Figure 1

TGF-β1 induces de novo synthesis of MMP-9 and cell migration in RBA-1 cells. (A) Time and concentration dependence of TGF-β1-induced MMP-9 expression. Cells were incubated with various concentrations of TGF-β1 for the indicated time intervals. The conditioned media were collected and analyzed by gelatin zymography. The levels of TGF-β1-induced MMP-9 expression were quantified (right panel). (B) Cells were treated with TGF-β1 (15 ng/ml) for the indicated time intervals. Total RNA was collected and analyzed by RT-PCR. (C) Cells were pretreated with actinomycin D (Act. D, upper panel) or cycloheximide (CHI, lower panel) for 1 h and then incubated with TGF-β1 for 16 h. (D) Cells were pretreated with or without Act.D (1 μM) or CHI (1 μM) before exposure to TGF-β1 for 6 h. The conditioned media and total RNA were collected and analyzed by gelatin zymography (C) and RT-PCR (D). (E) For cell migration, cells were plated on 6-well plates and grew to confluence. Cells were manually scratched with a pipette tip, and then incubated with or without TGF-β1 (15 ng/ml) for 48 h after pretreatment with MMP-2/9 inhibitor (2/9i, 3 μM) for 1 h. Phase contrast images of RBA-1 cells were taken at 48 h in response to TGF-β1. Data are expressed as mean ± SEM (A) or mean (B, C, D) of three independent experiments (n = 3). *P < 0.05; ^#P < 0.01, as compared with the cells exposed to vehicle (A, B) or TGF-β1 (C, D) alone. The image represents one of three similar experiments (n = 3).