FIG. 4.

HSV-1 infection of HaCaT cells upon silencing of Rac1, Cdc42, or RhoA. HaCaT cells were transfected with Rac1-siRNA, Cdc42-siRNA, RhoA-siRNA, or nonspecific control siRNAs, followed by infection with HSV-1 (20 PFU/cell) at 72 h posttransfection. (A) Total protein extracts were prepared, and proteins were resolved on SDS-PAGE gels (15%). Expression of the Rho GTPases was analyzed with mouse anti-Rac1, mouse anti-Cdc42, or mouse anti-RhoA antibodies. Staining of PDI or VDAC was used as a loading control. (B) At 2 h p.i., cells were fixed and costained with TRITC-conjugated phalloidin (red) and mouse anti-ICP0 (green) visualized with Alexa Fluor 488-conjugated anti-mouse immunoglobulin G. Single stainings and merged images of confocal projections are shown. (C) The number of infected cells upon transfection of either of the specific or nonspecific siRNAs was determined by counting about 1,000 cells in at least two independent experiments. Results are mean ± standard deviation values. (D) At 36 h p.i., the virus titer was determined in cells transfected with either of the specific or nonspecific siRNAs in two independent experiments. Open bars indicate the titer of CRV, and filled bars indicate the titer of cell-associated virus (CAV). Bar, 50 μm.