Ca 074 me

The mouse carotid arterial endothelial cells were isolated and characterized as described earlier [20 (link), 21 (link)]. For the TMAO stimulation, cells were treated with TMAO (30 μm) and then incubated for overnight. In case of inhibitors used, the cells were pretreated with 1 mmol/L Z-WEHD-FMK (WEHD; R&D Systems, Minneapolis, MN), cathepsin B inhibitor Ca-074Me (5 μM, Sigma), potassium channel blocker glibenclamide (Glib, 10 μM, Sigma) or ROS scavenger N-acetyl-L-cysteine (NAC, 10 μM, Sigma) for 30 min.

For isolation of hepatic stellate cells, 24 week old female or male mice were used. Briefly; mice were anesthetized by ketamine/xylazine injection and perfused in situ through the inferior vena cava with sequential Pronase E (0.4 mg/ml) and Collagenase D (0.8 mg/ml) solutions. Liver was removed and digested in vitro with Collagenase D (0.5 mg/ml), Pronase E (0.5 mg/ml) and DNAse I (0.02 mg/ml). After 20 minutes, tissue was filtered through a 70 μm mesh. Cells were separated using a Nycodenz gradient centrifugation. The HSC were sorted by gating for tomato positive signaling or seeded into plastic tissue culture flasks in DMEM containing fetal serum, and incubated at 37 °C with CO2 overnight. In order to deplete any macrophage contamination, HSC layer was treated with clodronate liposomes (5mg/ml) for 4 h at 37°C as previously described (15 (link)). The next morning, the culture medium was changed and inflammasome activation in HSC was induced with LPS (1 μg/ml) for 3 h and either adenosine triphosphate (ATP) (5 mM) for 1 h or with 4-hydroxytamoxifen (4-OH Tamoxifen), the active metabolite of tamoxifen, for 24 h. In addition, HSC were co-incubated with CA-074 –Me (20 μM) (Sigma Aldrich) to inhibit Cathepsin B activity. In an independent experiment, HSC were stimulated with TGF-β (100 ng/ml) for 4 h.