Figure 5.

LPS induces VCAM-1 expression via c-Src/NF-κB. (A) RASFs were pretreated with helenalin (HLN) and then incubated with LPS for 6 h. (B) Cells were transfected with a dominant negative mutant of MyD88, NIK, IKKα or IKKβ, and then treated with 100 μg·mL⁻¹ LPS for 6 h. The protein expression of VCAM-1 was determined. (C) Cells were treated with LPS (100 μg·mL⁻¹) for the indicated time intervals. (D) Cells were pretreated with the indicated inhibitors and then incubated with LPS for 1 h. Cytosol or nuclear extracts were examined by Western blotting. Lamin A and GAPDH were used as marker proteins for nuclear and cytosolic fractions respectively. (E) NF-κB nuclear translocation was identified by immunofluorescence staining. (F) RASFs were pretreated with the indicated inhibitors and then incubated with LPS for 1 h. Nuclear extracts were subjected to EMSA. (G) Cells were transfected with NF-κB reporter gene, treated with LPS (100 μg·mL⁻¹) for the indicated time intervals. (H) Cells were pretreated with the inhibitors for 1 h, and then incubated with LPS for 1 h. NF-κB promoter activity was determined. (I) Cells were transfected with c-Src siRNA, treated with LPS (100 μg·mL⁻¹) for the indicated time intervals. The phosphorylation of IKKα/β were determined by Western blot. All analyses were performed on samples from three RA patients. Results are representative of three independent experiments. In G, *P ≤ 0.05; ^#P ≤ 0.01 versus vehicle alone. Values in A and H are the mean ± SEM. *P ≤ 0.05; ^#P ≤ 0.01 versus LPS alone.