Adamts4 sirna

HMVEC-Ls were seeded on glass coverslips (Lab-Tek chamber slide, Thermo Fisher Scientific) at a concentration of 5 × 10 4 cells/well. After monolayer confluency was confirmed, control or ADAMTS4 siRNA (Horizon Discovery Ltd.) was transfected. After 24 h of culture, cells were stimulated with LPS for 24 h. After washing three times with PBS at room temperature, cells were fixed in chilled acetone for 5 min, and then stained by allophycocyanin-conjugated anti-human CD138/syndecan-1 antibody (BioLegend) for 30 min. After washing, cells were mounted with DAPI and Prolong Gold antifade reagent (Thermo Fisher Scientific) and covered with coverslips. A total of six fluorescence images were obtained at random positions for each sample from three independent experiments using an all-in-one fluorescence microscope BZ-X810 (Keyence, Osaka, Japan), and fluo-rescence intensities were automatically calculated using a hybrid cell-count application in .BZ-X Analyzer software (Keyence).

Boyden chambers were used for permeability evaluation; HMVEC-Ls were seeded on Transwell Inserts (1.0 µm pore; Falcon ® Transparent PET Membrane) at a concentration of 1 × 10 5 cells/well. The cells were cultured for 2 days. After monolayer confluency was confirmed, control or ADAMTS4 siRNA (Horizon Discovery Ltd., Waterbeach, UK) was transfected. After overnight culturing, cells were stimulated with or without LPS (10 ng/mL) for 24 h. To measure dextran leakage in human HMVEC-Ls, 100 µg/mL of assay solution containing FITC-labeled dextran (Molecular weight 3000, MERCK, Darmstadt, Germany) was added to each upper chamber, and after 20 min of incubation at 37 • C, the fluorescence intensity of the medium from the lower chamber was measured at 485-538 nm using a SpectraMax M2/M2e multi-mode plate reader (Molecular Devices, San Jose, CA, USA).