FIGURE 4.

Overexpression of spliced XBP1 inhibited TNF-α-induced ICAM-1 and VCAM-1 and leukocyte adhesion via suppression of NF-κB activation in HREC. Overexpression of spliced XBP1 was achieved by infection of HREC with adenovirus encoding with the spliced form of XBP1 (XBP1S; Ad-XBP1S) at a multiplicity of infection of 20 for 48 h. Adenovirus encoding GFP (Ad-GFP) were used as control. After infection with Ad-GFP and Ad-XBP1S, HREC were treated with 10 ng/ml TNF-α for 0–24 h. Cells were harvested for biochemical assays (A, B, D, E, and F) or used for leukocyte adhesion assay (C). A and B, expression of ICAM-1 (A) and VCAM-1 (B) was determined by Western blot (IB) analysis and semiquantified by densitometry. C, THP-1 monocytes were co-cultured with HREC for 3 h, and adherent monocytes were counted from three different visual fields and expressed at mean ± S.D. C, a, Ad-GFP; C, b, Ad-GFP+TNF-α; C, c, Ad-XBP1S; C, d, Ad-XBP1S+TNF-α. D, phosphorylation NF-κB p65 (Ser⁵³⁶) was determined in HREC after treatment with TNF-α for 0.5–24 h. E, agarose oligonucleotide pulldown assay. Nuclear extract from cells was incubated with agarose beads coated by the NF-κB consensus sequence. After intensive wash, bound NF-κB was determined by Western blot analysis. Level of nuclear membrane protein P62 in the input was used as loading control. F, the transcriptional activity of NF-κB was quantified using ELISA-based TransAM NF-κB activity assay. *, p < 0.05; **, p < 0.01 versus Ad-GFP; †, p < 0.05; ‡, p < 0.01 versus Ad-GFP+TNF-α.