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. Author manuscript; available in PMC: 2017 Feb 1.
Published in final edited form as: J Immunol. 2015 Dec 30;196(3):1412–1418. doi: 10.4049/jimmunol.1501886

FIGURE 1. TWIST1 binds to the C3 promoter.

FIGURE 1

(A) A schematic representation of TWIST1 binding site on the C3 promoter (−63 to −83). (B) PCR-amplification products using primers encompassing TWIST1 binding site on DNA template prepared by ChIP with TWIST1 antibody or control non-immune IgG were detected by agarose gel electrophoresis and ethidium bromide staining. (C) Results of qRT-PCR using primers encompassing TWIST1 binding site on the DNA templates prepared by ChIP with TWIST1 antibody or control IgG (n=3, p= 0.0017). (D) A schematic representation of reporter constructs generated by cloning the luciferase gene 3′ to the C3 promoter with either wild type (C3wt) or mutant (C3mut) TWIST1 core binding site. Four nucleotides in the TWIST1 core binding site were altered in C3mut (CAGC → TTTT). (E) HeyA8 ovarian cancer cells were co-transfected with plasmid containing C3 promoter constructs (pC3wt or pC3mut), plasmid containing TWIST1 cDNA, and plasmid containing Renilla luciferase. Promoterless luciferase plasmid (pGL3) was used instead of plasmids with C3 promoter constructs in the control group. Luciferase activity decreased by 66% in HeyA8 cells co-transfected with pC3mut plasmid as compared to that with pC3wt (n=3, p=0.001)