Th secondary anti-mouse IgG-AF488 (1:1000) in PS for 1 h, and the nuclei were stained by Hoechst 33258 (1:10000) for high-content automated microscopy. This method (referred to as Pinda/perm HA) efficiently distinguishes between the endocytosed and the non-internalized particles. In control samples, the antibody staining was done exclusively either in PS (perm Pinda/perm HA) or in BS (Pinda/HA). In cells following the perm Pinda/perm HA procedure, the endocytosed virus particles could not be distinguished from the non-internalized particles. In Pinda/HA cells, only the noninternalized particles were detected. 3. Acidification (EA assay). The cells were permeabilized with PS for 30 min at RT. The cells were then incubated with mouse monoclonal A1 antibody in PS (1:1000) for 2 h, washed with PBS, and incubated with secondary anti-mouse IgG-AF488 (1:1000) in PS for 1 h together with either DRAQ5 (1:1000) or Hoechst 33258 (1:10000) in 11967625 PS. 4. Fusion (EF assay). IAV stocks were diluted in PBS to 0.1 mg/ml and labeled for 1 h at RT with R18 and SP-DiOC18 (3) at final concentrations of 0.4 mM and 0.2 mM, respectively. The labeled virus particles were filtered through a 0.22 mM-pore filter (Millipore) and stored at 4uC in the dark till use. After internalization and fixation, nuclei were stained with either DRAQ5 (1:1000) or Hoechst 33258 (1:10000) in BS. 5. Uncoating (EU assay). The cell membrane was stained with WGA-AF647 as described above. The cells were permeabilized with PS for 1315463 30 min at RT, and incubated with purified mouse monoclonal antibody HB64 in PS (1:250) for 2 h to stain the viral M1. The cells were washed with PBS, followed by incubation with secondary anti-mouse IgG-AF488 (1:1000). Nuclei were stained with Hoechst 33258 (1:10000). 6. Nuclear import (EI assay). The cells were permeabilized with PS for 30 min at RT, and incubated with mouse monoclonal antibody HB65 (hybridoma supernatant) in PS (1:10) for 2 h to stain the Autophagy incoming viral NP. The cells were washed with PBS, followed by incubation with secondary anti-mouse IgG-AF488 (1:1000). Nuclei were stained with either DRAQ5 (1:1000) or Hoechst 33258 (1:10000). 7. Infection. Newly synthesized NP was detected as described in 6.High-Content Analysis of IAV Entry EventsImage AcquisitionFor high-resolution imaging, specimen on coverslips from 24well plates were mounted on a glass slide with Immu-mount (Thermo Scientific) and viewed on a Zeiss LSM 510 laser scanning confocal microscope. Both 1006 and 636 objectives (1.4 numerical aperture and 161 binning) were used to acquire images. Automated image acquisition of 96-well Matrix plates was performed with a 206objective (0.75 numerical aperture and 161 binning) using Molecular Devices ImageXpress Micro imaging system. From each well, 9 images (363) were acquired for each channel.Supporting InformationFigure S1 IAV binding in the neuraminidase and inhibitor mocktreated cells. A549 cells were treated with 0.25 units/ml neuraminidase at 37uC for 4 h, followed by EB assay. Images were acquired with a confocal microscope. The HA of IAV was stained with Pinda antibody (green), and the cell membrane was stained with WGA (blue). (TIF) Figure S2 Western blot showing the protein amount of ATP6V1B2 in the cells treated with AllStars and ATP6V1B2 siRNAs. b-actin actin was used as loading control. (TIF) Figure S3 IAV infection in AllStars negative andImage AnalysisImage analysis steps were performed using the CellProfiler program [16]. The analysis of all s.Th secondary anti-mouse IgG-AF488 (1:1000) in PS for 1 h, and the nuclei were stained by Hoechst 33258 (1:10000) for high-content automated microscopy. This method (referred to as Pinda/perm HA) efficiently distinguishes between the endocytosed and the non-internalized particles. In control samples, the antibody staining was done exclusively either in PS (perm Pinda/perm HA) or in BS (Pinda/HA). In cells following the perm Pinda/perm HA procedure, the endocytosed virus particles could not be distinguished from the non-internalized particles. In Pinda/HA cells, only the noninternalized particles were detected. 3. Acidification (EA assay). The cells were permeabilized with PS for 30 min at RT. The cells were then incubated with mouse monoclonal A1 antibody in PS (1:1000) for 2 h, washed with PBS, and incubated with secondary anti-mouse IgG-AF488 (1:1000) in PS for 1 h together with either DRAQ5 (1:1000) or Hoechst 33258 (1:10000) in 11967625 PS. 4. Fusion (EF assay). IAV stocks were diluted in PBS to 0.1 mg/ml and labeled for 1 h at RT with R18 and SP-DiOC18 (3) at final concentrations of 0.4 mM and 0.2 mM, respectively. The labeled virus particles were filtered through a 0.22 mM-pore filter (Millipore) and stored at 4uC in the dark till use. After internalization and fixation, nuclei were stained with either DRAQ5 (1:1000) or Hoechst 33258 (1:10000) in BS. 5. Uncoating (EU assay). The cell membrane was stained with WGA-AF647 as described above. The cells were permeabilized with PS for 1315463 30 min at RT, and incubated with purified mouse monoclonal antibody HB64 in PS (1:250) for 2 h to stain the viral M1. The cells were washed with PBS, followed by incubation with secondary anti-mouse IgG-AF488 (1:1000). Nuclei were stained with Hoechst 33258 (1:10000). 6. Nuclear import (EI assay). The cells were permeabilized with PS for 30 min at RT, and incubated with mouse monoclonal antibody HB65 (hybridoma supernatant) in PS (1:10) for 2 h to stain the incoming viral NP. The cells were washed with PBS, followed by incubation with secondary anti-mouse IgG-AF488 (1:1000). Nuclei were stained with either DRAQ5 (1:1000) or Hoechst 33258 (1:10000). 7. Infection. Newly synthesized NP was detected as described in 6.High-Content Analysis of IAV Entry EventsImage AcquisitionFor high-resolution imaging, specimen on coverslips from 24well plates were mounted on a glass slide with Immu-mount (Thermo Scientific) and viewed on a Zeiss LSM 510 laser scanning confocal microscope. Both 1006 and 636 objectives (1.4 numerical aperture and 161 binning) were used to acquire images. Automated image acquisition of 96-well Matrix plates was performed with a 206objective (0.75 numerical aperture and 161 binning) using Molecular Devices ImageXpress Micro imaging system. From each well, 9 images (363) were acquired for each channel.Supporting InformationFigure S1 IAV binding in the neuraminidase and mocktreated cells. A549 cells were treated with 0.25 units/ml neuraminidase at 37uC for 4 h, followed by EB assay. Images were acquired with a confocal microscope. The HA of IAV was stained with Pinda antibody (green), and the cell membrane was stained with WGA (blue). (TIF) Figure S2 Western blot showing the protein amount of ATP6V1B2 in the cells treated with AllStars and ATP6V1B2 siRNAs. b-actin actin was used as loading control. (TIF) Figure S3 IAV infection in AllStars negative andImage AnalysisImage analysis steps were performed using the CellProfiler program [16]. The analysis of all s.