Ab128936

STAV-AB cells were seeded on cover slips in 24-well plates and fixed, after 24 h, in 4% PFA for 15 min at room temperature. Cells were permeabilized using 0.1% Triton in PBS (PBST) for 15 min. Blocking was performed for 1 h with 3% Bovine Serum Albumin (BSA) in PBST. Co-staining was then performed with a combination of antibodies against SDC1 (1:500, rabbit monoclonal anti-SDC1, ab128936, Abcam, Cambridge, UK) and either EWSR1 (1:200, rat monoclonal anti-EWSR1/EWS, ab252829, Abcam) or FUS (1:200, mouse monoclonal anti-TLS/FUS, ab154141, Abcam, Cambridge, UK) in 0.1% BSA in PBST. The following secondary antibodies were used: goat anti-rabbit 555 for SDC1, goat anti-rat 488 for EWSR1 and goat anti-mouse 488 for FUS detection (ThermoFisher, Stockholm, Sweden). Cells were mounted and imaged through a Zeiss LSM800 confocal microscope (Stockholm, Sweden). Images were imported into Image J.

Lung tissue sections were deparaffinized with xylene and dehydrated with ethanol. The antigen was retrieved before immunofluorescence, and MLMECs were grown on a Confocal Dish (Dianrui, Shanghai, China) with proper treatment. Cells were fixed with 4% paraformaldehyde and ruptured using 0.1% Triton X-100. After blocking with 5% donkey serum, cells were prepared for immunofluorescence. Both were incubated with goat anti-mouse primary antibody to SDC1 (1:100; af3190, R&D Systems). They were then incubated with secondary antibodies conjugated to Alexa Fluor-488 (1:100; A12398, Thermo Scientific). For immunocytochemistry, HUVECs were incubated with rabbit anti-human primary antibody to SDC1 (1:100; ab128936, Abcam). They were then incubated with secondary antibodies conjugated to Alexa Fluor-488 (1:100; ab150077, Abcam). For the detection of heparan sulfate, MLMECs/HUVECs were incubated with primary mouse IgM to F58-10E4 (1:100; 370255-S, Amsbio) and then with secondary antibodies conjugated to Alexa Fluor-488 (1:1000; A10680, Thermo Scientific). Cell nuclei were counterstained with 4,6′-diamidino-2-phenylindole (DAPI). After staining, the cells were observed under a confocal laser scanning microscope SP8 (Leica Microsystems, Wetzlar, Germany), and images were recorded with LAS AF Lite 2.6.0 (Leica Microsystems, Wetzlar, Germany).

HEK293T hACE2 and VeroE6 cells were treated with 30 μM KSL-128114 or DMSO for 6 h, and then fixed in 4% formaldehyde. Cells were then either permeabilized in 0.5% Triton X-100 or left unpermeabilized. Then, cellular expression of ACE2 or Syndecan-1 were detected by rabbit monoclonal antibodies (ACE2, Novus bio, NBP2-67692, 1:500, Syndecan-1, abcam, ab128936 2 μg/mL) and secondary donkey anti-rabbit Alexa Fluor 488 (1:2000, Thermo Fisher Scientific). Cellular fluorescence was measured using a TROPHOS plate RUNNER HD.

We performed the multiplex immunofluorescence experiments by using the CD8 anti‐human antibody (Ab) (ab101500), EPCAM anti‐human Ab (Abcam, ab223582), cytokeratin‐8 anti‐human Ab (Abcam, ab53280), cytokeratin‐19 anti‐human Ab (Abcam, ab76539), collagen‐IV (COL4A1) anti‐human Ab (Abcam, ab214417), SDC1 anti‐human Ab (Abcam, ab128936), CD3 anti‐human Ab (Abcam, ab16669), CD56 anti‐human Ab (Abcam, ab75813), CCL22 anti‐human Ab (Abcam, ab23772) and 4′,6‐diamidino‐2‐phenylindole staining solution (Abcam, ab228549). Following the manufacturer's instructions (Akoya, Opal Polaris 7 Color Automation IHC Detection Kit), we scanned the slides with the Akoya Vectra Polaris Automated Quantitative Pathology Imaging System and quantified the results by using Akoya Inform software.

Protein from exosomes or renal tissues was extracted using radio-immunoprecipitation assay buffer and quantified by bicinchoninic acid protein (Pierce). The protein was boiled with loading buffer at 95 °C for 5 min (40 µg/well), separated by 10% polyacrylamide gel electrophoresis, and transferred to a polyvinylidene fluoride membrane. The membrane was blocked by 5% bovine serum albumin for 1 h, added with primary antibodies CD9 (1:1000, ab92726), CD63 (1:1000, ab134045), syndecan-1 (1:1000, ab128936), heparanase-1 (1:1000, ab59787) (all from Abcam, Cambridge, MA, USA), CD81 (1:500, sc-166029, Santa Cruz Biotechnology, CA, USA), RUNX1 (1:1000, 39000, Active Motif, Carlsbad, CA, USA), and GAPDH (1:1000, Beyotime Institute of Biotechnology, Shanghai, China) at 4 °C overnight, then incubated with the corresponding secondary antibody (1:3000, MT-BIO, Shanghai, China) and developed by chemiluminescence reagent. GAPDH was used as the endogenous control. Bio-rad Gel Doc EZ imager (Bio-rad, CA, USA) was utilized for development. The gray value was analyzed by Image J software.

Total proteins were extracted from tissues using RIPA Lysis Buffer (Beyotime, Haimen, Jiangsu, China). Equal amounts of the protein were separated using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to a polyvinylidene fluoride membrane, followed by overnight incubation with primary antibodies at 4°C. Primary antibodies against the following proteins were used: Pi3k (1:300, 20584-1-AP, Proteintech), Jak2 (1:4,000, ab108596, Abcam), Gng7 (1:800, GTX65584, GeneTex), Jun (1:1,500, 66313-1-Ig, Proteintech), IL-6 (1:1,000, ab9324, Abcam), Met (1:1,500, ab51067, Abcam), Pdgfra (1:1,000, ab203491, Abcam), Camk2b (1:1,000, ab34703, Abcam), Ptgs2 (1:1,000, ab15191, Abcam), Sdc1 (1:1,000, ab128936, Abcam), and Gapdh (1:10,000; SAB2100894; Sigma–Aldrich; Merck KGaA). Target protein expression was normalized to Gapdh expression.