Fig. 5: GII.4 entry is sensitive to factors controlling endo-lysosomal homeostasis and induces membrane wounding and subsequent wound repair mechanisms.

a GII.4 replication in the presence of PIKfyve inhibitor YM201636 at 1 h (black) and 24 h (red) at 37 ^oC. b GII.4 replication in the presence of vacuolin-1 (a lysosomal exocytosis inhibitor) at 1 h (gray) and 24 h (purple). c GII.4 replication in the presence of acid sphingomyelinase (ASM, amitriptyline) and neutral sphingomyelinase (NSM, GW4869) inhibitors at 1 h (black) and 24 h (blue). d Cell injury determination using propidium iodide (PI) uptake assay. Right panel: Graph quantitating membrane injury calculated by counting number of PI spots (red) counterstained with DAPI (blue) when HIEs were incubated with VLPs (ROI = 10), VLP + Ca²⁺ (ROI = 12) and media (ROI = 10). Significance was calculated using two-way ANOVA, Tukey’s multiple comparisons test. e Immunofluorescence staining showing GII.4-induced lysosomal exocytosis represented by the presence of LAMP-1 on the apical cell surface in media-treated cells compared to VLP-treated cells. LAMP-1 (red) and VP1 (green) colocalization was detected using mouse anti-LAMP-1 mAb and Gp Syd-pAb. (n = 3 HIE replicates). f VP1, ALIX and Gal-3 colocalization in VLP-treated and media-treated cells (1 h at 37^oC) using confocal microscopy. VP1 (green), ALIX (red) and gal-3 (white) were detected using Gp Syd-pAb, rabbit anti-ALIX pAb and rat-anti-gal3. Right panel: Graph showing colocalization between VP1, gal-3 and ALIX as estimated by Pearson Correlation Coefficient using EzColocalization (ROI = 16, black for VLP- and ROI = 17, blue for media-treated HIEs). Error bars indicate mean ± SD and P values were calculated using two-way ANOVA, Šídák’s multiple comparisons test. All the experiments were repeated independently three times with similar results. Error bars for a–c indicate mean ± SD calculated using 2 HIE replicates for 1 h and 3 HIE replicates for 24 h with two technical replicates/sample. P values were calculated using one-way ANOVA, Dunnett’s multiple comparisons test by comparing replication at 24 h to untreated control. Source data are provided as a Source Data file.