Journal: Journal of Virology
Article Title: Porcine Sapovirus-Induced Tight Junction Dissociation via Activation of the RhoA/ROCK/MLC Signaling Pathway
doi: 10.1128/JVI.00051-21
Figure Lengend Snippet: Inhibition of the RhoA/ROCK/MLC pathway restores the gate and fence functions of TJs in PSaV-infected polarized epithelial cells. Confluent LLC-PK cells grown on Transwell filters were untreated or treated with RhoA inhibitor CT04 (2 μg), ROCK inhibitor Y27632 (20 μM), or MLC inhibitor blebbistatin (10 μM) for 1 h at 37°C and then mock inoculated or inoculated with PSaV Cowden strain (MOI of 50) in the presence (A) or absence (B) of GCDCA for 5, 30, or 90 min. (A and B) Paracellular flux of FD4 and FD70 was measured in the apical-to-basolateral direction. The amount of FITC-dextran diffused to the basolateral side of the monolayer was normalized to the average obtained from control LLC-PK cells. As a positive control, confluent LLC-PK cells were treated for 10 min with 1.8 mM EGTA. All experiments were performed in triplicate. Data are presented as means ± standard errors from three independent experiments. Differences were evaluated using a one-way ANOVA. *, P < 0.05; **, P < 0.001; ***, P < 0.0001. (C and D) The distribution of the membrane fluorescent marker BodipyFL-C 12 -sphingomyelin-BSA (5 nmol/ml) loaded onto the apical surface of LLC-PK cells was determined via z-sectioning using a confocal microscope. All experiments were performed in triplicate; panels C and D show representative results.
Article Snippet: Afterward, they were labeled on the apical side for 10 min on ice with a 5-nmol/ml solution of the BodipyFL-C 12 -sphingomyelin-BSA complex (Molecular Probes).
Techniques: Inhibition, Infection, Positive Control, Marker, Microscopy
