Dual glo luciferase assay systems kit

The HCR assay was performed as described previously (Russo et al., 2018 (link)). The plasmids carrying the reporter genes (pShuttle MCS for luciferase and pRL SV40 for renilla) were previously treated with different doses and wavelengths of UV-radiation to generate DNA lesions. 2 × 10⁴ cells were plated in 96-well plates and transfected with the UV-damaged plasmids. The repair of UV-promoted lesions was associated with reactivation of luciferase expression by the Dual-Glo Luciferase Assay Systems kit (Promega), where the luminescence was detected in a GloMax^® luminometer (Promega). The luminescence associated to the plasmid without radiation treatment was considered as 100% of repair.

Promoter assays were performed using Dual-Glo Luciferase Assay Systems kit (Promega) according to manufacturer’s protocol. Briefly, ~5 x 10⁵ HEK293 cells grown in 12-well plates (Corning Inc.) were transiently transfected with pGL3-CA9p promoter plasmids (wild-type or mutant) in the presence of vector control or flag-tagged BZLF1 (wild-type) or ΔTAD-BZLF1 expressing plasmids. 36 h post-transfection, cells were washed with 1 x PBS and suspended in 100 μl of 1 x passive lysis reagent (PLB). After removing the cell debris, 20 μl of the cell lysate was mixed with 100 μl of Luciferase Assay Reagent (LAR), and luminescence was measured in Synergy H1 microplate reader (BioTek).