Gal4-based luciferase reporter analysis

Gal4 DNA binding domain (DBD)–based constructs were generated by replacing the NLS-GFP region of NLS-GFP, NLS-GFP-S100A8, or NLS-GFP-S100A9 with Gal4DBD cloned from pCAG-GBP1-10gly-Gal4DBD (plasmid #49438, Addgene). For the Gal4-based reporter assays, Gal4-based constructs (1 μg), pUAS-luc2 (1 μg; plasmid #24343, Addgene), and pCMV-RL (100 ng; catalog #E2261, Promega) were electroporated together into human breast MCF7 cells by the 4D-Nucleofector System with the SE Cell Line 4D-Nucleofector X Kit (Lonza). Forty-eight hours after the electroporation, Firefly and Renilla luciferase activities were measured with the Dual-Glo Luciferase Assay System (Promega). Luciferase activity is calculated as Firefly normalized by Renilla.