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. 2018 Feb 2;68(2):347–358. doi: 10.1136/gutjnl-2017-315348

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TonEBP-dependent stimulation of COX-2 requires transcription factor YY1. (A) TonEBP versus COX-2 mRNA abundance in tumours from 25 patients with HCC. (B) Immunohistochemical images of COX-2 and TonEBP in consecutive hepatic sections from two representative patients with HCC. (C) Immunoblots of livers from TonEBP^+/Δ (+/Δ) and their TonEBP^+/+ littermates (+/+) from figure 2A. (D) Immunoblots of HepG2 cells transfected with siRNA followed by treatment with IL-1β. (E) Serum PGE₂ levels in PBS-treated (n=5) or DEN-treated mice (n=8 for each genotype) from figure 2. Mean+SEM. *P<0.05 compared with TonEBP ^+/+. ^#P<0.05 compared with PBS-injected mice. Serum PGE₂ levels in mean+SEM. *P<0.05 compared with DEN-injected TonEBP^+/Δ animals. (F) Lysates of HepG2 cells treated with IL-1β or not (con) as indicated were immunoprecipitated (IP) with normal IgG, anti-YY1 IgG (YY1), normal serum (S) or anti-TonEBP serum (T). The immunoprecipitates and cell lysates were immunoblotted as indicated. (G) HepG2 cells were transfected with siRNA indicated followed by transfection with pGL3 (vector), COX-2 (COX-2 promoter reporter in pGL3) or ΔYY1 (COX-2 whose YY1 binding site was disabled by mutagenesis) (n=3). (H) HepG2 cells were treated with IL-1β as indicated. ChIP was performed using anti-TonEBP IgG, anit-YY1 IgG or normal IgG. YY1 binding region in the COX-2 promoter was quantified using RT-qPCR. Means of two independent experiments are shown. (I) HepG2 cells were transfected with siRNA and treated with IL-1β. ChIP was performed with anti-YY1 IgG. Means of two independent experiments are shown. (J) Nuclear extracts were prepared from HepG2 transfected with siRNA followed by treatment with IL-1β as indicated. Electrophoretic mobility shift assay was performed (see supplementary material and methods). Anti-YY1 IgG or normal IgG was used to supershift the YY1–DNA complex (right). (K) Lysates of HepG2 cells treated with IL-1β were immunoprecipitated. Lysates and immunoprecipitates were immunoblotted for p300 and YY1. (L) Cells transfected with siRNA were immunoprecipitated as above. (M) MEFs from TonEBP^+/+ (+/+) and TonEBP^Δ/Δ mouse (Δ/Δ) were immunoprecipitated. ChIP, chromatin immunoprecipitation; COX-2, cyclo-oxygenase-2; DEN, diethylnitrosamine; HCC, hepatocellular carcinoma; IL, interleukin; MEF, mouse embryo fibroblast; mRNA, messenger RNA; PBS, phosphate-buffered saline; PGE₂, prostaglandin E₂; siRNA, small interfering RNA; TonEBP, tonicity-responsive enhancer-binding protein.