Figure 2. Role of cofilin in TGEV-mediated rearrangement of actin filaments.

A. Levels of p-cofilin increase early in TGEV infection. Cells were fixed at the indicated time points, stained with anti-p-cofilin antibody, and observed by confocal microscopy. Scale bar = 10μm. B. The distribution of p-cofilin and F-actin early in TGEV infection. At 30 mpi, cells were fixed and stained with anti-p-cofilin antibody, followed by Dylight 488-conjugated goat anti-rabbit IgG. F-actin was stained with phalloidin-TRITC (Red). Scale bar = 10μm. C. Cofilin shRNA block the destruction of actin cytoskeleton caused by TGEV infection. Cells were incubated with cofilin shRNA for 48h, cells were infected with TGEV for 1 h, F-actin was stained with phalloidin-TRITC (Green), Scale bar = 10μm. D. Cofilin shRNA verification. Cells were transfected with three cofilin shRNAs. After 48 h incubation, the expression of cofilin was verified by Western blotting. E. Cofilin shRNAs block TGEV entry. Cells were transfected with lentiviral vectors expressing cofilin shRNAs. After 48 h incubation, cells were infected with TGEV for 1 h and entry of TGEV was evaluated by real-time PCR. F. Verification of expression of WT cofilin and mutant cofilins S3A and S3E. Cells were treated with lentiviral particles expressing WT cofilin or mutant cofilins S3A and S3E. After 48 h incubation, expression of p-cofilin and cofilin were verified by Western blotting. G. The WT cofilin and mutant cofilins S3A and S3E block TGEV entry. Cells were treated with lentiviral particles expressing WT cofilin or mutant cofilins S3A and S3E. After 48 h incubation, cells were infected with TGEV for 1 h and entry of TGEV was evaluated by real-time PCR. H. ARP2/3 inhibitor CK548 promotes TGEV entry. IPEC-J2 cells were pretreated with CK548 at different concentrations at 37°C for 1 h, for details concerning binding and entry assays, see materials and methods. Statistical significance was assessed by Student's t-test. Differences were considered significant at (*) 0.01 < p < 0.05, (**) p < 0.01. Scale bar = 20 μm. TGEV at a multiplicity of infection of 2 (MOI = 2).