Renilla glo luciferase assay system

A549 cells transfected with the pDENV-Luc replicon were plated at 15,000 cells/well in 96-well plates, and immediately treated with R1479, and/or, pyrimidine de novo synthesis inhibitors, and/or salvage inhibitors in complete DMEM medium supplemented with 20 μM uridine without antibiotics (Marceau et al, 2016 (link)). Forty-eight hours after drug treatment, cells were lysed and subjected to Renilla luciferase detection, which was measured using the Renilla-Glo Luciferase Assay System (Promega) according to the specifications of the manufacturer.

Madin–Darby canine
kidney (MDCK) epithelial cells, human alveolar epithelial (A549) cells,
and human embryonic kidney 293T (293T) cells were obtained from the
American Type Culture Collection (ATCC, Manassas, VA, USA). Production
of the MDCK cell line stably expressing the WSN HA protein was previously
described.^{24 (link)} MDCK, A549, and 293T cells
were cultured in Dulbecco’s modified Eagle’s medium
(DMEM) (Gibco, Carlsbad, CA, USA) supplemented with 10% fetal bovine
serum (FBS) (HyClone, South Logan, UT, USA) and 1% penicillin–streptomycin
(P/S) (Gibco). All cells were grown at 37 °C and 5% CO₂. Transfection of DNA was performed in Opti-MEM I-reduced serum medium
(Opti-MEM) (Gibco) with Lipofectamine LTX (Invitrogen) in A549 cells
according to the manufacturer’s specifications. For measurement
of luciferase production in reporter assays, the Dual-Luciferase Reporter
Assay System (Promega, Madison, WI, USA) was used. The Renilla-Glo
Luciferase Assay System was used in the high-throughput primary screen
and confirmation screen (Promega).

For DENV inhibition assays (Fig. 3c), human A549 cells were seeded into 24-well plates at a density of 20,000 cells/well and incubated for 24 h. The cells were treated with DMEM containing GSK983 at the indicated concentrations for 4 h at 37 °C. Growth medium containing GSK983 was removed and cells were incubated for 1 h with DENV-Luc (no GSK983) at 37 °C. Following DENV-Luc incubation, cells were washed with 1x PBS and treated with fresh DMEM containing GSK983 at the indicated concentrations. Cells were incubated at 37 °C for an additional 72 h. Where specified, the growth medium was supplemented with 1 mM uridine or 1mM deoxycytidine. DENV-Luc replication was monitored by the production of Renilla luciferase, which was measured using the Renilla-Glo Luciferase Assay System (Promega) according to the specifications of the manufacturer.
For the accompanying cell viability assay (Fig. 3d), A549 cells were seeded into 24-well plates at a density of 20,000 cells/well incubated for 24 h at 37 °C. Cells were then treated with GSK983 at the indicated concentration for 72 h. Where specified, the growth medium was supplemented with 1 mM uridine or 1 mM deoxycytidine. Following 72 h treatment, cells were harvested and the density of viable cells was determined by flow cytometry (FSC/SSC) using a BD Accuri C6 Flow Cytometer.