Small-molecule STING inhibitor (C-176) preparation

Powdered n-(4-iodophenyl)-5-nitrofuran-2-carboxamide (C-176) (Enamine, Cat. No. EN300-6503757) was dissolved in dimethyl sulfoxide (DMSO) (Biochemical, Cat. No. BIOD0231) to achieve a final concentration of 10mM. Aliquots of stock solution were stored at -20°C until required.

Cell culture

BV2 cells were cultured in Dulbecco’s modified eagle medium (DMEM) (Gibco, Cat. No. 11995-065) containing 5% foetal bovine serum (FBS) (Interpath, Cat. No. SFBSNZ5) and 1% penicillin-streptomycin (Life Technologies, Cat. No. 15070063) at 37 °C/5% CO2. Cells were plated in a 6-well plate at a density of 1 x 106 cells per well. Cells were pre-treated for 30 minutes in media containing 0.1-2µM C-176 or a DMSO vehicle. Following pre-treatment, c-di-GMP (Invivogen, Cat. No. tlrl-nacdg) was added to a final well concentration of 10-20µg/mL for 8 hours. Control wells were treated with an equivalent volume of endotoxin-free water. Following treatments, culture medium was removed, and wells were gently rinsed twice with ice cold phosphate-buffered saline (PBS) (Life Technologies, Cat. No. 18912014). Cells were scraped in PBS using a handheld cell scraper and placed in a cold Eppendorf tube. Samples were centrifuged at 3000g at 4°C for 10 minutes. Supernatants were removed and cell pellets stored at -80°C until required.