Figure 1. Human vaginal epithelial cells internalize semen exosomes.

(A) Purified human semen-derived exosomes (SE, 100µg/ml) were labeled with PKH67Green, added to E6/E7 transformed V428 human vaginal epithelial cells and incubated at 37°C for 3 h. Unbound exosomes were removed by washing and cells were fixed in 2% paraformaldehyde. Uptake of green fluorescent exosomes into V428 cells was detected by confocal microscopy. DAPI was added to cells to detect the nucleus of the cells (blue). Exosomes fused with vaginal cells present with diffuse green fluorescence and intact exosomes endocytosed into cells present with punctate green fluorescence. Scale bar: 30µm. (B) Internalization of PKH67Green-labeled PBS vehicle by V428 human vaginal epithelial cells analyzed by confocal microscopy. DAPI labels cell nucleus (blue). (C) Uptake of PKH67Green-labeled PBS vehicle or 25, 50 or 100 µg/ml of PKH67Green-labeled SE into V428 cells at 24 h post exposure was examined by FACS analysis. (D) Uptake of PKH67Green-labeled PBS vehicle or SE into VK2 human vaginal epithelial cells at 1 h, 3 h and 6 h post exposure was examined by FACS analysis. The latest PKH67Green treated PBS control time point is indicated on the histograms in Figures 1D; there was no change in the MFI of these control samples for earlier time points (not shown). (E) Vesicle uptake efficiency in V428 cells incubated for 24 h with PKH67Green-labeled PBS vehicle or 25 µg/ml of PKH67Green-labeled blood exosomes (BE), liposomes (LIPO) or SE were examined by FACS analysis. (F) Uptake efficiency of PKH67Green-labeled PBS vehicle or PKH67Green-labeled vesicles including BE, LIPO and SE into VK2 human vaginal epithelial cells at 24 h post exposure was examined by FACS analysis. (G) Uptake of PKH67Green-labeled PBS vehicle or 25 µg/ml of PKH67Green-labeled SE into V428 cells pretreated with endocytosis inhibitor Dynasore or macropinocytosis inhibitor EIPA at 24 h post exposure was examined by FACS analysis.