FIG 13.

E-Syt1 and E-Syt3 knockdown increases cell-to-cell fusion in infected cells. HeLa cells grown on coverslips were mock transfected (A) or treated for 48 h with each of the different siRNA pools targeting E-Syt1 (B), E-Syt3 (C), or E-Syt1 and E-Syt3 (D). They were subsequently infected at an MOI of 2 with wild-type HSV-1 for 14 h. To visualize cell-to-cell fusion, the cells were fixed and incubated with the membrane marker Alexa Fluor 647-labeled WGA, a lectin that binds to N-acetyl-d-glucosamine and sialic acid present on the cell surface and internal glycoproteins. The stained cells were processed for fluorescence microscopy and randomly (blindly) photographed in order to determine the presence of syncytia, defined as single cells containing two or more nuclei. For clarity, the outlines of some of the cells are indicated by dashed lines. Quantification was done by counting 200 cells per condition in each of 2 independent experiments. The reported values represent averages from these independent experiments, and the error bars indicate the standard errors of the mean. The fluorescence images show typical examples for each condition. Scale bars, 10 μm.