A COX-2-luc plasmid was synthesized to measure COX-2 luciferase activity. A region extended from −459 bp to +9 bp of the COX-2 promoter gene was cloned into the pGL3-basic vector (purchased from Promega Corporation, Fitchburg, WI, USA). Subsequently, HaCaT cells were transfected with the COX-2-luc plasmid, and the COX-2 luciferase activity was determined using the luciferase assay (Promega Corporation). Following various treatments, 5 μL of the supernatant was added to 50 μL luciferase assay solution, and luminescence was measured using a Fluoroskan Ascent FL luminometer (Thermo Fisher Scientific). All experiments were performed at least three times.
The fragment sequence of the COX-2 promoter region was as follows: GACGTACAGACCAGACACGGCGGCGGCGGCGGGAGAGGGGATTCCCTGCGCCCCCGGACCTCAGGGCCGCTCAGATTCCTGGAGAGGAAGCCAAGTGTCCTTCTGCCCTCCCCCGGTATCCCATCCAAGGCGATCAGTCCAGAACTGGCTCTCGGAAGCGCTCGGGCAAAGACTGCGAAGAAGAAAAGACATCTGGCGGAAACCTGTGCGCCTGGGGCGGTGGAACTCGGGGAGGAGAGGGAGGGATCAGACAGGAGAGTGGGGACTACCCCCTCTGCTCCCAAATTGGGGCAGCTTCCTGGGTTTCCGATTTTCTCATTTCCGTGGGTAAAAAACCCTGCCCCCACCGGGCTTACGCAATTTTTTTAAGGGGAGAGGAGGGAAAAATTTGTGGGGGGTACGAAAAGGCGGAAAGAAACAGTCATTTCGTCACATGGGCTTGGTTTTCAGTCTTATAAAAAGGAAGG.
The fragment sequence of the COX-2 promoter region was as follows: GACGTACAGACCAGACACGGCGGCGGCGGCGGGAGAGGGGATTCCCTGCGCCCCCGGACCTCAGGGCCGCTCAGATTCCTGGAGAGGAAGCCAAGTGTCCTTCTGCCCTCCCCCGGTATCCCATCCAAGGCGATCAGTCCAGAACTGGCTCTCGGAAGCGCTCGGGCAAAGACTGCGAAGAAGAAAAGACATCTGGCGGAAACCTGTGCGCCTGGGGCGGTGGAACTCGGGGAGGAGAGGGAGGGATCAGACAGGAGAGTGGGGACTACCCCCTCTGCTCCCAAATTGGGGCAGCTTCCTGGGTTTCCGATTTTCTCATTTCCGTGGGTAAAAAACCCTGCCCCCACCGGGCTTACGCAATTTTTTTAAGGGGAGAGGAGGGAAAAATTTGTGGGGGGTACGAAAAGGCGGAAAGAAACAGTCATTTCGTCACATGGGCTTGGTTTTCAGTCTTATAAAAAGGAAGG.