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

Figure 4.

Figure 4

S100A10 and A2t are involved post-HPV entry into HeLa cells, and in the absence of S100A10 and A2t, HPV is redirected to the lysosome and degraded. (a) WT, S100A10 KO, and A2t KO cells were treated with HPV16 pseudovirions (PsVs) (5 μg/1E6 cells) for 1 h at 4 °C and measured via flow cytometry. Cells were then incubated for 4 h at 37 °C to promote early internalization, and remaining cell surface-bound HPV was measured via flow cytometry. The difference between the amount of surface-bound HPV at 0 h and 4 h serves as a measure of internalization. Mean fluorescent intensity (MFI) of WT was set to 100% and results show the mean ± s.d. for 3 independent replicates (N = 9). (b) pHrodo is a pH-dependent fluorophore that fluoresces in low pH endosomes. Cells were treated with pHrodo-labeled HPV16 (pHrodo-HPV16) VLPs (5 μg/1E6 cells) for 4 h at 37 °C and then measured via flow cytometry. Results show the mean MFI ± s.d., relative to WT HeLa (N = 3), and are representative of at least 3 independent replicates. (c) WT, S100A10 KO, and A2t KO cells were pre-treated with either vehicle control or chloroquine, a lysosome acidification inhibitor for 16 h at 37 °C. Cells were then washed on ice and treated with HPV16 pseudovirions (PsVs) (5 μg/1E6 cells) for 1 h at 4 °C, washed, and incubated for 4 h at 37 °C to promote early internalization. Cells were harvested via in-plate lysis and image shows western blot for HPV16 L1 capsid protein (CAMVIR-1 Ab). Full length HPV L1 is visible as a single band (55 kDa), and lysosome-cleaved HPV L1 (~20 kDa) appears as a double band. Western blot is representative of 3 independent experiments. Western blot images were cropped. See full-length blots with molecular weight standards in Supplementary Fig. 1c. (d) Quantification of western blot band intensity from 3 independent replicates measured in Image StudioTM Lite. Results show mean band intensity ± s.d. (N = 3, normalized actin and relative to WT). Statistics: 1-way ANOVA with Dunnett’s multiple comparison test – ns = not significant, *P < 0.05, ****P < 0.0001.