FIG 3.

The feedback loop between miR-155, Pdcd4, and AP-1 in SAS cells. (a and d) Dual-luciferase reporter activity of wild-type BIC promoter and BIC promoter mutated at the AP-1 or NF-κB binding site in the presence of pcDNA3.1(−) or pcDNA-Pdcd4 in SAS and HEK293T cells (n = 3). (b and e) Expression of miR-155 normalized to that of U6 upon overexpression of Pdcd4 in SAS and HEK293T cells (n = 3). Insets show Western blots of Pdcd4 and β-actin in SAS and HEK293T cells in the presence of pcDNA3.1(−) or pcDNA-Pdcd4. (c and f) AP-1–luciferase activity detected by 4× AP-1 binding sites cloned upstream of a pGL3-luciferase plasmid in the presence of pcDNA-Pdcd4 and pcDNA3.1(−) in SAS and HEK293T cells (n = 3). (g) Dual-luciferase reporter activity of wild-type BIC promoter and BIC promoter mutated at the AP-1 or NF-κB binding site in the presence of pcDNA3.1(−) or pcDNA-BIC in HEK293T cells (n = 3). (h) Fold expression of miR-155 upon ectopic expression of miR-155 in HEK293T cells (n = 3). The inset shows a Western blot for Pdcd4 and β-actin in HEK293T cells in the presence of pcDNA3.1 and pcDNA-BIC. (i) AP-1–luciferase activity detected by 4× AP-1 binding sites cloned upstream of the pGL3-luciferase plasmid in the presence of pcDNA-BIC and pcDNA3.1(+) in HEK293T cells (n = 3). (j) Luciferase activity of wild-type BIC promoter and BIC promoter separately mutated at the AP-1 or NF-κB binding site in the presence of pLCE and pLCE–miR-155 sponge in SAS cells (n = 3). (k) Expression of miR-155 normalized to that of U6 upon ectopic expression of pLCE and pLCE–miR-155 sponge in SAS cells (n = 3). The inset shows a Western blot for expression of Pdcd4 and β-actin in SAS cells upon ectopic expression of pLCE and pLCE–miR-155 sponge. (l) AP-1 activity detected by a 4× AP-1–luciferase construct in the presence of pLCE and pLCE–miR-155 sponge in SAS cells. Values are expressed as the means ± SD (*, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001; ns, not significant).