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.