Figure 5.

CtBP mediated the trans-repressive activity of HDGF. A, Reporter gene construct G5-SV-LUC (0.5 ug) and pRL-CMV (50 ng) were cotransfected with (left panel) or without GBD-HDGF (right panel) and increasing amounts of CtBP expression construct (0.5, 1, 2 ug) as indicated in the figure in G7 cells, the dual-luciferase assay was performed as described in Fig 2. *P < 0.05 by one-way ANOVA control versus CtBP (Left panel). B, CtBP alone does not repress SMYD1 gene promoter activity. SMYD1 promoter reporter SMYD1-LUC (1ug) was cotransfected with 50 ng pRL-CMV and increasing amount of CtBP expression construct (1, 2, 3 ug) into G-7 cells. The dual-luciferase reporter assay was performed as described in Fig 3. C, HDGF expression constructs (2ug) were cotransfected with 0, 3 or 4 ug Flag-CtBP in G7 cells for 48 hours, real-time PCR were performed as described in Fig 3 to measure the mRNA levels change of SMYD1 gene. D, 1 ug GFP-HDGF was cotransfected with 1 ug of either conrol or specific miR targeting CtBP expression vectors into MCF-7 cells which cultured in 6 wells plate using lipofectamine 2000 reagent. 72 hours after transfection, the protein lysate were subjected to SDS-PAGE and further western blotting using specific antibodies against CtBP, GFP or Actin as control. Control 1 used empty GFP vector instead of GFP-HDGF. E, Total RNA extracted from MCF-7 cells described in 4D was subjected to real-time PCR. The mRNA levels of both SMYD1 and CtBP in control 1 (GFP vector plus control miR vector) were set to 1.