Western blot assay for EMT markers:

HCE-T (200,000 cells/well) were plated in a 4-well chamber slide (Millipore, #PEZGS0416), and incubated for 30 hours at 37°C for the following experimental groups: (i) RPMI; (ii) No NETs; (iii) NETs; and (iv) NETs + Heparin (100 IU/mL). After incubation, the medium was removed and cells were lysed with 100 µL of RIPA-buffer for 5 min on ice, then cells were scraped and collected in an e-tube. The collected supernatant was centrifuged at 5000 rpm for 10 min at 4°C to extract protein from cells. The protein concentration was determined by the Bradford protein assay using the Bio-Rad protein assay kit (Bio-Rad, #5000002). 500 ng of total protein was added per lane and then loaded in ProteinSimple (ProteinSimple, Santa Clare, CA, U.S.A.) The following primary antibodies were used: (i) α-SMA (1:100, Novus, NBP2–33006), (ii) β-Catenin (1:1000, Abcam, ab119801), (iii) CCN2 (1:50, Santa Cruz, sc-365970); (iv) E-Cadherin (5 µg/mL, R&D systems, MAB1838), and (5) Vimentin (1:100, Novus, NBP1–92687). ProteinSimple capillary electrophoresis immunoassay was performed according to the manufacturer’s instructions. Data analyses were performed using the Compass software (ProteinSimple) on Wes™.