FIG 9.

Integrin αvβ3 is important for cell-to-cell spread. (a) CaSki cells were transfected with the indicated siRNA and 72 h later were infected with HSV-2(G). Plaques were visualized by immunostaining and representative images are shown. (b) To further assess the impact of integrin αvβ3 on cell-to-cell spread, a modified infectious center assay was performed in which “donor” cells were labeled with Mitotracker Orange, infected with HSV-2(333)ZAG, treated with a low-pH citrate buffer at 1 h p.i. to inactivate any extracellular virus. At 4 h p.i., the donor cells were trypsinized and plated at a ratio of 1:5 on “receiver” cells that had been transfected 72 h earlier with the indicated siRNA and then cocultured in medium containing pooled human immunoglobulin. The cocultures were incubated for 12 h, washed, fixed, and mounted in anti-fade reagent with DAPI to stain nuclei (blue). Representative images from 10 different fields and a minimum of 2 independent experiments are shown. Upper left, DAPI channel; upper right, GFP channel; lower left, Mitotracker Orange; and lower right, merge.