Bottom). When the cells were not permeabilized at all during staining

Bottom). When the cells were not permeabilized at all during staining (Pinda/HA), only the non-internalized virus particles were detected. It was observed that in the non-permeabilized cells (Pinda/HA), WGA stained both the cell membrane and the nucleus after fixation, resembling the WGA staining pattern of the permeabilized cells. This indicated that the fixation procedure allowed WGA to access the cytoplasm of the cells. However, the HA1 antibody did not stain viral HA, the ectodomain of which is located in the lumen of endosomes. This observation demonstrated that the EE assay distinguished the endocytosed versus noninternalized virus particles.Detection of the Acid-induced Conversion of HA (EA Assay) and Viral Membrane Fusion (EF Assay)When IAV is exposed to a pH below 5.5, the HA undergoes an irreversible conformational change that can be detected using a monoclonal antibody A1 (EA assay) [9]. When cells with internalized viruses were subjected to IIF using the A1 antibody, the Title Loaded From File labeled HA was, as expected, localized exclusively in the perinuclear vacuoles (Sense 59TGTGGGAATCCGACGAATG-39 and antisense 59- GTCATATGGTGGAGCTGTGGG-39 for N-Cadherin; sense 59CGGGAATGCAGTTGAGGATC-39 and Figure 1d, Table S1c). Conversion of HA to the acid-induced conformation was inhibited by using siRNAbased depletion of ATP6V1B2, a subunit of the vacuolar-ATPase (vATPase) required for endosome acidification (Figure 1d, bottom). Western blotting of AllStars and ATP6V1B2 siRNA-treated cells showed significant decrease of ATP6V1B2 protein expression in the cells treated with ATP6V1B2 siRNA (Figure S2). To monitor fusion 1315463 between the IAV envelope and cellular membranes (EF assay), we used a lipophilic dye-based fluorescence dequenching assay using R18 (red) and SP-DiOC18 (green, fixable). In the labeled virus, the green fluorescence is suppressed by both self-quenching of DiOC18 and fluorescent resonance energy transfer (FRET) from DiOC18 to R18, whereas the red fluorescence from R18 is partly self-quenched [10]. Labeled virus was allowed to enter cells for 1.5 h, and then fixed. When viewed by confocal microscopy, the cells showed numerous perinuclear vacuoles with both red and green fluorescence. This indicated that viral fusion had occurred (Figure 1e, Table S1d). Cells in which acidification of endosomes was inhibited following depletion of ATP6V1B2, only R18 fluorescence was detected, indicating that fusion did not take place (Figure 1e, bottom).Detection of IAV Binding to Cell Membrane (EB Assay) and Viral Endocytosis (EE Assay)To detect binding of viruses to cells (EB assay), we incubated purified IAV with cells at 4uC for 1 h. The cells had been transfected with scrambled control siRNAs called AllStars, which we used as a negative control throughout the study. Indirect immunofluorescence (IIF) using a rabbit polyclonal antibody (Pinda) against HA [6] was used to label the viruses (green), and a fluorescent marker (wheat germ agglutinin, WGA) to define the location of cells (blue) (Figure 1b, Table S1a). By confocal microscopy, the viruses could be visualized as spots distributed over the cells. In a control experiment, we treated the cells with neuraminidase prior to the EB assay. Neuraminidase hydrolyzes the glycosidic linkages between cellular surface glycoproteins and sialic acids, the latter being attachment factor for IAV. We observed almost no binding of IAV particles to the cell membrane of neuraminidase-treated cells, whereas viral binding was normal in the mock-treated cells (Figure S1). To detect endocytosis (EE assay), cells with bound viruses were warmed up to 3.Bottom). When the cells were not permeabilized at all during staining (Pinda/HA), only the non-internalized virus particles were detected. It was observed that in the non-permeabilized cells (Pinda/HA), WGA stained both the cell membrane and the nucleus after fixation, resembling the WGA staining pattern of the permeabilized cells. This indicated that the fixation procedure allowed WGA to access the cytoplasm of the cells. However, the HA1 antibody did not stain viral HA, the ectodomain of which is located in the lumen of endosomes. This observation demonstrated that the EE assay distinguished the endocytosed versus noninternalized virus particles.Detection of the Acid-induced Conversion of HA (EA Assay) and Viral Membrane Fusion (EF Assay)When IAV is exposed to a pH below 5.5, the HA undergoes an irreversible conformational change that can be detected using a monoclonal antibody A1 (EA assay) [9]. When cells with internalized viruses were subjected to IIF using the A1 antibody, the labeled HA was, as expected, localized exclusively in the perinuclear vacuoles (Figure 1d, Table S1c). Conversion of HA to the acid-induced conformation was inhibited by using siRNAbased depletion of ATP6V1B2, a subunit of the vacuolar-ATPase (vATPase) required for endosome acidification (Figure 1d, bottom). Western blotting of AllStars and ATP6V1B2 siRNA-treated cells showed significant decrease of ATP6V1B2 protein expression in the cells treated with ATP6V1B2 siRNA (Figure S2). To monitor fusion 1315463 between the IAV envelope and cellular membranes (EF assay), we used a lipophilic dye-based fluorescence dequenching assay using R18 (red) and SP-DiOC18 (green, fixable). In the labeled virus, the green fluorescence is suppressed by both self-quenching of DiOC18 and fluorescent resonance energy transfer (FRET) from DiOC18 to R18, whereas the red fluorescence from R18 is partly self-quenched [10]. Labeled virus was allowed to enter cells for 1.5 h, and then fixed. When viewed by confocal microscopy, the cells showed numerous perinuclear vacuoles with both red and green fluorescence. This indicated that viral fusion had occurred (Figure 1e, Table S1d). Cells in which acidification of endosomes was inhibited following depletion of ATP6V1B2, only R18 fluorescence was detected, indicating that fusion did not take place (Figure 1e, bottom).Detection of IAV Binding to Cell Membrane (EB Assay) and Viral Endocytosis (EE Assay)To detect binding of viruses to cells (EB assay), we incubated purified IAV with cells at 4uC for 1 h. The cells had been transfected with scrambled control siRNAs called AllStars, which we used as a negative control throughout the study. Indirect immunofluorescence (IIF) using a rabbit polyclonal antibody (Pinda) against HA [6] was used to label the viruses (green), and a fluorescent marker (wheat germ agglutinin, WGA) to define the location of cells (blue) (Figure 1b, Table S1a). By confocal microscopy, the viruses could be visualized as spots distributed over the cells. In a control experiment, we treated the cells with neuraminidase prior to the EB assay. Neuraminidase hydrolyzes the glycosidic linkages between cellular surface glycoproteins and sialic acids, the latter being attachment factor for IAV. We observed almost no binding of IAV particles to the cell membrane of neuraminidase-treated cells, whereas viral binding was normal in the mock-treated cells (Figure S1). To detect endocytosis (EE assay), cells with bound viruses were warmed up to 3.