Firefly luciferase assay system

The 293T (1.5 × 10⁵ cells/well) and Vero E6 (1.5 × 10⁵ cells/well) were plated in a 24-well plate, transfected with a total of 0.4 µg DNA or 0.3 µg RNA, cultured for 6–72 h, harvested, and washed with PBS. The cells were lyzed and assayed for the luciferase activity using the Firefly Luciferase Assay system (Promega) according to the manufacturer’s instructions, and an Opticomp Luminometer (MGM Instruments, Hamden, CT, USA).

Relative luciferase activity was determined by using Dual-Luciferase Reporter Assay System, Firefly Luciferase Assay System and Nano-Glo Luciferase Assay System (Promega Corp., Fitchburg, WI, USA) in a GloMax luminometer following cell lysis in Passive Lysis Buffer (Promega Corp., Fitchburg, WI, USA) according to manufacturer’s protocols.

We performed a luciferase assay in order to monitor the transcriptional activity of the NF‐κB in HHPC exposed to acidic bile and corresponding controls, with or without the pharmacologic inhibitor of NF‐κB, BAY 11‐7082. We used Firefly luciferase Assay system (Promega Corporation, Madison, WI, USA), Lipofectamine^® 2000 (Invitrogen™) and pGL4.32[luc2P/NF‐κB‐RE/Hygro] Vector, encoded with the firefly luciferase reporter gene (luc2P) driven by five copies of an NF‐κB enhancer element during the first 48 hour in culture, and control vector (pGL4.27[luc2P/minP/Hygro]), and in accordance with the manufacturer's procedure. Equal number of cells was transfected with NF‐κB or control luciferase vector. We performed triplicate assays for each treatment condition (bile with or without NF‐κB inhibitor and corresponding controls, at pH 4.0 and pH 7.0). At the end of treatments, luminescence was measured using a luminometer (Infinite^® M1000 PRO, TECAN) and i‐control™ software. We expressed NF‐κB activity as ratios of mean values [values for NF‐κB reporter (luc2P/NF‐kB‐RE), against the mean value for control (luc2P)] calculated in treated HHPC for each condition. Finally, we expressed the alterations of NF‐κB activity induced by BAY 11‐7082 as ratios of relative NF‐κB activity (with/without NF‐κB inhibitor). (Data were obtained from three independent experiments).

The 50–92 HA pseudotyped viruses (PVs) encoding firefly luciferase were generated in HEK 293 T cells and treated with exogenous NA (Sigma Aldrich) as previously described [32 (link), 33 (link)]. Then 50 µl of PV stock was mixed gently with an equal volume of pre-warmed MES pH buffer at the indicated pH in 1.5 ml Eppendorf tubes and incubated at 37 °C for 5 min before inoculation of 25 µl per well of confluent HEK 293 T cells in a white bottomed 96-well plate. After 24 hours cell media was removed, and cells were lysed in passive lysis buffer (Promega) before quantification using a firefly Luciferase Assay System (Promega) and a FLUOstar Omega plate reader (BMG Labtech).

Huh-7.5 cells were seeded on 96-well clusters and transfected with in vitro transcribed subgenomic HAV-FLuc replicon or a replication-incompetent mutant (González-López et al., 2018 (link); Yi and Lemon, 2002 (link)) using TransIT-mRNA transfection kit (Mirus Bio, #MIR2250) according to the manufacturer’s instructions. Cells were harvested in 1 × passive lysis buffer (PLB, Promega) at the indicated times post-transfection and luciferase activity was measured using a firefly luciferase assay system (Promega, #E1501). For HAV-NanoLuc assays, cells were lysed for 5–10 min in 1 × PLB and mixed with 1 × substrate for Oplophorus luciferase (NanoLight Technology, #325) according to the manufacturer’s instructions. All luciferase readings were obtained on a BioTek Synergy two microplate reader.