Optiprep

An 800-mL stack of the RuV TO-336WT-AG1 strain was mixed with 200 mL of PEG-it virus precipitation solution (5×; System Biosciences, Palo Alto, CA) and incubated at 4°C overnight. The mixture was centrifuged at 1,500 × g for 30 min at 4°C twice. The resulting precipitation was resuspended with PBS containing 65 mM ethylenediaminetetraacetic acid (PBS-EDTA). The suspension (9 mL) was overlaid on 15% sucrose in PBS (2 mL) and then centrifuged at 49,000 × g for 3 h at 4°C. The precipitation was resuspended in PBS-EDTA again, overlaid on a 10% to 50% five-step discontinuous gradient of OptiPrep (Abbott Diagnostics Technologies AS, Oslo, Norway), and then centrifuged at 175,000 × g for 3 h at 4°C. The visible band between 20% and 30% OptiPrep layers was collected as purified RuV. Three hundred microliters of the purified RuV (1 × 10⁸ FFU) was supplemented with 7.5 μL of 100 μM Vybrant DiD cell-labeling solution (Thermo Fisher Scientific) with mixing by a vortex mixer and incubated at room temperature for 1 h. Under these conditions, the DiD dye incorporated into the lipid envelope of RuV was self-quenched. Just before use, the DiD-labeled RuV was diluted with RPMI 1640 containing 10% FBS to a concentration of 2 × 10⁷ FFU/100 μL.

HSCs were isolated from normal livers of 350-g adult male Sprague-Dawley rats by sequential perfusion with collagenase and pronase, followed by discontinuous density centrifugation in 11.5% Optiprep (Life Technologies). HSCs were cultured on plastic in DMEM supplemented with penicillin 100 U/mL, streptomycin 100 μg/mL, l-glutamine 2 mmol/L, and 16% fetal calf serum and were maintained at 37°C in an atmosphere of 5% CO₂. Activated HSCs were generated by continuous culture of freshly isolated cells on plastic for 7 days.

We used OptiPrep [60% (w/v) Life Technologies, Inc.], which is a ready‐made solution of Iodixanol. The iodixanol gradient is more efficient in the separation of protein complexes and subcellular compartments because its osmolality and viscosity remain relatively constant with changes in the density of the gradient (Zhang et al., 1998). Iodixanol gradient analysis and determination of endoplasmic reticulum (ER), Golgi, and mitochondrial compartments in collected fractions were performed as described (Graham, 2002; Rezvani et al., 2009; Sane et al., 2014).
Briefly, cell lysates were spun at 13 000 g for 10 min at 4 °C, and then, supernatants were subjected to a BCA protein assay using a NanoDrop 2000 (Thermo Fisher Scientific). Supernatants were diluted to 2 mg·mL⁻¹, and 1 mL of extract was loaded onto a performed iodixanol (v/v) linear gradient (8–38%), as previously described (Sane et al., 2014). The gradients were spun at 28 500 r.p.m. (100 000 g) for 18 h using a Beckman SW41Ti rotor at 4 °C (Beckman Coulter Brea, CA, USA). Equal‐volume fractions (~ 0.6 mL) were collected from the top of each gradient and analyzed by WB. For the distribution of major subcellular markers in discontinuous iodixanol gradients, please see references (Rezvani et al., 2009; Sane et al., 2014).