FIG 2.

siRNAs targeting α5 and αv back cell-cell fusion triggered by hMPV F protein. (A) Knockdown of integrin α5 and αv expression by siRNA. Twenty picomoles of synthetic siRNA targeting human integrin subtype α5 or αv as well as control siRNA was transfected into Vero E6 cells in 24-well plates using Oligofectamine reagents according to the manufacturer's instructions. After 48 h posttransfection, the expression of α5 or αv was detected by Western blotting. (B) Syncytium formation induced by F protein of hMPV after knockdown of integrins α5 and αv. Vero E6 cells in 24-well plates were transfected with 20 pmol of synthetic siRNA targeting human integrin subtype α5 or αv as well as control siRNA. After treatment with siRNAs for 24 h, Vero E6 cells were transfected with 0.8 μg of pCAGGS-F using Lipofectamine Plus reagents and then subjected to pH pulses (pH 5.0). At 48 h, monolayers were fixed with methanol and stained with Giemsa. (C) Quantitation of syncytium formation after knockdown of integrins α5 and αv. The number of syncytia (≥4 nuclei in each syncytium) was counted under a microscope using six randomly selected fields in each siRNA-treated or untreated well. The mean number of syncytia per field was calculated for each treatment. The percent fusion for each siRNA treatment was normalized by the mean number of syncytia in cells transfected with pCAGGS-F without siRNA treatment. The data shown are averages for three independent experiments.