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. 2006 Jul 27;25(15):3714–3724. doi: 10.1038/sj.emboj.7601246

Figure 6.

Figure 6

NFATc1 directly regulates c-myc promoter activity. (A) Northern blot analysis demonstrating increased c-myc transcription following transient transfection of Panc-1 cells with an NFATc1 expression construct. Loading control: 28S rRNA. (B) Activities of a reporter gene construct containing the full-length (2778 bp) c-myc promoter sequence. Panc-1 cells were cotransfected with the reporter gene construct and increasing amounts of an NFATc1 expression construct or the NFATc1-specific siRNA#2 and treated with 1 μM CsA where indicated. Firefly luciferase reporter gene activities were normalized to Renilla luciferase activity and expressed as RLA. (C) Location of putative NFAT-binding sites (green bars) within the full-length c-myc promoter fragment. The magnified section shows the sequence of the TGFβ inhibitory element (TIE) with the NFA-T binding site as well as previously described binding sites for the Smad-3, Sp1 and E2F transcription factors. (D) DNA pulldown assays using double-stranded oligonucleotides representing the wild-type (TIE-wt; upper sequence) or mutated (TIE-M1; mutation indicated in lower sequence) TIE. Panc-1 cells were grown in the absence of serum and either left untreated or treated with 1 μM ionomycin for 1 h as indicated. DNA–protein complexes were collected from nuclear extracts by precipitation with streptavidin–agarose beads and analyzed by immunoblotting using an anti-NFATc1 antibody. (E) Activities of reporter gene constructs containing TIE fragments with wild-type (TIE-wt) or mutated (TIE-M1) NFAT-binding sites. Panc-1 cells were cotransfected with the reporter gene constructs and NFATc1 expression vectors as indicated. Firefly luciferase reporter gene activities were normalized to Renilla luciferase activity and expressed as RLA. (F) Activities of reporter gene constructs containing the full-length (2778 bp) c-myc promoter sequence carrying wild-type (c-myc-wt) or mutated (c-myc-M1) NFAT-binding sites within the TIE. Panc-1 cells were cotransfected with the reporter gene constructs and NFATc1 expression vectors as indicated. Firefly luciferase reporter gene activities were normalized to Renilla luciferase activity and expressed as RLA (for colour figure see online).