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. 2009 Jul 10;5(7):e1000512. doi: 10.1371/journal.ppat.1000512

Figure 3. Inhibition of clathrin assembly reduces the entry and trafficking of KSHV particles to the perinuclear region.

Figure 3

(A and D) HUVEC were treated with chemical inhibitors of clathrin assembly at the indicated doses for 1 h prior to inoculation with KSHV. Cells were inoculated with KSHV in the presence of inhibitors, fixed at 8 hpi (dextrose) or 2 hpi (chlorpromazine), and stained for Orf65+ viral particles (red) and nuclei (blue). (B and E) The total number of nuclei bearing at least one Orf65+ particle was determined and calculated as Orf65+ nuclei (%). In parallel experiments, cells were subjected to the same treatments and evaluated for viability by PI staining. (C and F) The total number of Orf65+ particles docked at each nucleus was determined. (G–H) Inhibition of clathrin assembly reduces the internalization of AlexaFluor 488-transferrin (G) but not AlexaFluor 488-CTB (H). HUVEC were pretreated with inhibitors of clathrin assembly for 1 h prior to exposure to AlexaFluor 488-transferrin or AlexaFluor 488-CTB. The reduced internalization and perinuclear accumulation of transferrin (green) compared to untreated control cells demonstrates the effective inhibition of clathrin-mediated endocytosis while the internalization and perinuclear accumulation of CTB (green) indicates that the inhibitors did not affect caveolae-mediated endocytosis.