c-Jun/AP-1 plays a critical role in LTA-induced MMP-9 expression. (A) Time dependence of LTA-induced proMMP-9 expression and activity. Cells were pretreated with tanshinone IIA (TSIIA, 0.1, 1, or 10 μM) for 1 h and then incubated with 50 μg/ml LTA for the indicated time intervals. Conditioned media were collected and assayed for proMMP-9 expression and activity by gelatin zymography. ProMMP-2 expression is shown as an internal control. (B) Cells were pretreated with TSIIA for 1 h and then incubated with 50 mg/ml LTA for 16 h. Total RNA was extracted and analyzed by RT-PCR. (C) Time dependence of LTA-stimulated c-Jun/AP-1 phosphorylation. RBA-1 cells were pretreated with TSIIA for 1 h and then incubated with 50 mg/ml LTA for the indicated time intervals. Phosphorylation of c-Jun was determined by western blot using an anti-phospho-c-Jun (p-c-Jun) antibody. (D) Cells were transfected with a c-Jun shRNA plasmid for 48 h, and incubated with LTA for 24 h. The cell lysates were assayed by western blot using an anti-c-Jun antibody and anti-GAPDH antibody as a control (upper panel). Conditioned media (CM) and cell lysates were analyzed by zymographic analysis and western blot using an anti-GAPDH antibody as a control (lower panel). Data are expressed as mean ± SEM (A-C) or mean (A, C, D) of three independent experiments (n = 3). #P < 0.01, as compared with the cells exposed to LTA alone. The figure represents one of three individual experiments.