Blebs induction upon ASFV entry in IPAM cells.

A) Field Emission SEM of mock-infected and infected cells. Cells were synchronously infected for 10 min (MOI 50) with E70 after serum starved for 24 h. Membrane perturbations are indicated by arrowheads. A magnification of the cell surface detail (boxes) is shown in the lower right panels. Bars: 1 µM. B) After synchronic infection at different times (E70, MOI 50), the cells were fixed and blebs formation (arrowheads) was analyzed by Phase Contrast Microscopy using a 63× objective. C) Blebbistatin treatment inhibits ASFV entry. Cells were treated with DMSO or Blebbistatin 60 min before the infection (Pre) or treated 60 min after virus addition (Post) and maintained during the infection, at indicated concentrations. After 16 hpi (Ba71V, MOI 1) equivalent amounts of protein were analyzed by immunoblotting with an anti-ASFV antibody. β-actin was detected as a load control. Fold induction was determined by densitometry (mean ±S.D) as shown in the graphic below. D) Rock1 colocalizes with ASFV in blebs (arrows). Cells were infected (Ba71V, MOI 50) and fixed at 30 min after infection. Cells were incubated with, anti-Rock1 (red), anti-p72 (green) and Topro3 (blue) to stain blebs, virus and nuclei, respectively. Images were taken by CLSM and represented as a maximum z-projection of x–y plane and Normasky. Magnifications of the bleb containing Rock1 and viruses (boxes) are shown in the corresponding bottom panels. S.D., standard deviations.