Figure 3. Rta activates MEK/ERK-AP-1 pathway, which is required for Rta-induced IL-6 expression. (A) An ERα inhibitor, MPP, an E2F inhibitor, HLM006474 (HLM), a MEK/ERK inhibitor, U0126, a JNK inhibitor, SP600125 (SP) and the solvent control dimethyl sulfoxide (DMSO), were used to treat HONE-1 cells transfected with a vector plasmid (V) or an Rta-expressing plasmid (R). IL-6 concentrations in the cell culture supernatants were quantified by ELISA. Protein expression of Rta and β-actin was examined using an immunoblot assay. (B) HONE-1 cells transfected with a vector plasmid (V) or an Rta-expressing plasmid (R) were treated with the solvent control DMSO or a MEK/ERK inhibitor, PD184352 (PD). The concentrations of IL-6 in the cell culture supernatants were quantified by ELISA. Protein expression of phosphorylated ERK1/2 (ERK-p), total ERK1/2 (ERK), Rta, and β-actin was detected using an immunoblot assay. (C) An AP-1 activation inhibitor, SR11302 (SR), and the solvent control DMSO were used to treat HONE-1 cells transfected with a vector plasmid (V) or an Rta-expressing plasmid (R). IL-6 concentration in the cell culture supernatants was quantified by ELISA. (D) HONE-1 cells co-transfected with an AP-1 reporter plasmid and the indicated effector plasmids (vector plasmid (V) or Rta-expressing plasmid (R)) were treated with SR11302 (SR) or the solvent control DMSO. (E) HONE-1 cells co-transfected with an AP-1 reporter plasmid and the indicated effector plasmids (vector plasmid (V) or Rta-expressing plasmid (R)) were treated with U0126 or the solvent control DMSO. (F) HONE-1 cells co-transfected with an AP-1 reporter plasmid and the indicated effector plasmids [vector plasmid (V) or Rta-expressing plasmid (R)] were treated with PD184352 (PD) or the solvent control DMSO. A dual luciferase reporter assay was used to detect the transcriptional activity of AP-1 proteins.