Figure 3. Effect of blocking de novo synthesis of PI(4,5)P₂ with 1-butanol on FMDV and VSV internalization.

(A) BHK-21 cells transfected (24 h) with PH-PLC-eGFP (green) were treated or not with 1.5% 1-butanol or 2-butanol for 5 min and then fixed and observed by confocal microscopy. Nuclei were stained using ToPro-3 (blue). Bar 10 µm. (B) Treatment with 1-butanol inhibits clathrin-dependent endocytosis. BHK-21 cells were treated as in (A) were incubated with fluorescent TF and processed as described in the legend of Fig. 1. Bar: 10 µm. (C) Reduction of the ability of cells to internalize FMDV and VSV upon 1-butanol treatment. Cells treated as in (A) were incubated with the different FMDV variants (C-S8c1 and MARLS) or VSV (MOI of 70 PFU/cell) for 25 min in the presence of the drugs. Bars represent the mean percentage of cells with internalized virions ± SD, normalized to the level of cells with internalized virions in control samples. At least 500 cells per coverslip were scored for each case (3 coverslips). Asterisks denote statistically significant differences (ANOVA P≤0.05).