Luciferase reporting system

The interaction between HIF-1α and its SLUG target gene was predicted using the JASPAR database (http://jaspar.binf.ku.dk/) and, subsequently, validated using the dual-luciferase reporter assay. Briefly, the promoter sequence of SLUG carrying the wild type and mutant binding sites was cloned into the dual-luciferase reporter vector, pGL3-basic. The full length sequence of HIF-1α was cloned into the pcDNA3.0 vector to force overexpression of HIF-1α. The SLUG-pGL3-basic and HIF-1α sequences was cotransfected into cells using Lipofectamine 2000. Relative luciferase intensities were detected with a microplate reader at 48 h posttransfection according to instructions provided by the manufacturer of the luciferase reporting system (Promega, Mannheim, Germany).

The interaction between miR-27b-3p and Foxo1 target was predicted using TargetScan Human (http://www.targetscan.org/vert_71/) and was validated using the dual-luciferase reporter assay [29 (link)]. The interaction between GSDMD and Foxo1 was predicated using JASPAR database (http://jaspar.binf.ku.dk/) (updated 2020) [30 ]. The luciferase vectors containing wild-type and mutant 3′UTR reporters of Foxo1, and the dual-luciferase reporter gene vectors of GSDMD promoter carrying the wild-type binding sites of Foxo1 were purchased from Vipotion (Guangzhou, China) and used for dual-luciferase reporter assay. miR-27b-3p mimics and negative control mimics were purchased from GenePharma and used with Lipofectamine 2000 for cellular transfections. The relative fluorescence intensities were detected 48 h after the cell transfection according to the manufacture's instruction of luciferase reporting system (Promega, Mannheim, Germany).