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. 2014 Mar 26;5(9):2664–2677. doi: 10.18632/oncotarget.1872

Figure 3. The inhibition of c-Jun/AP-1 by ISO mediated the suppression of cyclin D1 transcription.

Figure 3

(A) Cl41 cells were pretreated with ISO at the indicated dose for 30 min and then co-incubated with ISO and TPA/EGF (40 ng /ml) for 12 hours. RT-PCR was performed to determine cyclin d1 mRNA levels. β-actin was used as loading control. Data are representative of three independent experiments. (B) Cl41 cells stably transfected with cyclin D1-promoter luciferase reporter were pretreated with ISO in the indicated concentration for 30 min followed by co-incubation with ISO and TPA/EGF (40 ng /ml) for 24 hours. The luciferase activity was measured as described in “Materials and methods”. The results were presented as relative cyclin D1 promoter activity. The symbol (*) indicates a significant decrease as compared with Cl41 cells treated with TPA/EGF alone (P<0.05). (C) Cytoplasmic and nuclear extracts from Cl41 cells (treated with TPA/EGF (40 ng /ml) alone, or pretreated with ISO (50 μM) for 30 min and then co-incubated with ISO and TPA/EGF for 9 hours were prepared as described in “Materials and methods”, and then subjected to Western blot analysis with specific antibodies against Sp-1, c-Fos, p-c-Jun (Ser73), p-c-Jun (Ser63), c-Jun, Jun D, Jun B, p-NF-kB p65, NF-kB p50, NFAT1, and CREB, respectively. (D and E) Cl41 cells were treated with TPA/EGF (40 ng/ml), or pretreated with 50 μM ISO for 30 min and then co-incubated with ISO and TPA/EGF for indicated time periods. The p-c-Jun (Ser73), p-c-Jun (Ser63) and c-Jun expression levels were determined by Western blot analysis. (F) AP-1-luciferase reporter containing seven tandem AP-1 binding sites was stably transfected into Cl41 cells. The transfectants were treated with TPA/EGF (40 ng /ml) alone, or pretreated with ISO in the indicated concentrations for 30 min followed by co-incubation with ISO and TPA/EGF for 24 hours. The results were presented as relative AP-1 activity compared to that treated with TPA/EGF alone. The symbol (*) indicates a significant decrease as compared with the indicated cells treated with TPA/EGF alone (P<0.05). (G) Cyclin D1 promoter-driven Luciferase reporter (-963CD1) or Cyclin D1 promoter-driven Luciferase reporter with AP-1 binding site mutation (-963CD1 mt) were stably transfected into Cl41 cells. The transfectants were pretreated with ISO in the indicated concentrations for 30 min followed by co-incubation with ISO and TPA/EGF (40 ng /ml) for 24 hours. The luciferase activity was measured as described in “Materials and methods”. The results were presented as relative cyclin D1 promoter activity compared to that treated with TPA/EGF alone. The symbol (*) indicates a significant decrease as compared with the indicated cells treated with TPA/EGF alone (P<0.05).