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Uptake of HSV-1 into dynasore-treated keratinocytes.

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posted on 20.02.2013, 13:53 by Elena Rahn, Philipp Petermann, Mei-Ju Hsu, Frazer J. Rixon, Dagmar Knebel-Mörsdorf

Primary human keratinocytes and HaCaT cells were pretreated with 120 or 80 µM dynasore, and correspondingly with 1.2% or 0.8% DMSO for 30 min at 37°C followed by 15 min at 4°C to precool the cells. Cells were incubated with HSV-1 (1500 PFU/cell) for 1 h at 4°C followed by incubation at 37°C. (A) Infected primary human keratinocytes were fixed and prepared for electron microscopy at 10 min (a, b, e, f), or 30 min at 37°C (g, h). As control, DMSO-pretreatd cells were incubated with HSV-1 (1500 PFU/cell) for 60 min at 4°C (c, d). Bar, 0.2 µm. (B) At 2 h at 37°C infected primary human keratinocytes were fixed and costained with TRITC-phalloidin (red) to visualize F-actin and mouse anti-ICP0. Single immunofluorescence analyses are shown. Bar, 40 µm. (C) Percentages of particles on surface, and particles inside including free cytoplasmic capsids and enveloped particles in vesicles are shown in DMSO- or dynasore-treated primary keratinocytes at 10 and 30 min at 37°C. In two independent experiments 108 (DMSO) and 122 (Dynasore) particles in total were evaluated for the 10 min time point and in one experiment 52 (DMSO) and 62 (Dynasore) particles were analyzed for the 30 min time point. (D) Infected HaCaT cells pretreated with DMSO or dynasore were fixed and prepared for electron microscopy at 10 min at 37°C (a–c). Bar, 0.2 µm. (E) Percentages of particles on surface, and particles inside are shown in DMSO- or dynasore-treated HaCaT cells at 10 min at 37°C. In two independent experiments 78 (DMSO) and 76 (Dynasore) particles in total were evaluated. Results are mean ± standard deviation values.

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