Matrigel basement membrane matrix matrigel

Recombinant human TNF-α protein and human IL-1β/IL-1F2 Quantikine ELISA kits were purchased from R&D Systems (Minneapolis, MN, USA). ATP, apyrase, anti-β-actin (#A2066) antibody, and 4′,6-diamidine-2′-phenylindole dihydrochloride (DAPI) were obtained from Sigma-Aldrich (St. Louis, MO, USA). Anti-NLRP3 (#ab214185), anti-NLRC4 (#ab99860), and anti-cleaved caspase-1 (#ab179515) antibodies and human VEGF-A ELISA kits were purchased from Abcam (Cambridge, United Kingdom), and anti-ASC (#13833) antibody was obtained from Cell Signaling Technology (Beverly, MA, USA). TOPscript One-step RT PCR Drymix was purchased from Enzynomics (Daejeon, Korea). Negative control siRNA (control siRNA) and NLRP3, NLRC4, ASC, caspase-1, and P2Y₂R siRNA were obtained from Bioneer (Daejeon, Korea), and Lipofectamine 3000 reagent, TRIzol reagent, and anti-P2Y₂R (#PA1-46150) antibody were purchased from Thermo Fisher Scientific (Waltham, MA, USA). Hybond-P⁺ polyvinylidene difluoride membrane was obtained from GE Healthcare (Chicago, IL, USA), and Clarity Western enhanced chemiluminescence (ECL) substrate was purchased from Bio-Rad (Hercules, CA, USA). BD Matrigel basement membrane matrix (Matrigel) was obtained from BD Bioscience (Franklin Lakes, NJ, USA), and cell culture inserts for 24-well plates (8.0 µm, Translucent PET membrane) were purchased from Corning (Corning, NY, USA).

For Transwell® assays, 5 × 10⁴ cells were seeded per well post‐treatment into 6.5 mm diameter, 8 μm pore‐size Transwells® (Corning BV, Thermo Fisher). For migration assays, the underside of the wells was coated with fibronectin (Sigma‐Aldrich) at 10 μg/ml or TGFβ1 LAP (Sigma‐Aldrich) at 1 μg/ml final concentration. For invasion assays, the upper surface of the wells was coated with 70 μl of BD Matrigel Basement Membrane matrix (Matrigel™; BD Biosciences):media (1:2 ratio). After 16 h (migration) or 72 h (invasion), cells that had migrated/invaded were trypsin/EDTA harvested and counted using an automated cell counter CASY (Scharfe Systems, Midland, Ontario, Canada) as published previously 20.