Figure 7

Inhibition of the RhoA/ROCK/MLC pathway restores the gate and fence functions of tight junction in rotavirus-infected polarized epithelial cells. MDCK monolayers grown on transwell filters were untreated or treated with the RhoA inhibitor (CT04), ROCK inhibitor (Y27632), or MLC inhibitor (blebbistatin) for 1 h at 37 °C and then mock-inoculated or inoculated with RVA strain (a) DS-1 or (b) NCDV (MOI = 10) for 5, 60, or 120 min. (a and b) Paracellular flux of 4-kDa FITC-dextran (FD4) and 70-kDa FITC dextran (FD70) was measured in the apical to basolateral direction. The amount of FITC dextran diffused to the basolateral side of the monolayer was normalized by the average obtained from control MDCK cells. As a positive control, MDCK monolayers were treated for 10 min with 1.8 mM EGTA. (c and d) Distribution of the membrane fluorescent marker BodipyFL-C12-sphingomyelin/BSA (5 nmol/ml) loaded onto the apical surface of MDCK cells was determined by z-sectioning using a confocal microscope. All experiments were performed in triplicate; c and d panels show representative results. Data are presented as the mean ± standard error of the mean from three independent experiments. Differences were evaluated using a one-way ANOVA. *p < 0.05; **p < 0.001; ***p < 0.0001.