Figure 5.

Pinocytosis is reduced, clathrin-mediated endocytosis is unaffected, and HPV endocytosis remains clathrin-independent in S100A10 KO and A2t KO cells. (a) Bovine serum albumin (BSA) is taken up via pinocytosis. Here, 10μg/mL of fluorescein isothiocyanate-conjugated BSA (FITC-BSA) was added to pre-cooled WT, S100A10 KO, and A2t KO cells at 4 °C, transferred to 37 °C, and analyzed via flow cytometry at 0.5, 1.5, and 3 h. Results show mean fluorescent intensity (MFI) ± s.d. (N = 3) relative to WT HeLa. (b) Transferrin is taken up via clathrin-mediated endocytosis. 10μg/mL of Alexa Flour® 488-conjugated transferrin (transferrin-AF488) was added to cells and analyzed as described in (a). Results show mean fluorescent intensity (MFI) ± s.d. (N = 3) relative to WT HeLa. (c) HPV16 endocytosis is shown to be clathrin-independent. HPV16 pseudovirions (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 clathrin (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 clathrin-HPV16 colocalization was measured as Mander’s coefficient using the JACoP plugin for Fiji. Representative results are shown as the mean ± s.d. (N = 11 images). Statistics (a,b): repeated measures ANOVA (rANOVA) with Dunnett’s multiple comparisons test. Statistics (d): 1-way ANOVA w/ Dunnett’s multiple comparisons test. ns = not significant.