Cells were washed with phosphate-buffered saline (PBS) and fixed in 3.7% formaldehyde in PBS for 30 min. For detection of sialic acid residues on the surface of cells, apical monolayers were blocked with 3% bovine serum albumin (BSA; Merck, Darmstadt, Germany) in PBS for 30 min and then incubated with 5 μg/mL fluorescein isothiocyanate (FITC)-conjugated
Sambucus nigra lectin (SNA; Vector Laboratories, Burlingame, CA, USA) for 1 h. To confirm the specificity of lectin binding, monolayers were treated with 50 mU Vibrio cholerae ACP-196 ic50 neuraminidase (VCNA; Roche, Almere, Netherlands) for 1 h prior to fixation and then examined with a rapid-scanning confocal laser microscope (Nikon Corp, Tokyo, Japan). Flow cytometry Approximately 106 cells transfected with control
or ST6GAL1 siRNAs were scraped from the culture surface and washed twice with PBS containing 10 mM glycine, and then washed once with buffer 1 (50 mM Tris–HCl, 0.15 M NaCl, 1 mM MgCl2, 1 mM MnCl2, 1 mM CaCl2, pH 7.5). Cells were blocked with 3% BSA-PBS for 1 h on ice and washed in the same manner as described above. After centrifugation, the cell pellet was incubated with FITC-conjugated SNA at room temperature for 30 min, then washed and fixed with 1% paraformaldehyde. After another three washes with PBS, mean fluorescence click here intensities were determined on a fluorescence-activated cell sorter (FACS) Calibur flow cytometer (BD, San Jose, CA, USA) by counting a minimum of 10,000 events. Receptor specificity of virus strains To study the receptor-binding properties of the virus strains used, we enzymatically modified chicken red blood cells (CRBCs) to express either sialic acid (SA)-α2,6-Galactose (Gal) or SAα2,3Gal as previously described [38, 39] with minor modifications. Briefly, SA was removed from 100 μL of 10% CRBCs using 50 mU VCNA at 37°C for 1 h. Subsequent resialylation was performed using
50 μL of 0.5 mU α2,3-(N)-sialyltransferase (Calbiochem, La Jolla, Sucrase CA, USA) or 125 μL of 2 mU α2,6-( N)-sialyltransferase (Japan Tobacco, Shizuoka, Japan), and 1.5 mM cytidine monophospho-N-acetylneuraminic (CMP) sialic acid (Sigma-Aldrich) at 37°C for 30 or 60 min, respectively. Receptor specificity of the virus strains was then determined using standard hemagglutination assays with the modified CRBCs. Influenza virus challenge of ST6GAL1-siRNA transduced epithelial cells All challenge experiments were carried out at a multiplicity of infection (MOI) of 0.01 for 1 h in the presence of N-p-Tosyl-L-phenylalanine JNK inhibitor chloromethyl ketone (TPCK)-trypsin (Sigma-Aldrich). Viral supernatants were harvested at various time points post-infection for TCID50 assays. To obtain dose–response curves, a dilution series of siRNAs were added to cells in 96-well plates in triplicate. Cells were challenged and supernatants were examined as described above .