Dual luciferase assay

Cells were plated in 12-well plates overnight, and the plasmids were transfected by using Lipofectamine 3000 (Invitrogen, Carlsbad, CA, USA). The nucleolin expression plasmid and luciferase reporter plasmids of Sp1-targeted genes were normalized to 1 μg per well. At 4 h after transfection, the transfection mix of each well was replaced with complete media containing different concentrations of phloretin (0, 20, 50, and 100 μM) for 24 h. Cells were then harvested and lysed for dual-luciferase assay according to the kit instruction (Beyotime Biotechnology). Relative luciferase units and fold inductions (compared with relative luciferase of control) were calculated by using the measured luciferase values.

Human TLR4 promoter reporters were the kind gifts from Prof Michael Rehli at the University of Regensburg Medical School. HCT116 cells were seeded in a 24-well plate and were separately transfected with the TLR4 promoter reporter (TLR4-E, mPU.1_0, mPU.1_1, mPU.1_2, mPU.1_5, and pGL3-Basic) using Lipofectamine^TM 3000 Transfection Reagent (Thermo Fisher Scientific) for 8 h, and then treated with or without PA (50 μM) in the presence of 1% fatty acid-free BSA for 48 h. Cells were lysed with 100 μL of lysis buffer and 20 μL of cell lysate was subjected to Dual Luciferase Assay (Beyotime Biotechnology). The firefly luciferase signals were normalized to that of Renilla luciferase by EnVision Mutilabel Reader. The luciferase reporter activity was calculated, all values were expressed as fold induction relative to basal activity.