Figure 1

Protein 14-3-3ɛ increases MMP-2 gene expression in NIH3T3 fibroblast cells. (A) Construction of stable cell lines expressing the 14-3-3ɛ gene. Cell lysates of NIH3T3 cells, either stably expressing the 14-3-3ɛ gene tagged with HA epitope (lane 2 and 3) or empty pMSCV-puro vector (lane 1), were immunoblotted with anti-HA antibody. Gelatinolytic activities of MMP-2 and MMP-9 proteins and their expressions were analyzed in 14-3-3ɛ-expressing and pMSCV-puro-expressing NIH3T3 cells by gelatin zymogram assay (B) and Western blot analysis (C). Conditioned media for gelatin zymography and cell lysates were obtained at the indicated times. (D) Expressions of MMP-2 mRNA were analyzed by RT-PCR analysis in control NIH3T3 cells and 14-3-3ɛ-expressing cells. (E) NIH3T3 cells were transiently transfected with the full-length MMP-2 promoter (pGL-1659/+57-luc) in the presence or absence of the 14-3-3ɛ gene. Cells were subsequently incubated for the indicated times, and luciferase activities were normalized on the basis of β-galactosidase expression to adjust for variations in transfection efficiency. Results are mean values ±SD of three independent experiments.