TWIST1 interacts with RELA and induces NF-κB transcriptional activity through the TWIST1 WR domain. (A) IL8 promoter-driven relative luciferase activities of SKBR3 and MCF7 RELA KD cells transfected with or without TWIST1. Cells were transfected with control shRNA (shGFP) or shRNA against RELA (shRelA) 24 h before the transfection of IL8 promoter construct with TWIST1 or pcDNA (vector) control followed by luciferase activity measurement 24 h later. Western blots (below) show the representative KD levels of RELA in these cell lines. (B) Relative luciferase activities in HEK293 cells 24 h post-transfection with TWIST1 and/or RELA. WT, IL8 WT promoter; ΔκB, ΔκB IL8 mutant promoter (shown in Figure 2A). (C) Co-IP experiments with α-RELA antibodies or IgG control. Antibodies were incubated with lysates collected from HEK293 cells 24 h post-transfection with RELA and WT or mutant TWIST1. Cells that were not transfected or transfected with WT TWIST1 only were used as negative controls. ‡, presence of Myc-tag on the TWIST1 proteins. (D) Relative luciferase activities driven by 3x NF-κB response elements in SKBR3 cells 24 h post-transfection with indicated constructs or treated with TNF (40 ng/ml). (E) Relative activities of luciferase driven by 3x NF-κB response elements in HEK293 cells 24 h post-transfection with RELA (50 ng) and WT or ΔWR TWIST1. The amounts of TWIST1 DNA constructs transfected are indicated below (ng). Total amount of DNA transfected was kept constant with sham vectors. (A), (B), (D) and (F) are from single representative experiments. Mean ± SD, n = 3, *P ≤ 0.05, ** P ≤ 0.01.