Activation of c-Jun/AP-1 is required for TNF-α-mediated VCAM-1 expression. (A) HCFs were pretreated with TSIIA (0.01, 0.1, or 1 μM) for 1 h, and then incubated with TNF-α for 16 h. The protein levels of VCAM-1 were determined by Western blot. (B) HCFs were pretreated with TSIIA (1 μM) for 1 h, and then incubated with TNF-α for 4 h. The mRNA expression (white bar) and promoter activity (gray bar) of VCAM-1 were determined by real-time PCR or promoter report assay, respectively. (C) HCFs were pretreated with TSIIA (1 μM) for 1 h, and then incubated with TNF-α for the indicated time intervals. The levels of phospho-c-Jun were determined by Western blot. (D) HCFs were transfected with either scrambled or c-Jun siRNA, and then incubated with TNF-α for 16 h. The levels of c-Jun and VCAM-1 protein were determined by Western blot. (E) HCFs were pretreated without or with TNFR nAb (10 μg/ml), Gö6976 (10 μM), edaravone (10 μM), DPI (10 μM), U0126 (1 μM), SP600125 (3 μM) for 1 h, or p38 siRNA transfection, and then incubated with TNF-α for the indicated time intervals. The levels of phospho-c-Jun were determined by Western blot. (F) HCFs were transiently cotransfected with pAP1-Luc and pCMV-Gal for 24 h, and then incubated with TNF-α for the indicated time intervals (F, left). The transfected cells were pretreated with TNFR nAb, Gö6976, DPI, edaravone (Eda) (F, middle), apocynin (APO), SB202190 (SB), SP600125 (SP), or TSIIA (F, right) for 1 h and then incubated with TNF-α for 2 h. The AP-1 transcription activity in the cell was determined as described in Section “Materials and Methods”. Data are expressed as mean ± SEM of three independent experiments (n = 3, Quantitative data of Figures 7C–E were presented in Supplementary Table S2). ∗P < 0.05; #P < 0.01, as compared with the cells exposed to TNF-α alone.