Sc 9989

Confluent HUVECs seeded on 24-well plates were subjected to the scratch-wound assay and then processed for PLA using the Duolink In Situ Red Mouse/Rabbit Starter Kit (DUO92101-1KT, Sigma-Aldrich) as described by the manufacturer’s protocol. To probe interactions between vinculin and VE-cadherin, cells were incubated with an anti-vinculin antibody raised in rabbit (V4139, Sigma-Aldrich) and an anti-VE-cadherin antibody raised in mouse (sc-9989, Santa Cruz Biotechnologies). In parallel, cells were also incubated with an anti-VE-cadherin antibody raised in goat (AF938, R and D Systems) and subsequently with an anti-Goat fluorescent-conjugated secondary antibody (A21447, Thermo Fisher Scientific) to label adherens junctions. To quantify co-localization of PLA signal at adherens junctions, high-resolution Z-stack images at multiple positions on the wound edge were acquired on a confocal Laser Point-Scanning Microscope 880 (Zeiss) equipped with the Zen black software with a Plan Apochromat 63x NA 1.40 oil DIC M27 objective. Briefly, PLA dots were quantified only at adherens junctions, using a similar approach to the co-localization studies described in the section ‘Immunostaining co-localization analysis’, using the VE-cadherin immunofluorescence staining to detect overlapping pixels between junctions and PLA signals.

VE-cadherin was detected using a mouse monoclonal antibody (sc-9989, Santa Cruz Biotechnology Inc., Dallas, TX, USA) at 1:100 dilution for immunofluorescence and at 1:2000 dilution for immunoblotting. Connexin43 (Cx43) was detected using rabbit polyclonal antibodies directed against amino acids 363–382 of human/rat Cx43 (C6219, SIGMA-Aldrich, Saint Louis, MO, USA) at 1:250 dilution for immunofluorescence and at 1:5000 dilution for immunoblotting. Immunoblotting for EV markers was performed using primary mouse monoclonal antibodies anti-flotillin-1 (sc-133153, Santa Cruz, CA, USA) diluted 1:200 and anti-CD63 (sc-5275, Santa Cruz, CA, USA) diluted 1:500. The secondary antibodies, AlexaFluor 488 goat anti-mouse IgG and horseradish peroxidase (HRP)-conjugated goat anti-rabbit or anti-mouse IgG antibodies, were obtained from Jackson ImmunoResearch (West Grove, PA, USA). Immunoblotting was performed as described earlier [32 (link),33 (link)].

HUVECs were grown to confluence on μ-slides coated with collagen and were treated with PT as indicated. Cells were directly fixed with Roti-Histofix (4 %, Carl Roth, Karlsruhe, Germany). For VE-cadherin analysis, cells were permeabilized with 0.2 % Triton X-100 (Sigma-Aldrich). Unspecific binding sites were blocked with 0.2 % BSA in PBS. Primary antibodies: mouse monoclonal anti-VE-cadherin (F-8) antibody (1:400, sc-9989, Santa Cruz Biotechnology), mouse monoclonal anti-PECAM-1 (JC70) antibody (1:400, sc-53411, Santa Cruz Biotechnology) and rabbit polyclonal anti-Collagen I + II + III + IV + V antibody (1:40, ab36064, Abcam). Secondary antibodies: Alexa Fluor 633-conjugated goat anti-mouse antibody (1:400, A21050) and Alexa Fluor 488-conjugated goat anti-rabbit antibody (1:400, A11008) from Life Technologies. HOECHST 33342 (1:10.000, 14533, Sigma-Aldrich) was used for nuclei staining. Images were obtained by a Leica DMI6000 B fluorescence microscope (Leica Microsystems, Wetzlar, Germany).

Cells were cultured on 24-well plates coated with rat collagen (100 µg/mL) until they formed confluent monolayers. After stimulation, cells were rinsed and fixed/permeabilized with 3.7% paraformaldehyde and 0.1% Triton X-100 for 10 min. After the washing steps, a monoclonal (F-8) mouse anti-VE-cadherin antibody (1:250, SC-9989, Santa Cruz Biotechnologies, Dallas, TX, USA) was added for 1 h at room temperature. Subsequently, a corresponding secondary antibody was incubated for another 1 h. An anti-fading agent was added. Images were taken with a Leica DMi8 inverted microscope (Wetzlar, Germany). VE-cadherin staining was quantified by dividing the cell circumference by the length of VE-cadherin staining at the cell border (% positive) using ImageJ(version 1.53k by Wayne Rasband and contributors, National Institutes of Health, Bethesda, MD, USA; https://imagej.nih.gov/ij/download.html, accessed on 27 May 2022).

Total proteins from PMVECs and lung tissues were isolated using radio-immunoprecipitation assay lysis (20–188, Merck, Zurich, Switzerland), quantified using BCA, boiled in sample preparing buffer to prepare samples, and separated using sodium dodecyl sulfate polyacrylamide gel electrophoresis. The separated proteins were transferred to PVDF membranes, which were sealed in 3% skimmed milk for 30 min and probed with diluted primary antibodies overnight at 4 °C. On the next day, PVDF membranes received washing and 1-h probing with HRP-coupled secondary antibody (1:5000, ab205719, Abcam) at room temperature. The used antibodies are as follows: Fcgr2b (1:1000, 550,270, BD Biosciences), vascular endothelial (VE)-cadherin (1:3000, sc-9989, Santa Cruz Biotechnology), β-catenin (1:3000, 13-8400, Invitrogen), Elk1 (1:1000, sc-365,876, Santa Cruz Biotechnology), GAPDH (1:5000, ab8245, Abcam).