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. 2009 Oct 7;83(24):12822–12832. doi: 10.1128/JVI.01744-09

FIG. 7.

FIG. 7.

Requirement of O3 for syncytium formation. (A) Fusion from within. BS-C-1 cells were infected with vO3-HAi in the absence and presence of IPTG for 18 h at 37°C. The cells were exposed to pH 5.5 or pH 7 buffer (not shown) for 2 min and incubated for an additional 3 h at 37°C in the presence of regular medium. The cells were fixed, stained with DAPI (4′,6-diamidino-2-phenylindole), and examined by phase-contrast and fluorescence microscopy to visualize DAPI and GFP. (B) Fusion from without. BS-C-1 cells were infected with vO3-HAi in the absence and presence of IPTG, and O3− and O3+ virions were purified, respectively. BS-C-1 cells on coverslips were incubated with 200 PFU per cell of O3− or O3+ virions for 1 h at 4°C, exposed to pH 5.5 buffer for 2 min, and incubated for 3 h in regular medium containing 300 μg/ml of cycloheximide at 37°C. The cells were fixed, stained with DAPI and phalloidin (Alexa fluor 594; fluorescent conjugated), and examined by fluorescence microscopy.