Gradient master

A linear sucrose gradient, 0.25–2.0 M sucrose in 20 mM HEPES at pH 7.4, as described previously38 (link), was prepared with a Gradient Master (BIOCOMP Model 107ip). Freshly prepared P100 pellet from either 400 mL of apical or basolateral culture medium was re-suspended in 1.1 mL of 2.5 M sucrose/HEPES solution and loaded onto the bottom of a sucrose gradient with a syringe. The gradient was then centrifuged at 100,000 × g for 18 h at 4 °C in a SW41 Ti rotor (Beckman Coulter). Fractions of 0.5 mL each were collected from the top of the centrifuge tube by using a Piston Gradient Fractionator (BIOCOMP). The density of individual fractions was calculated by use of a refractometer operated at 25 °C. The proteins were precipitated with trichloroacetic acid.

HEK 293 Trex 169 or C6.TAP [50 (link)] cell were uninduced or induced with 1 μg/ml of DOXY for 16 h. Thirty min prior to harvesting, the cells were treated with CHX (1 μg/ml). Cells were washed and lysed in lysis buffer supplemented with protease inhibitors (cOmplete, Mini, EDTA-free, Roche, 1 tablet in 10 ml of lysis buffer) (20 mM Tris HCl pH 7.5, 100 mM KCl, 5 mM MgCl₂, 1 mM CHX, 1 mM DTT, 0.1 mM EDTA) with DNAse I and NP-40 (0.03%) followed by trituration with a 25G needle. Cleared (19,000 g for 5 min at 4°C) cytoplasmic lysates were layered on top of sucrose density gradient (10–50% sucrose in lysis buffer) prepared by a Gradient Master (Biocomp) and resolved by centrifugation at 200,000 g for 90 min at 4°C. Absorbance (254 nm) composition within the gradient was measured during fractionation at 4°C using an Isco fractionator. Proteins from these fractions were extracted using methanol-chloroform extraction and subjected to immunoblotting analysis. Polysome profiling in higher salt condition was carried out with HEK 293 Trex 169 as described above except that the lysis buffer and sucrose density gradient contained 400 mM KCl.

17 Cl-1 cells were infected as described above. 10 min prior to harvesting, cells were treated with cycloheximide (100 μg/ml), washed with PBS and lysed in a buffer containing 20 mM Tris HCl pH 7.5, 100 mM KCl, 5 mM MgOAc, 0.375 mM CHX, 1 mM DTT, 0.1 mM PMSF, 2 U/μl DNase I, 0.5% NP-40, supplemented with protease and phosphatase inhibitors (ThermoFisher Scientific). Following trituration with a 26-G needle (ten passes), lysates were cleared (13,000 g at 4°C for 20 min) and the supernatants layered onto 12 ml sucrose density gradients (10–50% sucrose in TMK buffer: 20 mM Tris-HCl pH 7.5, 100 mM KCl, 5 mM MgCl₂) prepared in Beckman SW41 polypropylene tubes using a Gradient Master (Biocomp). Following centrifugation (200,000 g for 90 min at 4°C), fractions were prepared using an ISCO fractionator monitoring absorbance at 254 nm. Proteins were concentrated from fractions using methanol-chloroform extraction and subjected to immunoblotting analysis. Polysome profiling in higher salt conditions was carried out as described above except that the lysis buffer and sucrose density gradient contained 400 mM KCl.

For determination of sEV density on sucrose gradients, sEVs were labeled with the fluorescent red carbocyanine DiI (Invitrogen, Carlsbad, CA, USA) at a concentration of 1.0 mmol/L in 50 mM trehalose PBS and re-isolated at 110,000× g for 1.5 h according to established methods [16 (link)]. Flotation of DiI labeled sEVs on a continuous sucrose gradient (2.0–0.25 M sucrose, 20 mM HEPES/NaOH, pH 7.4) was performed similarly as previously described using a Beckman Coulter SW 41 rotor [16 (link),18 (link)]. For each gradient, 400 μg of sEV protein was loaded except for the primary melanocyte sEVs in which case 80 μg of protein was loaded. The primary melanocytes grow very slowly, requiring multiple months of culture to accumulate small amounts of sEV material. The gradient was produced using a Gradient Master (Biocomp Instruments, Fredericton, NB, Canada) and was spun, after loading sEVs, for >15 h at 100,000× g. Post centrifugation, 1 mL fractions were collected from the bottom up. The density of each fraction was calculated using a refractometer [18 (link)]. Two hundred microliters of each fraction were added to a black 96-well plate, and DiI sEV fluorescence detected using a Tecan M200 infinite pro microplate reader according to established methods [16 (link),18 (link)].

The SHL5e nucleosome (2.6 μM) was mixed with GATA3_znf (6.5 μM), in 240 μl of reaction buffer containing 13.3 mM Tris-HCl (pH 7.5), 100 mM NaCl, 3.3% glycerol, 1.3 mM DTT, 0.67 mM 2-mercaptoethanol, and 0.3 μM ZnSO₄. The reaction mixture was incubated for 2 h at 25 °C. After the incubation, the sample was purified and stabilized by the GraFix method^{45 (link)}. A gradient solution was formed with buffer A (10 mM HEPES-NaOH pH 7.5, 20 mM NaCl, 1 mM DTT, 1 μM ZnSO₄, and 5% sucrose) and buffer B (10 mM HEPES-NaOH pH 7.5, 20 mM NaCl, 1 mM DTT, 1 μM ZnSO₄, 20 % sucrose, and 3% paraformaldehyde), using a Gradient Master (BioComp). The GATA3_znf-nucleosome sample was applied onto the top of the gradient solution, and was centrifuged at 89,800 × g (27,000 r.p.m.) at 4 °C for 16 h, using an SW41 Ti rotor (Beckman Coulter). After the ultracentrifugation, fractions (0.6 ml each) were collected from the top of the gradient, and were analyzed by native-PAGE. The collected samples were desalted on a PD-10 column (GE Healthcare), equilibrated with 10 mM HEPES-NaOH (pH 7.5) buffer containing 1 mM DTT and 1 μM ZnSO₄, and were concentrated with an Amicon Ultra centrifugal filter unit (Millipore).