Dynein and dynactin function in early HSV-1 infection of NHDFs. (A) NHDFs were treated with DMSO solvent control or 200-µM ciliobrevin D for 1 h. Cultures were then infected with HSV-1 at MOI 30 for 3 h in the continued presence of inhibitor. Samples were then fixed and stained for Tyr-tubulin (red) or the major capsid protein ICP5 (green). Nuclei were stained with Hoechst. Representative widefield images are shown. (B) NHDFs were treated with control nontargeting, EB1, or CLIP-170 siRNAs and then fixed and stained for Tyr-tubulin (red) or DIC (green). Representative widefield images are shown. Arrowheads point to concentrations of DIC and Tyr-tubulin at centrosomes. Boxes highlight examples of flecked patterns of DIC staining at the cell periphery in control samples, which appear more diffuse in NHDFs depleted of either EB1 or CLIP-170. (C) NHDFs were treated with control, EB1, CLIP-170, or DCTN1 siRNAs and then infected with HSV-1 at MOI 5 for 6 h. Whole cell extracts were analyzed by WB with the indicated antibodies. Poly A–binding protein served as loading control. (D) NHDFs treated with control or DCNT1 siRNAs were infected with HSV-1 at MOI 20 for 4 h. Fixed samples were stained for Tyr-tubulin (red) or the capsid protein ICP5 (green). Nuclei were stained with Hoechst (blue). Representative widefield images are shown.