Luciferase Assay of LGALS1 Regulation

The luciferase reporter experiments were performed using the Dual-Luciferase Reporter Assays System (Promega Corporation, Madison, WI, USA). RPE cells were transfected with the pGL4 vector containing one of three fragments spanning nucleotides –500 bp to +67 bp from the LGALS1 transcription start site (promoter region; pGal), +450 bp to +1750 bp (enhancer region; pGal+AP-1), and AP-1 site (TGACTCA)-mutated enhancer region (pGalΔAP-1), as previously described.¹⁴ (link)^,15 (link) Mutation in two HRE sites in the LGALS1 promoter region (CACGC to CAAAC, positions at –441 bp to –437 bp and –427 bp to –423 bp)⁸ (link)^,12 (link) was synthesized and sequenced by Integrated DNA Technologies (Coralville, IA, USA), and subcloned into the pGL4 vector (pGalΔHRE). The pRL-CMV Renilla luciferase plasmid (Promega) was used as internal control. Cells were transfected with plasmid DNA using Lipofectamine LTX with Plus Reagent (Thermo Fisher Scientific) following the manufacturer's protocols.