Additional file 3.
Figure S2. IL8 promoter activation is independent of mutations in the TWIST1 bHLH DNA binding domain. (A) Relative luciferase activities of SKBR3, MCF7 or BT549 cells 24 h post-transfection with IL8 WT or ΔE-box mutant promoter constructs together with TWIST1 or pcDNA vector control. WT, WT IL8 promoter; ΔE-box, ΔE-Box IL8 mutant promoter. (B) Relative luciferase activities of SKBR3 and MCF7 cells co-transfected with IL8 promoter reporter plasmid and WT, R118C, ΔWR, S144R K145E ΔWR or ΔbHLH (complete removal of the bHLH domain) mutant TWIST1. (C) Western blot of nuclear TWIST1 in SKBR3 and MCF7 cells that were transfected with or without TWIST1 and/or IκBSR. (D) Relative luciferase activities of IL8 promoter in BT549 cells stably transduced with shRNAs against TWIST1 and transiently transfected with either shGFP or shRelA (24 h). In A, B, D, data shown are from single representative experiments. Mean ± SD, n = 3, *P ≤ 0.05, ** P ≤ 0.01. (E) Western blot of cytoplasmic and nuclear RELA in MCF10A and MCF10ATw cells. Cyto., cytoplasmic; nu., nuclear. (F) Immunoprecipitation of nuclear RELA in HEK293 cells that were transfected with indicated constructs. Normal rabbit IgG was used as controls. (G) ChIP assays using α-TWIST1 antibodies with solublized chromatin collected from HEK293 cells transfected with vector control, TWIST1, or TWIST1 and RELA for 48 h.
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Li et al. BMC Biology 2012 10:73 doi:10.1186/1741-7007-10-73