Figure 4

Inhibition of the RhoA/ROCK/MLC signaling pathway affects rotavirus infectivity and viral protein expression. MDCK cells were pretreated with non-cytotoxic concentrations of RhoA inhibitor (CT04), ROCK inhibitor (Y27632), or MLC inhibitor (blebbistatin) for 1 h at 37 °C and then infected with RVA strain DS-1 or NCDV (MOI = 10) for 12 h. (a) Total viral RNA was determined by real-time RT-PCR. (b) Virus titers were determined by cell culture immunofluorescence assay using cell lysates produced by three cycles of freezing and thawing; the results are expressed as fluorescent focus forming unit (FFU). (c and d) Viral VP6 protein was detected by western blot analysis. GAPDH was used as a loading control. The intensity of VP6 relative to GAPDH was determined by densitometric analysis and is indicated above each lane. All experiments were performed in triplicate. Representative images of different gels from each group are presented. 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.