Extracellular matrix protein 1 induces matrix metalloproteinase 9 transcription. (A) At 24 hours after seeding, cells were incubated with serum-free medium for a further 24 hours. Supernatant medium from each cell line was reacted with matrix metalloproteinase 9 (MMP9) substrate, and relative fluorescence units were determined at 480 to 620 nm. Cont, Control; shC, Control short-hairpin RNA; shE, Extracellular matrix protein 1 short-hairpin RNA; TR, Trastuzumab-resistant; Vec, Vector. (B) MMP9 mRNA levels were determined by real-time PCR using primers specific for MMP9 (*P < 0.05). Each cell line was transfected with an MMP9 promoter luciferase reporter construct. After 48 hours, cells were harvested, and the lysates were analyzed by dual-luciferase assay (*P < 0.05, ***P < 0.0005). (C) At 24 hours after seeding, each cell line was treated with recombinant human MMP9 (rhMMP9; 20 ng/ml), incubated further for 48 hours, and lysates from each cell were analyzed by Western blotting. EGFR, Epidermal growth factor receptor; ERK, Extracellular signal-regulated kinase; MUC1, Mucin 1; WT, Wild type. (D) Schematic model showing the role of extracellular matrix protein 1 (ECM1) in cell signaling. EGF, Epidermal growth factor.