Piston gradient fractionator

Polysome profiling assay was performed as previously described^{33 (link)}. Colorectal cancer cell line HT29, either MG-132 treated or untreated, were harvested and lysed on ice with lysis buffer (20 mM Tris HCl pH 7.4, 5 mM MgCl₂, 100 mM NaCl, 100 µg/mL cycloheximide, 1% Triton X-100, 40 U/mL RNasin and protease inhibitor cocktail). The cell extracts were centrifuged at 12,000 g 4 °C for 10 min, and then layered on a 10–50% sucrose gradient (composed of 25 mM Tris HCl pH 7.4, 5 mM MgCl₂ and 100 mM NaCl) and centrifuged at 4 °C in a SW41Ti Beckman rotor for 3.5 h at 35,000 rpm. Absorbance at 260 nm was recorded by using Piston Gradient Fractionator (Biocomp, USA). Polysomal fractions (fractions 13–22) were pooled, and RNA was isolated using the RNA extraction Kit as described above for further analysis.

Nuclease-treated bulk rabbit reticulocyte lysate (Green Hectares) was spun down at 20,000 RPM for 15 min to remove debris, nuclei, and mitochondria.⁶⁰ Clarified lysate was filtered with a 0.22 μm filter (Millipore). The supernatant was loaded on to a 30% sucrose cushion (20 mM Tris-HCl pH 7.5, 2 mM MgOAc₂, 150 mM KCl, 30% w/v sucrose) and ultracentrifuged for 17.5 h at 36,000 RPM (50.2 Ti rotor) at 4°C to obtain a ribosomal pellet.^{61 (link)} The pellet was washed and resuspended in a buffer containing 20 mM Tris-HCl pH 7.5, 6 mM MgOAc₂, 150 mM KCl, 6.8% w/v sucrose, 1 mM DTT, 1 μL RNasin from Promega (Cat N2618). To remove non-resuspended particles, the resuspended pellet was centrifuged again at 10,000 g for 10 min at 4°C and the supernatant was isolated. 15–30% sucrose gradients were prepared using a buffer containing 20 mM Tris-HCl pH 7.5, 2 mM MgOAc₂, 150 mM KCl, and either 15% or 30% w/v sucrose using the Gradient Master (Biocomp). Gradients were cooled to 4°C before use. Pellet supernatant was loaded onto gradients and ultracentrifuged at 19,100 RPM for 17.5 h (SW-28 rotor) at 4°C. Gradients were fractionated using the Piston Gradient Fractionator and Fractionator Software v8.04 (Biocomp), monitoring for absorption at 260 nm. Fractions corresponding to pure 80S ribosomes were pooled and concentrated to an A₂₆₀ of 95 using an Ultra-15 centrifugal filter unit with a nominal molecular weight limit of 100 kDa (Amicon). This concentrated stock was subsequently diluted to 250 nM using buffer containing 20 mM Tris-HCl pH 7.5, 2 mM MgOAc₂, 150 mM KCl and 20 μL aliquots were flash frozen in liquid nitrogen and stored at −80°C.

Frozen mitochondria were thawed on ice and resuspended in 2 volumes of mitochondrial lysis buffer [25 mM HEPES–KOH, pH 7.4, 150 mM KCl, 50 mM MgOAc, 2% Triton (v/v) X-100, 2 mM DTT, EDTA-free protease inhibitor (Pierce), 50 μg/ml LZD] and mixed via inversion, followed by homogenization with 5 strokes of a Dounce homogenizer. The sample was then rotated on a nutator at 4°C to complete the lysis. The lysed sample was then clarified by centrifugation at 30 000 × g, 4°C, for 20 min in a TLA 100.3. The supernatant was carefully removed and the pellet discarded. This centrifugation step was then repeated to ensure clarification. The clarified mitochondrial lysate was then loaded onto a sucrose cushion [1 M sucrose (34%, w/v); 20 mM HEPES–KOH, pH 7.4, 100 mM KCl, 20 mM Mg(OAc)₂, 1% (v/v) Triton X-100, 2 mM DTT, 50 μg/ml LZD] in a 2.5:1 ratio of lysate to sucrose cushion. The samples were then centrifuged at ∼231 550 × g in a TLA 100.3 rotor (66 000 rpm) to pellet the mitoribosomes. The supernatant was discarded and the pellets rinsed gently several times with resuspension buffer (50 mM HEPES–KOH, pH 7.4, 250 mM KCl, 12.5 mM Mg(OAc)₂, 5 mM DTT, 50 μg/ml LZD). The pellets were then resuspended in a total of 170 μl of resuspension buffer and loaded onto a 15%–30% sucrose gradient (prepared in resuspension buffer using a BioComp Gradient Master^™) to isolate mitomonosomes. The samples were centrifuged at 19 500 rpm for 18.5 h in an SW41 rotor. The gradient was then fractionated using a BioComp Piston Gradient Fractionator^™ with UV260 monitoring. Two hundred microliter fractions were collected and the fraction corresponding to the 55S peak was collected and used for cryo-EM sample preparation.

Sucrose gradients ranging from 10 to 50% in a solution containing 20 mM Tris-HCl (pH 7.5), 100 mM NaCl, 5 mM MgCl₂, and 50 μg/ml cycloheximide were used for polysome analysis as previously described using a tilted tube rotation method on a gradient station equipped with a Piston Gradient Fractionator and a Gradient Master from BioComp (Fredericton, Canada; Palam et al., 2011 (link); Teske et al., 2011a (link)). MEF cells were cultured in DMEM in the presence or absence of 1 μM TG for 6 h. Before harvesting, cells were incubated in culture media containing 50 μg/ml cycloheximide for 10 min at 37°C. Cells were rinsed twice with chilled phosphate-buffered saline (PBS) containing 50 μg/ml cycloheximide and then lysed with 500 μl of cold lysis buffer consisting of 20 mM Tris (pH 7.5), 100 mM NaCl, 10 mM MgCl₂, 0.4% Nonidet P-40, 50 μg/ml cycloheximide, and EDTA-free protease inhibitor cocktail tablet (Roche, Indianapolis, IN). The lysates were sheared with a sterile syringe with a 23-gauge needle, incubated on ice for 10 min, and clarified at 8000 × g for 10 min. A 400-μl amount of supernatant was layered atop the sucrose gradients, which were subjected to centrifugation in a Beckman SW41Ti rotor at 40,000 rpm for 2 h at 4°C. Sucrose fractions and the resulting polysome profiles for each sample were then collected using a Piston Gradient Fractionator and a 254-nm ultraviolet monitor with Data Quest software.
To investigate specific mRNA transcript shifts during stress, 10 ng/ml firefly luciferase control RNA (Promega, Madison, WI) was added to each pooled sample before RNA isolation, allowing for measurements of the relative amounts of the transcript of interest to be normalized to an exogenous RNA control (Palam et al., 2011 (link); Teske et al., 2011a (link)). Samples were then immediately mixed with 750 μl of TRIzol Reagent LS and RNA isolation and cDNA generation performed as described later. To calculate percentage total gene transcript for the seven fractions, 2^(−ΔΔCT) was summed for each treatment group, and the 2^(−ΔΔCT) value for each fraction was considered as a percentage of the total. This calculation serves to omit changes in the levels of transcript abundance between treatment groups. All polysome profiles and mRNA shifts depicted are representative of three independent biological replicates. The percentage shift was calculated as (percentage total mRNA in fractions 5–7 during ER stress) – (percentage total mRNA in fractions 5–7 during no stress).