Alexa 647

The cultured cells were fixed with 4% paraformaldehyde for 30 min. Samples were incubated with 5% BSA for 1 h. For the transfection efficiency of ACE2, an immunofluorescence assay was performed, as previously described [26 (link)], using an anti-ATPase antibody (EP1845Y, Abcam, Cambridge, UK), a plasma membrane loading control (Abcam), and a primary ACE2 antibody (E-11, Santa Cruz Biotechnology, Santa Cruz, CA, USA) all at a dilution of 1:400, at 4 °C overnight, followed by incubation with Alexa 647 and 488 conjugated antibodies (Abcam Ltd., Cambridge, UK), respectively. After immunolabeling, the samples were mounted with ProLong^® Gold Antifade Mountant containing DAPI (Cell Signaling Technology, Danvers, MA, USA). For pseudovirus entry, the infected cells were obtained following the method from 4.4. Zsgreen was introduced to the pseudovirus beforehand, while the F-actin component of the cell membrane was stained with fluorescent phalloidin. All of the images were visualized and obtained utilizing a confocal inverted laser microscope (Leica SP8) with 48× magnification. The emission wavelength was 405 nm for DAPI, 488 nm for Zsgreen, and 505 nm for F-actin/phalloidin.

Cells were kept in culture for 72 hours. Cells collected for flow cytometry were washed with 1x PBS, trypsinised, and resuspended in PBS-2% BSA-0.05% Tween® 20. One million (1 x 10⁶) cells were used for each labelling. The primary antibodies used were: anti-CD49f-PE (BD, Pharmigen, Franklin Lakes, NJ, USA); anti-EpCAM, anti-MUC1, anti-EGFR, anti-N-cadherin, anti-E-cadherin, anti-CD66a/c/e (Biolegend, San Diego, CA, USA); anti-EpHB4 and anti-claudin-3 (R&D Systems, Inc., MN, USA); anti-vimentin, anti-claudin-4, anti-ALDH1, anti-FGFR; secondary antibodies were Alexa488 and Alexa647 (Abcam, Cambridge, UK). Unlabelled cells and with only secondary antibody labelling were included in each independent assay and considered autofluorescent. Dead cells and debris were excluded from the analysis. Cells were analyzed in a FACs-Aria flow cytometer (BD Bioscience).

Frozen sections were incubated with rabbit monoclonal anti-ZO-1 (ABCAM), followed by incubation with anti-rabbit IgG conjugated to Alexa 594 (1:400), Alexa 488, and Alexa 647 (1:400) (Abcam, Cambridge, MA, USA). The sections were stained with 4′,6-diamidino-2-phenylindole (DAPI) (Life Technologies, Molecular Probes, Carlsbad, CA, USA). Fluorescent images were collected using confocal Leica SP5 microscopy.

The cells were cultured on sterile glass coverslips in six-well plates and fixed with PBS containing 3.7% paraformaldehyde for 15 min at room temperature. Cells were permeabilized with PBS containing 0.2% Triton X-100. Indicated antibodies were incubated overnight at 4 °C. Secondary antibodies were coupled to Alexa 647 (Abcam). Nuclear counterstaining was performed with DAPI (Invitrogen). Additionally, all images were acquired using Leica TCS SP5 Confocal microscope (Leica, Wetzlar, Germany).

For the double-staining experiments, formalin-fixed, paraffin-embedded synovial sections from patients with RA (n = 4) were incubated with primary antibodies against S100A11 (diluted 1:100; Abcam, Cambridge, UK), CD68 (diluted 1:100; Abcam, Cambridge, UK), CD3 (diluted 1:100; Abcam, Cambridge, UK), vimentin (diluted 1:50; Sigma-Aldrich, St. Louis, MO, USA), and CD20 (diluted 1:50; Abcam, Cambridge, UK) before being incubated with secondary antibodies coupled to Alexa 488 or Alexa 647 (diluted 1:200) (Abcam, Cambridge, UK). The samples were then analysed using a BX53 microscope with a DP80 Digital Microscope Camera and CellSens Standard Software (Olympus, Philadelphia, PA, USA). S100A11 positivity in different cell populations was quantified in six randomly selected high-power fields per patient by two experienced researchers in a blinded manner and expressed as percentage of S100A11-positive cells.

PBMCs (250,000/coverslip) were added in 100μL PBS on poly-L-lysine pre-coated coverslips (BioCoat™) and left to adhere 30 min at 37°C. After attachment coverslips were washed in PBS and fixed 10 min at RT using 4% PFA. Cells were blocked using 3% BSA and antibody detection of CD4 (R&D systems, #AF-379-NA) and Alexa 647 conjugated CD8 (Abcam, #ab196193) performed prior permeabilization 10 min using 0.2% Triton x-100 and subsequent primary antibody incubation targeting HIF-1α (Novus Biologicals, #NB-100-449) and HIF-1β (Abcam, #ab2771). Secondary antibodies, Alexa Fluor 488 (Invitrogen, #A32731), Alexa Fluor 568 (Invitrogen, #A-11004), and Alexa Fluor 647 (Invitrogen, #A-21447) were incubated 1h at RT. Cells were counterstained using DAPI and mounted with Prolong Gold Antifade reagent (Thermofisher). Images were acquired using Nikon single point scanning confocal microscope with 60x/1.4 oil objective. All samples were analysed in two technical replicates and detection threshold was set using secondary antibody controls. Image analysis was performed in Imaris (Bitplane) using detection of Nucleus, Cell and Vesicles detection according to pipeline in Figures S4A and S4B. Graphical representation was performed in Imaris and statistical analysis was performed using Mann-Whitney U-test in Prism 8.4.3 (GraphPad Software) (significance level, p<0.05).