E cadherin

Nasal epithelial cells from welders were stained with PAFR primary antibody (1:200, Abcam, Cambridge, UK) for 1h with shaking at room temperature (RT). In order to analyse PAFR expression specifically in epithelial cells, a marker for epithelial cells, E-cadherin (primary antibody used at 1:100, Abcam, Cambridge, UK) was concurrently used. Cells were then washed and stained with secondary antibodies conjugated to Alexa Fluor 488 for PAFR expression (1:3000, Abcam, Cambridge, UK) and conjugated to APC for E-cadherin expression (1:1500, Abcam, Cambridge, UK) for 30min with shaking at RT. Cells were washed and analysed as described before. An isotype control for PAFR was used to exclude any non-specific staining. The resulting median florescence intensity was measured.
Cell lines were stained with PAFR primary antibody (1:200, Abcam, Cambridge, UK) for 1h with shaking at room temperature (RT). Cells were then washed and stained with secondary antibody conjugated to Alexa Fluor 488 (1:3000, Abcam, Cambridge, UK) for 30min with shaking at RT. Cells were washed and analysed as described before. The resulting median florescence intensity was measured.

Mouse ephrin-A1 (R&D Systems), VE-cadherin (Abcam), and E-cadherin (Abcam) ELISA kits were used to detect the presence of soluble ephrin-A1 ligand, VE-cadherin, and E-cadherin, respectively, in the plasma of mice. Mouse ephrin-A1 ELISA kits were also used to detect soluble ephrin-A1 present in MBMEC culture supernatants post-stimulation as described in detail in a previous section. Mice were euthanized with isoflurane and whole blood was collected into heparinized tubes and centrifuged at 1000g for 15 minutes at 4°C to isolate plasma. Plasma was diluted 1:10 for ELISA analysis of ephrin-A1 ligand, 1:800 for ELISA analysis of VE-cadherin, and 1:2000 for ELISA analysis of E-cadherin. Absorbance was measured at 450nm using a plate reader (Biotek) and plasma concentrations were determined using standard curves as per the manufacturer’s instructions. Human ephrin-A1 (Sino Biological) and human EphA2 (R&D Systems) ELISA kits were used to detect the presence of soluble ephrin-A1 ligand and soluble EphA2, respectively, in the plasma of humans and carried out according to the manufacturer’s instructions.

Bladder tissue sections were deparaffinized and hydrated. After blocking with BSA, the sections were incubated with primary antibodies (1:200) against HIF-1α (Zenbio, 382600), E-cadherin (Abcam, ab231303), N-cadherin (Cell Signaling Technology, 14215S), Vimentin (Zenbio, R22775), ZO-1 (Proteintech, 21773-1-AP), NLRP3 (Zenbio, 381207) and caspase1 (Proteintech, 22915-1-AP) overnight at 4 °C. SVHUC cells were cultured on coverslips in 24-well plates at a density of 60%. After treatment with hypoxia, the cells were fixed in 4% paraformaldehyde for 15 min and permeabilized with 0.05% Triton X-100. After blocking with BSA, the cells were incubated with primary antibodies (1:200) against HIF-1α (Zenbio, 382600), collagen I (Zenbio, 501352), E-cadherin (Abcam, ab231303), Vimentin (Zenbio, R22775), NLRP3 (Zenbio, 381207) and IL1-β (Abcam, ab283818) at 4 °C overnight. On the second day, tissue sections and cell slides were incubated with a secondary antibody (Alexa Fluor^® 488 or Alexa Fluor^® 594) at room temperature in the dark for 45 min. Then, the sections were stained with DAPI for 10 min.

The cell protein was extracted with RIPA reagent (Beyotime), and protein concentration was assessed with BCA reagent (Beyotime). After the protein was separated by 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE), the protein was transferred to nitrocellulose membranes (Millipore, Billerica, MA, USA), and sealed in 5% skimmed milk for 1 h. And then, the membranes were overnight at 4 °C and incubated with GAPDH (1:5000, Abcam, Cambridge, UK), E-cadherin (1:1000; Abcam), CyclinD1 (1:1000; Abcam), Cleaved-casp-3 (1:1000; Abcam), Bcl-2 (1:1000; Abcam), MMP9 (1:1000; Abcam), E-cadherin (1:1000; Abcam). After the membranes were treated with the second antibody (Abcam), the protein bands were observed by ECL assay (Millipore).