Fig 1.
Results of transfection into HK-2 cells of siRNA against claudin-2, occludin or ZO-1.
A: Immunoblots using cells treated with 10, 25, 50 of 100 nM of indicated siRNA for 72 h. Each lane contained protein from a different Transwell filter support of HK-2 cells. Immunoblots were treated with anti-claudin-2 (~22 kDa), anti-occludin (~65 kDa) or anti-ZO-1 (~210 kDa). B: Immunofluorescence microscopy of HK-2 monolayers with junctional localization of claudin-2, occludin and ZO-1 depleted by siRNA transfection. Magnification, x40.
Fig 2.
Effects of claudin-2, occludin and ZO-1 siRNA transfection on expression of other tight junction proteins in HK-2 cells.
A: Immunoblots with antibodies to claudin-2, occludin, or ZO-1 using cells transfected with 25 nM indicated siRNAs for 72 h. GAPDH was the loading control. B: Densitometric analysis showing claudin-2, occludin, or ZO-1 siRNA significant decreased claudin-2 protein. C: Overexpression of occludin protein induced by claudin-2 siRNA transfection. D: Expression of ZO-1 protein was not affected in HK-2 cells by claudin-2 or occludin siRNA transfection. Quantification of data is % volume difference in protein expression, expressed as mean ± SD from 3 independent experiments. *P < 0.05 by Student’s t-test for unpaired data.
Fig 3.
Effects of claudin-2, occludin or ZO-1 siRNA transfection on transepithelial electrical resistance (TER) in HK-2 cell monolayers.
TER was significantly decreased by claudin-2 siRNA transfection, but significantly increased by siRNA transfection against occludin or ZO-1. Data are means ± SD. *P < 0.05 by Student’s t-test for unpaired data.
Fig 4.
Effects of claudin-2, occludin and ZO-1 siRNA transfection on dextran permeability of HK-2 cell monolayers.
A: Flux of 4-kDa FITC-dextran, measured at 24 h, was significantly increased by transfection of siRNA against claudin-2 or occludin. B: Flux of 70-kDa FITC-dextran at 24 h measured epithelial permeability and was significantly increased by siRNA against claudin-2, occludin or ZO-1. Data are mean ± SD. *P < 0.05 by Student’s t-test for unpaired data.
Fig 5.
Effects of siRNA-induced multiple knockdown of tight junction proteins on transepithelial electrical resistance (TER) in HK-2 cell monolayers.
TER was significantly decreased by claudin-2/ZO-1 siRNA cotransfection, but significantly increased by occluding/claudin-2 siRNA co-transfection. When claudin-2, occludin, and ZO-1 were simultaneously inhibited, TER was suppressed. Data are mean ± SD. *P<0.05 vs. vehicle; by Student’s t-test for unpaired data.
Fig 6.
Effects of siRNA-induced multiple knockdown of tight junction proteins on dextran permeability in HK-2 cell monolayers.
A: Measurements of 4-kDa FITC-dextran flux at 24 h with significant increases with siRNA cotransfection against occludin and ZO-1 or claudin-2 and occludin/ZO-1. B: Flux at 24 hrs of 70 kDa FITC-dextran flux was used to measure epithelial permeability, and was significantly increased by transfection with all combinations of siRNA. Data are means ± SD. *P< 0.05 vs. vehicle; by Student’s t-test for unpaired data.