FIG 2.
NLRX-1 is required for RV-induced tight-junction breakdown. 16HBE14o− cells were transfected with NT or NLRX-1 siRNA and allowed to polarize for 48 h. (A) Cells were infected with either RV or sham control and incubated for 24 h, and then RT was measured. (B) Total RNA isolated from sham- or RV-infected cells was used to determine viral RNA copy numbers. (C and D) NP-40-insoluble fractions from cells infected with sham control or RV were subjected to Western blot analysis using antibodies to occludin or β-actin, and band intensities were quantified and expressed as fold change over β-actin. (E) NT or NLRX-1 siRNA-transfected cell cultures were infected with RV or sham control and incubated for 16 h. NTHI was added to the apical surface and incubated for an additional 4 h, and the number of bacteria in the basolateral chamber was determined by plating. (F) Total proteins from cells transfected with NT or NLRX-1 siRNA and infected with sham control or RV were subjected to Western blot analysis with antibody to NLRX-1 or β-actin to confirm knockdown of NLRX-1. The data in panels A, B, and D represent means and SD calculated from 3 or 4 independent experiments (*, P ≤ 0.05; ANOVA). The data in panel D represent medians with ranges from 3 independent experiments (*, P ≤ 0.05; ANOVA on ranks). The images in panels B and E are representatives of 3 or 4 independent experiments.