Journal: PLoS ONE

Article Title: Human Parechovirus 1 Infection Occurs via αVβ1 Integrin

doi: 10.1371/journal.pone.0154769

Figure Lengend Snippet: (A) Immunofluorescence microscopy of antibody-treated SW480 cells infected with HPeV-1. SW480 cells were treated with 15 μg/ml of anti-integrin antibodies for 1 h and subsequently infected with HPeV-1 at a MOI of 10. The cells were incubated with HPeV-1 for 1 h on ice followed by wash and incubation for 6 h at 37°C. The cells were fixed and stained with nuclear stain Hoechst (blue) and HPeV-1 specific antiserum (green). (B) Relative infection efficiency of HPeV-1 in integrin-treated cells. SW480 cells were processed as in Fig 2A. The infection efficiency of HPeV-1 in total of 10 000 cells was counted from microscopic images, and p values were calculated. Percentage of HPeV-1 infection in mock-treated (control) cells was set as 100%. Standard deviations are shown, and ** indicates p<0.001. The cells were imaged with a 10×objective.

Article Snippet: The function blocking β1 integrin antibody 6S6 (MAb 2253) was from Millipore and the β1 integrin activating antibody TS2/16 (sc-53711) was from Santa Cruz.

Techniques: Immunofluorescence, Microscopy, Infection, Incubation, Staining, Control

Journal: PLoS ONE

Article Title: Human Parechovirus 1 Infection Occurs via αVβ1 Integrin

doi: 10.1371/journal.pone.0154769

Figure Lengend Snippet: (A) HPeV-1 internalizes into GE11-β1 but not GE11-KO cells. GE11-KO and -β1 cells were inoculated with HPeV-1 at a MOI of 5 on ice for 1 h followed by incubation for 6 h at 37°C. The cells were fixed, permeabilized, and stained with appropriate stains or antibodies as described in Materials and Methods. Actin filaments are shown in red, HPeV-1 in green and β1 integrin in blue. The β1 integrin expression on the cell surface was also analyzed by flow cytometry (right panel). (B) Clinical HPeV-1 isolates with low passage numbers (152231, 152478 and 452252) act similarly to the prototype. The cells were treated as in (A). (C) CV-A9 infects both GE11-KO and GE11-β1 cells. The CV-A9 infection was performed similarly to the HPeV-1 assay, followed by staining with anti-CV-A9 antibody (green) and DAPI (blue). Microscopic imaging was performed with Zeiss LSM780 confocal microscopy using a Plan-Apochromat objectives (63× / 1.2 oil/water [panels A and B] or with 40×/ 1.2. oil/water [panel C]). Bar 10 μm.

Article Snippet: The function blocking β1 integrin antibody 6S6 (MAb 2253) was from Millipore and the β1 integrin activating antibody TS2/16 (sc-53711) was from Santa Cruz.

Techniques: Incubation, Staining, Expressing, Flow Cytometry, Infection, Imaging, Confocal Microscopy

Journal: PLoS ONE

Article Title: Human Parechovirus 1 Infection Occurs via αVβ1 Integrin

doi: 10.1371/journal.pone.0154769

Figure Lengend Snippet: (A and B) SW480 cells were treated with β1 integrin activating antibody (TS2/16) at 37°C for 1 h after which the cells were inoculated on ice with HPeV-1 at a MOI of 10 followed by incubation for 6 h at 37°C. The cells were fixed, permeabilized and stained. The relative infection efficiency of HPeV-1 was calculated from nine parallel images (totally 20 000 to 40 000 cells per each antibody concentration) obtained with Zeiss Axiovert 200M (10×objective). The percentage of infection of HPeV-1 in mock-treated (control) cells was set as 100%. The error bars indicate standard deviation, * indicates p<0.01 and ** indicates p<0.001. (C) Receptor clustering is not detected during HPeV-1 infection in SW480 cell line. The cells were incubated with activating anti-β1 antibody for 15 min after which a secondary antibody was added, and incubation was continued for another 15 minutes before fixing of the cells (positive control, left panel). HPeV-1 was allowed to bind to SW480 cells for 15 minutes before fixing and staining (middle panel). SW480 cells were incubated with β1 primary antibody for 15 minutes prior to fixation of the cells. Integrin β1 is shown in red, HPeV-1 in green and nuclei in blue. Microscopic imaging was performed using Zeiss LSM780 confocal microscopy using a Plan-Apochromat objective (63× / 1.2 oil/water). Bar 10 μm.

Article Snippet: The function blocking β1 integrin antibody 6S6 (MAb 2253) was from Millipore and the β1 integrin activating antibody TS2/16 (sc-53711) was from Santa Cruz.

Techniques: Incubation, Staining, Infection, Concentration Assay, Control, Standard Deviation, Positive Control, Imaging, Confocal Microscopy

Journal: PLoS ONE

Article Title: Human Parechovirus 1 Infection Occurs via αVβ1 Integrin

doi: 10.1371/journal.pone.0154769

Figure Lengend Snippet: GE11-β1 cells were inoculated on ice for 1 h with HPeV-1 at a MOI of 5 and incubated for 0 (non-permeabilized), 5 and 30 minutes (permeabilized) at 37°C before fixing and staining. HPeV-1 is shown in green and β1 integrin in red. The right panel shows the co-localization analysis performed by BioImageXD software. The software calculates co-localizing voxels between two channels (HPeV-1 and β1 integrin), and these (co-localizing voxels) are shown in white. Microscopic imaging was performed with Zeiss LSM780 confocal microscopy using a Plan-Apochromat objective (63× / 1.2 oil/water). Bar 10 μm.

Article Snippet: The function blocking β1 integrin antibody 6S6 (MAb 2253) was from Millipore and the β1 integrin activating antibody TS2/16 (sc-53711) was from Santa Cruz.

Techniques: Incubation, Staining, Software, Imaging, Confocal Microscopy