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. Author manuscript; available in PMC: 2010 May 27.
Published in final edited form as: Mucosal Immunol. 2008 Sep 17;2(1):33–42. doi: 10.1038/mi.2008.62

Figure 5.

Figure 5

Guanylate-binding protein-1 (GBP-1) downregulation inhibits epithelial barrier formation. (a) Downregulation of GBP-1 by siRNA is efficient and specific in SK-CO15 cells treated with interferon (IFN)-γ and tumor necrosis factor (TNF)-α. Western blot analyses show silencing of GBP-1 in cells transfected with GBP-1-specific siRNA, whereas cells transfected with siRNA specific for cyclophilin B (CyB) show no unspecific silencing. Tubulin is shown as a loading control. The immunofluorescent (IF) image shows specific removal of GBP-1 from the lateral cell membrane. Bar = 20µm. (b) SK-CO15 cells were transfected with siRNA specific for CyB or GBP-1 after 24 h exposure to IFN-γ and TNF-α and transepithelial electrical resistance (TER) was measured every 24 h in five independent experiments (n = 5). Cells transfected with GBP-1 siRNA showed significantly lower TER compared with cells transfected with CyB siRNA 96 h after transfection (*P < 0.05). (c) SK-CO15 cells were transfected with siRNA specific for CyB or GBP-1. After differences in TER were measured fluorescein isothiocyanate (FITC)-labeled 4kDa dextran was added to the apical side and 24 h later the flux was fluorimetrically assessed in four independent experiments (n = 4). Paracellular flux was increased more than twofold in cells transfected with GBP-1 siRNA compared with cells transfected with CyB siRNA (**P < 0.01).