Figure 1. Actin cytoskeleton dynamics are crucial for TGEV entry.

A. IPEC-J2 cells were incubated with TGEV (MOI = 2) at 4°C for 1 h, shifted to 37°C, and then fixed at the indicated time points. F-actin was stained with phalloidin-TRITC (Red) and observed by confocal microscopy. Scale bar = 20 μm. B. Electron microscopic analysis of ultrathin sections of IPEC-J2 cells infected with TGEV (MOI = 10), the white arrows indicated the podosome and lamellipodium. Scale bar = 150 μm. C. F-actin surround with TGEV particles. TGEV particles were labeled with fluorescent probe Dylight 594, IPEC-J2 cells were incubated with DyLight 594 labeled TGEV at 4°C for 1 h, then shifted to 37°C, fixed at 30 mpi and 60 mpi, F-actin stained with phalloidin (Green). Images were captured with a Zeiss LSM710 confocal laser-scanning microscopy system and rendered three-dimensional (3D) images. Scale bar = 10 μm. D. to F. Concentration-dependent inhibition of TGEV (MOI = 2) entry by cytoskeleton inhibitors. G. and H. Cells were incubated with TGEV (MOI = 2) at 4°C for 1 h, unbound virus removed, and cells were then incubated at 37°C. Levels of p-cofilin, cofilin and p-LIMK were measured by Western blotting using either mAb specific for p-cofilin, or pAb for p-LIMK, cofilin. I. and J. The amount of p-cofilin and cofilin were quantified. Statistical significance was assessed by Student's t-test. Differences were considered significant at (*) 0.01 < p < 0.05, (**) p < 0.01. All experiments were performed separately three times.