2.9. Forkhead Response Element (FHRE) Luciferase Assay.

The FHRE was cloned into the pGL3-Basic vector (Promega), which contains the Firefly luciferase gene directly downstream of the FHRE (pGL3-FHRE – a gift from Dr. Michael Greenburg, Boston MA; Addgene #1789). HaCaT cells were grown to ~60% confluency in 3.5 cm plates and transfected with either the FHRE vector or the pGL3-control firefly vector containing the SV-promoter (Promega) as described above. In order to control for transfection efficiency, cells were co-transfected of the pRL-SV40 vector containing the Renilla luciferase reporter gene (Promega). Cells were subjected to UVB irradiation 48 h after transfection. Cells were lysed in 1× Passive Lysis Buffer (Promega) at specific timepoints before and after UVB-exposure and then assayed using the Dual-Luciferase Kit as per the manufacturer’s instructions (Promega). Values listed are fold change in luciferase luminescence at the 1 h and 3 h post-UVB compared to their respective No-UVB control group. The data were normalized to a Renilla-driven luciferase plasmid.