Gcdca

We performed all infections in triplicate on 100% confluent 4-day-old differentiated HIE (J3 line) monolayers, except when specified that the J2 line was used. In some experiments, we pretreated monolayers with 1% sow bile included in the differentiation medium 48 h before infection. In other experiments, we differentiated HIE monolayers without pretreatment, and infected them in the presence of 500 μmol/L of glycochenodeoxycholic acid (GCDCA; Sigma, St. Louis, MO, USA) or with 500 μM GCDCA plus 50 μM ceramide.
To determine viral infectivity, we inoculated duplicate 96-well plates with 100 μL of fecal filtrate (Technical Appendix) at 1:10, 1:100, and 1:1000 dilution. After 1 h incubation at 37°C in 5% CO_2, we washed the monolayers twice with CMGF^– and added 100 μL of differentiation medium containing 1% sow bile, 500 μmol/L GCDCA, or 500 μM GCDCA plus 50 μmol/L ceramide to each well. For each set of infections, we immediately froze 1 plate at −70°C and incubated a duplicate plate at 37°C in 5% CO₂ for 72 h and froze it at −70°C. We used quantitative reverse transcription real-time PCR to determine the amount of norovirus RNA from input virus and from HIE monolayers at 1 hour postinfection (hpi) and at 72 hpi. Standard curve based on quantified RNA transcripts was included.

Cells were plated at 150,000 cells/cm² and 0.5 mM GCDCA (Sigma-Aldrich) was administered after 48 h of cell culture. The SOD mimetic MnTBAP (manganese (III) tetrakis (4-benzoic acid) porphyrin chloride) (Calbiochem, Darmstadt, Germany) and the mitochondria-targeted antioxidant MitoQ were administered at concentration of 0.1 mg/ml and 1 μM, respectively. Curcumin (diferuloylmethane), Quercetin (Sigma-Aldrich, Missouri, USA), the NOS inhibitor N^ω-Nitro-L-arginine methyl ester hydrochloride (L-NAME; Sigma-Aldrich) and the specific AP-1 inhibitor SR 11302 (Santa Cruz Biotechnology, California, USA) were used at 4 μM, 20 μM, 5 mM and 50 μM, respectively. Cells were harvested at different times according to the parameter under study. MitoQ was synthesized according to the procedure described by Kelso et al. [13 (link)].