Paxillin

AECs were grown in monoculture using 8-well plates or in co-culture with fibroblasts using Transwells. At the end of each experiment, cells were fixed with 4% paraformaldehyde followed by permeabilization and staining with primary antibodies for Paxillin (Abcam 1:100), ZO-1 (Life Technologies 1:100), E-cadherin (Cell Signalling 1:100), and β-catenin (Cell Signalling 1:100). The secondary antibodies used were Alexafluor 488, 555, and 647 (all from BioLegend, London, UK). Cellular F-actin was stained using TRICT-phalloidin (Millipore UK Limited, Watford, UK). Cell nuclei were counterstained with 4′,6-diamidino-2-phenylindole, dihydrochloride (DAPI) 1:1000 dilution (Millipore UK Limited, Watford, UK). Cells were imaged using an inverted fluorescence microscope (Leica DMI 6000B, Leica Microsystems, Milton Keynes, UK) or an inverted confocal microscope (Leica TCS-SP5 Confocal Microscope, Leica Microsystems, Milton Keynes, UK).

The cells were washed with PBS and fixed with 4% paraformaldehyde for 15 min at room temperature. Then, the cells were permeabilized with 0.05% Triton X-100 for 1 min and blocked with 10% BSA for 1 hr. The primary antibodies against ZYX (1 : 00, Proteintech), p-CREB1 (1 : 100, ImmunoWay), and paxillin (1 : 100, Abcam) were diluted with 1% BSA and incubated with the cells at 4°C overnight. Then, the cells were washed with PBS and incubated with Alexa Fluor-594 or -488 conjugated species-matched secondary antibodies. Finally, the cell nucleus were stained with DAPI, and the immunofluorescence images were captured and analyzed with a confocal microscopy.

Confocal microscopy was performed using an LSM 880 Confocal Microscope (Zeiss, Jena, Germany) or a Zeiss Observer spinning disc confocal microscope using standard protocols. Hoescht 33342 (ThermoFisher, Waltham, MA, USA) was used to stain nuclei. Antibodies used: ELTD1 (‘in house’ mouse mAb raised to the extracellular EGF domains, clone name 97.1); αSMA (1A4, Sigma, St. Louis, MO, USA); Alexa Fluor^® 568 Phalloidin (ThermoFisher, Waltham, MA, USA), Alexa Fluor^® 488 HA-Tag (Cell Signaling, Danvers, MA, USA); VE-Cadherin (BV13, ThermoFisher, Waltham, MA, USA), paxillin (Y113, Abcam, Cambridge, UK).

Cells were washed with PBS and lysed in Laemmli Sample Buffer (Bio-Rad) and further homogenized with a rotorstator homogenizer. Proteins were isolated and concentrations were determined using the BCATM Protein Assay Kit (Thermo Scientific). 80–120 µg proteins were loaded on a 12–15% sodium dodecyl sulfate-polyacrylamide gel. After electrophoresis, proteins were transferred to a PVDF Western Blotting membrane (Roche). Membrane were blocked with 5% nonfat dried milk (in TBST) for 2 h at room temperature and then incubated overnight at 4°C with Primary antibody (p-FAK, FAK, p-Src, Src, paxillin, vinculin, and talin were purchased from abcam, cleaved-caspase3, caspase3 and β-actin were purchased from Cell Signaling Technology).The membrane was subsequently washed with TBST (5 min×3) and incubated with horseradish peroxidase-conjugated secondary antibodies (Cell Signaling Technology) for 1 h at room temperature. After washing with TBST (5 min×3), bands were detected by enhanced chemiluminescence substrate (Applygen). Band intensities were normalized to its respective internal standard signal intensity. The experiment was repeated 6 times.

Antibody to PLOD2 for immunohistochemistry and Western blot was purchased from Proteintech (Wuhan, China). HIF-1α (PX-478) and FAK inhibitors (TAE226; Selleckchem; Shanghai, China) were resolubilized in DMSO. Matrigel was purchased from BD Biosciences (San Diego, CA, USA). The primary human antibodies used in this study were the following: rabbit anti-HIF-1α, anti-HIF-2α, anti-FAK-p³⁹⁷, anti-FAK, Paxillin (Abcam; Cambridge, MA, USA); mouse anti-β-actin (Beyotime, China).

Fibroblasts were grown on 24 well plates with glass coverslips in each well. After 4 h of BCM treatment, cells were fixed with 4% paraformaldehyde (PFA) for 10 min, permeabilized 10 min with 0.1% Triton X-100 (Sigma) and 30 min blocking with 0.1 M L-lysine monohydrochloride in PBS. Samples were incubated with Cx43 (Sigma C6219, 1:4000), ZO-1 (Invitrogen 339 100, 1:100), β-catenin (Abcam ab6302, 1:2000), α-tubulin (Sigma T6074, 1:1000), GM130 (BD transduction Laboratories 610 822, 1:200), phospho-Histone H3 (Ser10) (Milipore 09–797, 1:1000), vinculin (Sigma) and paxillin (Abcam) at room temperature for 1 h. Samples were washed with PBS, incubated with secondary antibodies for 1 h at room temperature, stained with DAPI and/or rhodamine-phalloidin, washed in PBS and mounted in Citifluor® (Glycerol/PBS solution, Citifluor Ltd). Immunofluorescence was imaged on a Leica SP8 upright confocal microscope. Optimal gain and offset were set and kept constant during image acquisition. Minimum of three regions were imaged of each biological replicate.