P4xm67 tk luc plasmid

Luciferase assays were performed with the dual luciferase assay kits (Promega, Madison, WI, USA) according to the manufacturer's instructions. In brief, p4xM67-TK-luc plasmid (Addgene plasmid 8688, Addgene, Cambridge, MA, USA) [32 (link)] containing four copies of the STAT-binding site (TTCCCGTAA) was transfected in 293T or MDA-MB-231 cells and then extracts were treated for 24 hours. EF.STAT3C.UBC.GFP and EF.STAT3DN.UBC.GFP (Addgene plasmids 24983 and 24984, Addgene, Cambridge, MA, USA) [33 (link)] were transfected into 293T or MDA-MB-231 cells, which were subjected to the luciferase assays. Luciferase assays were conducted in quadruplicate and independently repeated at least three times. Representative data were described as means ± standard deviations. For knockdown strategies, pSIH1-puro-STAT3 shRNA (Addgene plasmid 26596, Addgene, Cambridge, MA, USA) [34 (link)] was used.

Cells were seeded in 24-well plates and p4xM67-TK-luc plasmid (Addgene, Cambridge, MA, USA) was transfected in MDA-MB-231 cells by using Lipofectamine reagent (Invitrogen, Carlsbad, CA, USA). Cells were treated with TTB for 6 h, and then luciferase assay was performed by using dual-luciferase reporter assay kits (Promega, Madison, WI, USA) according to the manufacturer’s instructions. All transfections included the RLTK-Luc (kindly provide by Sang Hoon Kim) for transfection efficiency. For ELISA assay, cells were seeded in 6-well plates and treated with TTB. After 24 h, supernatants were harvested and secreted protein levels of VEGF, MMP-9, and IL-6 were performed with human VEGF and MMP-9 ELISA kits (R&D Systems, Minneapolis, MN, USA) and human IL-6 ELISA kit (BD Biosciences, San Jose, CA, USA) according to the manufacturer’s instructions.