10,000 cells were analyzed using FACS Canto II . order CX-4945 Bafilomycin A1 was used at 50 nM as a negative control. The experiment was repeated three times. For the lipid mixing assay, X-31 virus was labeled with R18/SP-DiOC18 . A549 cells were infected for 1 hour as above. For the acid-bypass assay, trypsinized A549 cells were bound with labeled X-31 virus for 45 min on ice, and subsequently incubated for 2 min at 37 in either pH 6.8 or pH 5.0 medium, and fixed. For FACS analysis the cells were harvested by trypsinization and 5,000 cells were analyzed. For microscopy analysis, cells were grown on coverslips, infected, fixed in 4 R547 formaldehyde, and examined using a Zeiss LSM 510 confocal fluorescence microscope. BafA was used as a negative control. BafA blocks endosome acidification, blocking the low pH-induced conformational change of HA, and lipid mixing of R18/SP-DiOC18 labeled influenza virus in late endosomes. A 50 ��L aliquot of X-31 virus at 0.08 mg/mL was incubated with DMSO, 211, or 136 for 1 hour at room temperature. Trypsin was added to the samples. Acidified samples were acidified to pH 5.0 for 5 minutes by addition of 50mMcitrate pH 3.0 100 mM NaCl, reneutralized with 100 ��Mtris pH 10.0 100 mMNaCl, and left at 37 for 15 minutes to allow digestion to occur. For samples that were not acidified, an equivalent volume of 10 mM HEPES pH 7.5 100 mMNaCl was added to the samples. Trypsin digestion was stopped by addition of 2 mM4- benzenesulfonyl fluoride hydrochloride for 15 minutes. Samples were mixed with nonreducing loading buffer and loaded onto a 10 polyacrylamide gel for SDS-PAGE. The gel was fixed and stained with the Pierce Silver Stain Kit and imaged with a ccd based gel imager . Compound P25H2 was previously found to inhibit cell infection of multiple influenza virus strains with high potency . Derivatives of P25H2 were synthesized and tested for anti-influenza activities in plaque reduction assays . Fig. 1A shows the structure of a particularly potent derivative, 136. A less potent derivative with a similar structure is used for a control because it inhibits virus only at a much higher concentration than 136 . Using X-31 viru
