Figure 4. MEK-ERK pathways are important for Emdogain-stimulated MG-63 cell-mediated degradation of type I collagen. (A) MG-63 cells were pre-treated for 30 min with U0126 (10 µM) and then incubated in the presence or absence of 100 µg/mL Emdogain for 20 hrs. Cells were cultured on glass coated with 25 µg/mL of the quenched fluorescence substrate, DQ-collagen I. Degradation of type I collagen (green fluorescence) was detected by confocal microscopy (fluorescence: excitation, 488 nm; emission, 530 nm). Pictures were taken at 40x magnification (a–f). (B) The concentrated conditioned media prepared from unstimulated MG-63 cells (lane 1), U0126 (10 µM) (lane 2), Emdogain-stimulated MG-63 cells (lane 3), and Emdogain-stimulated MG-63 cells cultured in the presence of U0126 (10 µM) (lane 4) were separated on 8% SDS-PAGE. The membranes were blotted with anti-MMP-1 antibody and visualized with a Super Signal west pico chemiluminescent substrate. Molecular-weight markers (kDa) are shown in the left column. The same concentrated conditioned media were separated and transferred onto a membrane. (C) The membranes were blotted with anti-MMP-3 antibody and visualized with a Super Signal west pico chemiluminescent substrate. Molecular-weight markers (kDa) are shown in the left column. Quantification of MMP-1 (B) or MMP-3 (C) (upper panel) was performed densitometrically with NIH image software. The peak heights of each density are depicted as percent of maximum value (lower panel). These data are representative of more than 3 independent experiments.