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. 2018 Aug 3;8:11642. doi: 10.1038/s41598-018-30051-2

Figure 6.

Figure 6

S100A10 knockout (KO) and A2t KO inhibit the progression of HPV16 from early endosomes to multivesicular endosomes (MVEs). After binding to the cell surface, HPV is endocytosed into early endosomes. (a) Here, HPV16 pseudovirions (PsVs) (0.5 μg/1E6 cells) were added to pre-cooled cells grown on chamber slides, bound for 1 h at 4 °C, washed, and incubated for 1 h at 37 °C. Cells were then fixed with 4% PFA and immunostained for EEA1 (red) to mark early endosomes, HPV16 (green), and nuclei were stained with DAPI (blue). A single confocal slice is shown in each representative image. Scale bar = 10 μm. (b) Quantification of extent of EEA1-HPV16 colocalization was measured as Mander’s coefficient M2 using the JACoP plugin for Fiji. Representative results are shown as the mean ± s.d. (N = 11 images, ≥2 cells/image). (c) Post-entry, HPV travels from early endosomes to MVEs, and directly interacts with CD63, a marker for MVEs. Cells were treated as described in (a), incubated for 7 h at 37 °C, fixed with 4% PFA, and immunostained for CD63 (red) and HPV16 (green), and nuclei were stained with DAPI (blue). A single confocal slice is shown in each representative image. Scale bar = 10 μm. (d) Quantification of extent of CD63-HPV16 colocalization was measured as Mander’s coefficient using the JACoP plugin for Fiji. Representative results are shown as the mean ± s.d. (N = 10 images). Statistics (b,d): 1-way ANOVA with Dunnett’s multiple comparisons test – *P < 0.05, **P < 0.01, ****P < 0.0001.