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Figure 2


Fig. 2. Promoter analysis of cyclophilin B. (A) Sequence analysis of the CypB promoter with the Genomatix MatInspector program. Two putative ERSE elements of the CypB promoter are underlined. (B) Luciferase reporter assay of CypB promoter. Cells were incubated with 1 µM Tg or 10 µg/ml Tm for 24 hours in proliferation condition. Control were the cells without Tg or Tm treatment. The data are expressed as means ± s.d. from five independent experiments. §P<0.05 versus untreated pGL3 basic; *P<0.05 versus untreated pCypB/400; #P<0.05 versus untreated pCypB/400. (C) Mutation analysis of the ERSE consensus-like sequence. Luciferase assay was conducted using pCypB/400-based luciferase reporter constructs containing an ERSE mutated promoter. 1, pGL3 basic vector; 2, pCypB/400; 3, CCAAg mutant construct; 4, aaccT mutant construct; 5, CtTGG mutant construct; 6, aGTGG mutant construct; 7, pCypB/400 containing ERSE consensus sequence. The data are expressed as the means ± s.d. obtained from five independent experiments. *P<0.05 versus Tg-treated pCypB/400; #P<0.05 versus Tm-treated pCypB/400. (D) Activation of the CypB promoter by ATF6 or XBP1. Luciferase assay was conducted with pCypB/400 and one of the following constructs: pcDNA, inactive ATF6, active ATF6, unspliced XBP1, spliced XBP1, or ATF6-siRNA. The data are expressed as the means ± s.d. obtained from five independent experiments. *P<0.05 versus inactive ATF6 transfection; #P<0.05 versus unspliced XBP1 transfection; **P<0.05 versus active ATF6 transfection. pcDNA was employed as a negative control. All luciferase data are shown with the means ± s.d. relative to the basal activity of pCypB/400 (B-D). (E) EMSA and supershift EMSA of the CypB ERSE motif. Nuclear extracts prepared from H9C2 cells exposed to Tm for 24 hours or left unexposed, were incubated with the CypB-ERSE probe or a mutated probe (CGTtt) after preincubation with no antibody or with antiserum specific to ATF6. (F) ChIP assays with antibodies against ATF6, XBP1 or NF-Y along with extracts of H9C2 cells exposed to Tg or Tm for 24 hours or left unexposed. Input, sample representing amplification from a 1:100 dilution of total input chromatin. IgG: pre-immune serum. (G) Western blot with antibodies against ATF6, NF-Y or Lamin B using nuclear extracts. When exposed Tg and Tm, active ATF6 was increased whereas NF-Y remained unchanged. Lamin B was used as a loading control for nuclear extracts.