Skip to main content
. 2010 Dec 7;286(6):4912–4921. doi: 10.1074/jbc.M110.199729

FIGURE 3.

FIGURE 3.

Pharmaceutical and genetic inhibition of XBP1 splicing attenuated the protective effect of ER stress preconditioning in HREC. A and B, HREC were incubated with 0.1 μg/ml tunicamycin (TM) in presence or absence of quinotrierixin (QT) (0.001–1 μm) for 8 h. A, spliced XBP1 was determined by RT-PCR (upper panel) and Western blot analysis (lower panel) using specific antibody against spliced form of XBP1 (XBP1S). B, protein level of XBP1S was semiquantified by densitometry. **, p < 0.01 versus control; ‡, p < 0.01 versus tunicamycin. C and D, HREC were preincubated with 0.1 μg/ml tunicamycin with or without 0.1 μm quinotrierixin for 8 h followed by treatment with 10 ng/ml TNF-α for 24 h. Expression of ICAM-1 (C) and VCAM-1 (D) were determined by Western blot analysis and semiquantified by densitometry. **, p < 0.01 versus control; ‡, p < 0.01 versus TNF-α; #, p < 0.05 versus tunicamycin+TNF-α. E, in vitro leukocyte adhesion assay. HREC were preincubated with 0.1 μg/ml tunicamycin with or without 0.1 μm quinotrierixin for 8 h followed by treatment with 10 ng/ml TNF-α for 4 h, and then THP-1 monocytes were added and co-cultured for 3 h. Adherent monocytes were counted per visual fields and expressed as mean ± S.D. E, a, Control; E, b, TNF-α; E, c, TNF-α+TM; E, d, TNF-α+TM+QT. **, p < 0.01 versus control; ‡, p < 0.01 versus TNF-α; #, p < 0.05 versus tunicamycin+TNF-α. F, HREC were pretreated with 0.1 μg/ml tunicamycin with or without 0.1 μm quinotrierixin for 8 h followed by TNF-α treatment for 4 h. Phosphorylation of NF-κB p65 subunit (Ser536) was determined by Western blot analysis. G–J, HREC were transfected with XBP1 siRNA for 48 h. The knockdown efficiency was determined by protein level of XBP1S in the presence of MG-132 (10 μm) (G). Expression of ICAM-1 (H) and VCAM-1 (I) were determined by Western blot analysis. J, XBP1 siRNA-tranfected cells were pretreated with 0.1 μg tunicamycin for 8 h, followed by TNF-α treatment for 4 h. Phosphorylation of NF-κB p65 (Ser536) was determined. Ctrli, control siRNA; XBP1i, XBP1 siRNA.