Human il1β

Primary human ventricular cardiac fibroblasts were purchased from PromoCell (PromoCell C-12375; PromoCell GmbH, Germany). Cells were cultured in DMEM containing 10% FBS and 1% Pen/Strep, and were kept in an incubator maintained at 5% CO₂. Cells were plated in a 96-well plate at 5,000 cells/well and then grown in 10% FBS for 24 h, serum starved for 24 h, and then treated with the following conditions for 96 h: 0%FBS control media, 0%FBS media with 20 ng/mL TGFβ1 (Cell Signaling Technology, 8915LC), and 0% FBS media with 10 ng/mL human IL1β (Cell Signaling Technology, 8900SC). Cells were then fixed in 4% PFA in PBS for 30 min, permeabilized and blocked for 1 h in a solution containing 3% BSA and 0.2% Triton, and then stained overnight at 4°C with a 1:500 primary Anti-Collagen I antibody (Abcam, ab34710). After an overnight incubation, cells were washed 3 × in PBS and stained with 1:5,000 Dapi, 1:1,000 Phalloidin CruzFluor 647 stain (Santa Cruz Biotechnology, sc-363797), 1:250 α-Smooth Muscle Actin preconjugated antibody (Sigma-Aldrich, C6198), and 1:1000 Goat-anti-Rabbit (secondary for Anti-Collagen I) (ThermoFisher Scientific, A-11034).

Total protein extraction was performed using the M-PER mammalian protein extraction reagent (Thermo Fisher Scientific, Inc.). The concentration of proteins was measured with the bicinchoninic acid (BCA) assay kit (Sigma-Aldrich). Western blot analysis, including gel electrophoresis and transfer, was conducted by means of the Mini-PROTEAN Tetra Cell and Mini Trans-Blot electrophoretic transfer cell system (Bio-Rad Laboratories, Hercules, CA, USA)^{15 (link)}. The membranes were immunoblotted with specific primary antibodies (1:500–1000) against p-p65, p65, p-JNK, JNK, p-IRF3, IRF3, p-TBK1, TBK1, p-ERK, ERK, p-p38, p38, Iκ-Bα, and human IL-1β (Cell Signaling Technology Inc., Danvers, MA, USA); ATF3, COX2, caspase 1, and β-actin (Santa Cruz Biotechnology Inc., Dallas, TX, USA); iNOS (BD Biosciences); IL-6 and mouse IL-1β (R&D Systems Inc.); TNF-α (Thermo Fisher Scientific, Inc.); or NLRP3 (Adipogen, San Diego, CA, USA) with gentle shaking at 4 °C overnight. Next, the membranes were rigorously washed with PBST and incubated with a peroxidase-conjugated anti-mouse or anti-rabbit immunoglobulin G antibody (1:1000) for 2 h. The proteins were detected by means of a SuperSignal West Pico ECL solution (Thermo Fisher Scientific, Inc.) and visualized on a Fuji LAS-3000 system (Fujifilm, Tokyo, Japan) or ChemiDoc Touch Imaging System (Bio-Rad Laboratories).