°C and subjected to ultracentrifugation (121,355 × g_avg 3.5 h, 4°C) in SW-40 rotor. Gradients were fractionated using Gradient Station (Biocomp) and absorbance at 254 nm was monitored using a Bio-Rad detector.

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.

Polysome profiles were prepared from four independent biological samples of control cells and siRNAG5 cells as described [21 (link)]. Briefly, HEK293 cells (~ 2 × 10⁷ cells per gradient) were incubated with 0.1 mg/ml cycloheximide (Merck) for 10 min on ice to block ribosomes in the elongation step. Then, cells were washed twice with 0.1 mg/ml cycloheximide in cold PBS, lysed in buffer A (15 mM Tris–HCl pH 7.4, 80 mM KCl, 5 mM MgCl₂, 0.1 mg/ml cycloheximide), supplemented with 1% (v/v) Triton X-100, 40 U/ml RNase OUT (Thermo Fisher Scientific), and protease inhibitors (Complete mini, Roche), and centrifuged 10 min at 14,000g, 4 °C. The clear supernatants were loaded into a linear 10–50% (w/v) sucrose gradient in buffer A, centrifuged at 39,000 rpm SW40 Ti rotor 2 h 15 min at 4 °C. Sucrose gradients were fractionated using a gradient fractionation system (ISCO UA-5 UV monitor) measuring the absorbance at A260 to record the polysome profile. Twelve fractions (1 ml) were collected from each gradient.