Dual luciferase report assay

Firefly luciferase reporter pGL4 plasmids expressing the wild-type or mutated interferon stimulated responsive element (ISRE) CXCL10 promoter were provided by David Proud [33 (link)–35 (link)]. The β-gal reporter control plasmid was used to normalize the transfection efficiency. HepG2 were cultured in 12-well plates and co-transfected with β-gal and either pGL4 empty vector or CXCL10 promoter luciferase reporter constructs using lipofectamine 2000 (Invitrogen). Serum-free medium was replaced with growth medium after 6h. 24 h after transfection, cells were induced by IFN-γ or infected with either Adψ5, AdIRF-2, or combo for 24 h. 48 h after transfection, the cells were lysed. Relative luciferase and β-galactosidase activities were measured with the reporter lysis buffer and luciferase substrate (Promega). The relative luciferase unit (RLU) was measured using the Dual-Luciferase Report Assay (BioTek, Winooski, VT, USA).

The pMIR-REPORTTM miRNA expression reporter vector system (Applied
Biosystems) was used to evaluate miRNA regulation and the β-gal reporter
control plasmid was used to normalize the transfection efficiency [19 (link)]. Huh7 and Hep3B cells were cultured in
a 12-well plate and transfected with 200 ng β-gal combined with 500 ng of
the pMIR-REPORT empty vector or pMIR-IRF-1–3′UTR plasmid.
Furthermore, the cells were co- transfected with the
pMIRIRF-1–3′UTR plasmid and 50 pmol of the miR-301a mimic,
inhibitor and its NC (Ambion), respectively. miRNA NC was used to normalize the
total volume for transfection. Serum-free medium was replaced with growth medium
after 6 h. Relative luciferase and β-galactosidase activities were
measured with the reporter lysis buffer and luciferase substrate (Promega,
Madison, WI, USA). The cells were lysed 48 h after transfection. The relative
luciferase unit (RLU) was measured using the Dual-Luciferase Report Assay
(Bio-Tek, Winooski, VT, USA).

The β-gal reporter control plasmid was used to normalize the transfection efficiency. Huh-7 and Hepa1–6 were cultured in 6-well plate and co-transfected with β-gal and either pGL3 empty vector Basic (Sigma) or pGL3 PD-L1 promoter luciferase reporter using lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA). 24 h after transfection, the cells were induced by IFN-γ for 6 h or infected with AdLacZ, AdIRF-1, or AdIRF-2 for 24 h before relative luciferase and β-galactosidase activities were measured with the reporter lysis buffer and luciferase substrate (Promega, Madison, WI, USA). Serum-free medium was replaced with growth medium after 6 h. The relative luciferase unit (RLU) was measured using the Dual-Luciferase Report Assay (BioTek, Winooski, VT, USA).