Examination of the membrane permeability during membrane fusion. Staining of 293CD4 cells cotransfected with the Env expression vector and pKcTac-Halo with membrane-impermeable ligand, Alexa Fluor 488 (AF488, shown in green) in the presence (+) or absence (-) of the fusion inhibitor, C34 (2 μM). The names of the expression vectors used are shown in left. Staining and image capturing were performed as described in Methods. For the HIV-1 Env with its native MSD images were captured at 16 h post transfection. The similar assay was performed for CD22MSD mutants at 44 h post transfection. PI: images with propidium iodide staining as described in Methods. Merge: merged images of bright field with AF488 and PI labelings. The bar indicates 40 μm.