Sw41 rotor

Cycloheximide (Sigma C7698) was added to growing culture to a final concentration of 0.1 mg/mL and shaken for 2 minutes. Cultures were quickly poured over ice, spun down, and washed twice with ice-cold Polysome Lysis Buffer (PLB; 20 mM HEPES-KOH pH 7.4, 2 mM MgAc, 0.1 M KAc, 0.1 mg/mL Cycloheximide, 1% TritonX-100). For lysis, cells were vortexed with glass beads and PLB supplemented with 1 mM PMSF and 1X EDTA-free protease inhibitors (Roche 11836170001). Extracts were clarified at 21,000 × g for 20 minutes.
For absorbance profiling in 2 mM Mg, 10–25 OD₂₆₀ units were loaded onto 10%–50% sucrose PLB gradients, followed by centrifugation at 35,000 rpm in a Beckman SW41 rotor for 3 hours. For gradient profiling in high salt, 4 M KCl was added to cell extract (10 OD₂₆₀ units) immediately prior to centrifugation to raise the final concentration to 0.8 M KCl. Extract was then loaded onto 10%–40% sucrose PLB gradients containing 0.8 M KCl and centrifuged at 35,000 rpm in a Beckman SW41 rotor for 3 hours. For gradient profiling in EDTA, 60 OD₂₆₀ units of extract made with PLB were loaded onto 10%–30% sucrose PLB gradients containing 25 mM EDTA. Gradients were centrifuged at 18,000 rpm in a Beckman SW28 rotor for 16 hours. All gradients were fractionated from the top down using a Biocomp Gradient Station (Biocomp Instruments) with continual monitoring of absorbance at 254 nm.

Polysome profiling was performed from cytosolic and membrane fraction lysates. Lysates were thawed at 25 °C and cleared for 5 min at 12000 g. 4 A260 units of cytosolic fractions and 2-4 A260 units of membrane fraction were loaded onto linear 15 % −45 % sucrose gradients and centrifuged for 2 h at 273865 g at 4 °C in a Beckman SW41 rotor. Gradients were prepared by underlying 45% sucrose dissolved in polysome buffer 1.0 (20 mM Tris-HCl pH 7.5, 10 mM MgCl₂, 100 mM KCl, 0.1 mg/ml CHX) a 15% sucrose solution and mixed using a Gradient Master. Lysates were treated with 20 U RNAse I (Ambion) per A260 unit of extract for 5 min at 25 °C. Digestion was stopped through the addition of 10 U RNAse Inhibitor SUPERase-In (Ambion) per 20 U of RNAse I. Treated extracts were loaded onto linear 10 % − 50 % sucrose gradients prepared in polysome buffer 2.0 (20 mM Tris-HCl pH 7.5, 10 mM MgCl2, 100 mM NH4Cl, 0.1 mg/ml CHX) and centrifuged for 3 h at 209,678 g at 4 °C in a Beckman SW41 rotor. Monosome peaks were collected and subjected to hot phenol RNA extraction in the presence of 1% SDS.

An aliquot of VLPs was absorbed to a glow-discharged 300-mesh formvar/carbon film, stained with 2% phophotungstic acid (PTA) (Electron Microscopy Sciences), and examined by transmission electron microscopy (HT7700, Hitachi). For immunogold labeling, the pellets overlaid with 5–20% sucrose gradient in PBS, followed by ultracentrifugation at 247,606×g (SW41 rotor, Beckman) and 4°C for 90 min. Each fraction was first diluted three folds in PBS, then ultracentrifuged at 65,000×g (SW41 rotor, Beckman) and 4°C for 5 h and resuspended in PBS [41] (link). Gradient-purified particles were absorbed to a glow-discharged 300-mesh nickel grid for 10 min. After blocking with 3% BSA in Tris-buffer saline, grids were incubated for 1 h at room temperature with anti-E mAbs (1H10-6-7) in Tris-buffered saline. Grids were then washed three times with Tris-buffered saline and incubated at room temperature for 1 h with goat anti-mouse antibody conjugated with 6 nm gold particles (Electron Microscopy Sciences) and diluted 1: 20 in Tris-buffered saline. Grids were washed with deionized water and stained as described above.

Huh7 cells were seeded in 60 mm dishes and transfected with siNSP or siRPS5. After 48 h, cells were treated with 100 μg/ml of cycloheximide for 10 min at 37°C. Cells were washed once with ice cold PBS-containing cycloheximide and hypotonic buffer (5 mM Tris–HCl pH 7.5, 1.5 mM KCl, 5 mM MgCl₂, 100 μg/ml cycloheximide). Cells were harvested in ice-cold lysis buffer (5 mM Tris–HCl pH 7.5, 1.5 mM KCl, 5 mM MgCl₂, 100 μg/ml cycloheximide, 1 mM DTT, 200 U/ml RNAsein, 200 μg/ml tRNA, 0.5% Triton-X-100, 0.5% sodium deoxycholate, 1× protease inhibitor cocktail), incubated on ice for 15 min and KCl concentration in the lysate was adjusted to 150 mM. Cells were centrifuged at 3000g for 8 min at 4°C. Supernatant was processed immediately or flash frozen and stored at −70°C for later use. Lysate equivalent of 200 μg RNA was loaded on to a 15–50% sucrose gradient and centrifuged at 36 000 rpm for 2 h at 4°C in SW41 rotor (Beckman). We used the Density Gradient Fractionation System (ISCO) to fractionate the gradients at a flow rate of 0.75 ml/min with the UV-detector sensitivity set at 1.0. To study the level of 40S and 60S subunits lysate treated with 100 mM EDTA was loaded on 5–30% sucrose gradient containing 25 mM EDTA and centrifuged at 38 000 rpm for 3.5 h at 4°C in SW41 rotor (Beckman) as described by Huang et al. (31 (link)).

Plasma-membrane-enriched fractions (PMEFs) was isolated as previously described^{14 (link)}. Briefly, cells were lysed in buffer A (5 mM Tris–HCl pH 7.4, 1 mM EGTA, 1 mM DTT and 320 mM sucrose). Extracts were passed through a 26G needle five times and centrifuged at 1000 g for 10 min at 4 °C. The supernatant was kept, and the pellet was quickly vortexed in the presence of original volume of lysis buffer and centrifuged at 1000 g for 10 min at 4 °C. The two supernatants were pooled and centrifuged at 24,000 g for 20 min at 4 °C in a Beckman SW41 rotor. The supernatant was discarded, and the pellet was resuspended in 12 ml of buffer B (5 mM Tris–HCl pH 7.4, 1 mM EGTA and 1 mM DTT), and centrifuged at 24,000 g for 30 min at 4 °C in a Beckman SW41 rotor. The supernatant was discarded. The pellet was aliquoted and processed for both RNA and protein extractions.

Following differential ultracentrifugation, EVs were fractionated by OptiPrep density gradient ultracentrifugation (100,000 × g, 18 h, 4°C) using a SW41 rotor (Beckman Coulter, Fullerton, CA, USA) through a continuous 5% to 40% OptiPrep (Sigma-Aldrich, D1556) gradient. Fractions (1 mL) were collected from the top of the gradient for further analysis and density was verified by measuring the mass of a 100 µL aliquot of each fraction. Fractions of EV-specific density were then pooled together and subsequently concentrated via ultracentrifugation (100,000 × g, 4 h, 4°C) through a 20% w/v sucrose cushion in a SW41 rotor (Beckman Coulter, Fullerton, CA, USA). The resulting supernatant was discarded, and the EV pellet was resuspended in PBS -/-.