Figure 5

Mechanisms involved in the tumor suppression role of YY1. (a) Effects of YY1 on MUC4, ErbB2, MEF2C, MMP10 and MAPK (Erk1/2, JNK and p38) signaling pathways were assayed by western blotting. BXPC-YY1 indicates YY1overexpressing BXPC-3 cells; BXPC-Vector indicates BXPC-3 cells transfected with a control vector; BXPC-YY1 shRNA indicates YY1 knockdown BXPC-3 cells; BXPC-Scramble shRNA indicates BXPC-3 cells transfected with a vector expressing Scramble shRNA. GAPDH was used as the internal control. (b) Bands were quantified by densitometry. Histograms of the ratio (phosphorylated/constitutive form) for Erk1/2, JNK and p38 MAPK kinases are shown. (c) BXPC-YY1 shRNA cells were transfected with MMP10 siRNA or negative control siRNA. 12 h after transfection, cell migration assays were performed. The upper chambers were seeded with various cell lines. The membranes of the chambers were stained with 0.1% crystal violet. Scale bar, 100 μm. (d) Quantification of the data from Figure 5c. OD values from three independent experiments were assessed. (e) Luciferase activities of MMP10 promoter-transfected BXPC-Scramble shRNA or BXPC-YY1 shRNA cells treated with MUC4 siRNA or various inhibitors of signal transduction molecules. MUC4 blockage, the ErbB2 inhibitor (AG825, 25 μM) and the p38 inhibitor (SB203580, 10 μM) significantly inhibited YY1 knockdown-stimulated luciferase activity in BXPC-YY1 shRNA cells transfected with the MMP10 promoter. (f) The YY1 knockdown-stimulated luciferase activity could be significantly inhibited by MEF2C blockage. Luciferase activity of MMP10 promoter in BXPC-YY1 shRNA cells was significantly decreased when the presumed MEF2C binding site (nucleotides −881 to −890) was mutated. (*represents p < 0.05, **represents p < 0.01, # represents p < 0.001, when compared to the control group).