Phosphate buffered saline (pbs)

HUVEC and BJ cells seeded on glass bottom in multiwell plate were fixed in p-formaldehyde at 4% v/v in PBS, (Lonza; Basilea, Swiss), permeabilized with Triton X-100 at 0.5% v/v in PBS (Lonza; Basilea, Swiss) and then blocked with goat serum at 20% v/v in PBS (Lonza; Basilea, Swiss). Incubation with the respective antibodies against VE-cadherin (mouse monoclonal, 1:100; Santa Cruz Biotechnologies, Dallas, TX, USA), FAP1α (rabbit polyclonal, 1:100; Santa Cruz Biotechnologies, Dallas, TX, USA) were performed for O/N at 4 °C. F-actin detection was evaluated by Phalloidin-FITC (5 μg/mL, Sigma-Aldrich; Saint Louis, MO, USA) for 30 min, at RT in the dark. The staining with conjugated secondary antibodies (1:500, antimouse and antirabbit), the nuclei with DAPI (1:1000) and the subsequent confocal microscope analysis and quantifications were performed as described in [62 (link),63 (link)]. In particular, the final images were generated using Adobe Photoshop CS4, version 11.0. Quantifications were performed from multichannel images obtained using a 63× objective using ImageJ, marking either the cell perimeter or the nucleus as the region of interest and calculating integrated densities per area from the appropriate channel. A minimum of 50 cells were analyzed for each data set; the obtained mean value has been used to compare experimental groups.

PBS-washed cells were fixed in 4% PFA solution (Santa Cruz Biotechnology) at RT for 30 min. The cells were then permeabilized with 0.05% Triton X-100 (BioShop) in PBS (Lonza) for 5 min, washed 3 times with PBS, incubated with 0.25% glycine for 30 min, washed 3 times with PBS (Lonza) and kept in blocking solution (10% goat serum, Sigma-Aldrich) for 1 h. After overnight incubation with rabbit anti-human α-SMA (Abcam) antibody, the washing and addition of goat anti-rat Alexa Fluor 488 antibody (for detection of α-SMA) and DAPI (for detection of nuclei) was performed. The images were acquired using a Nikon Eclipse Ti or Leica DMi8 microscope with Leica DFC7000 GT fluorescent camera.

Cells were fixed with 4% paraformaldehyde in PBS (Lonza) for 30 min at room temperature and permeabilized with 0.1% Triton X-100 in PBS for 10 min. Alternatively, for immunostaining of glutamylation (GT335) and polyglutamylation (polyE), cells were fixed and permeabilized with cold methanol for 5 min at −20 °C. Then, after blocking with 3% BSA for 30 min, cells were immunostained with polyE antibody at 4 °C overnight, then with polyE and GT335 together at 4 °C overnight, followed by secondary antibody staining at room temperature for 1 h, and finally with anti-α-tubulin Alexa Fluor 647 at room temperature for 2.5 h. For staining of lipid droplets with LD540 dye, cells were incubated with 0.1 μg ml⁻¹ LD540 in PBS for 5 min. Cells were mounted using Fluoromount-G (SouthernBiotech) and imaged using a Zeiss LSM880 confocal microscope in Airyscan mode equipped with a 63 × 1.4 NA Plan-Apochromat oil objective (Carl Zeiss). Images were acquired using ZEN software (Carl Zeiss) and processed with ZEN software or Fiji (NIH).

The aerial parts of the plants were harvested 5 days post agroinfiltration and homogenized in two buffer volumes of PBS (Lonza), supplemented with cOmplete^™ EDTA-free protease inhibitor (Roche). The crude homogenate was incubated for 1 h, at 4°C, with shaking and then filtered through four layers of Miracloth^™ (Merck). The crude plant sap was then clarified by sequential centrifugation steps; twice at 15,344 × g for 20 min and then again at 17,000 × g for 20 min. The supernatant was vacuum-filtered through a 0.45 µM Stericup-GP device (Merck Millipore) and applied to a Galanthuis nivalis lectin (GNL) column (Sigma) with a 0.5–1 ml/min flow rate. The column was sequentially washed with 100 ml of 0.5 M NaCl and then 100 ml of PBS (Lonza). The proteins were eluted in 1 M methyl α-D-manno-pyranoside (MMP) (Sigma), buffer exchanged into PBS and then concentrated using a Vivaspin Protein Concentrator with a 30 kDa cut-off (GE Healthcare). The purified proteins were quantified using the DC^™ Protein Assay (Bio-Rad).

Calu-3 and Vero cells were seeded in 100 mm dishes, washed twice with phosphate-buffered saline (PBS; Lonza), and infected with SARS-CoV-2 at a MOI of 10 or 0.1 for 1 h. Next, the viral inoculum was removed, and infected cells were washed thrice with PBS and then maintained in DMEM supplemented with 2% FBS (Serana) and 1% penicillin/streptomycin (Gibco) in a 5% CO2 incubator at 37 °C. The supernatant was collected at 0, 1, 2, 4, 12, 16, 24, and 36 hpi, and titration was performed by plaque assay in Vero cells. All SARS-CoV-2-related experiments were performed at a BSL3 facility at Korea University (Seoul, Korea).