Fig. 5

Knockdown of galectin-3 reduces the nuclear import of the vRNP complex. a, b Galectin-3-knockdown and vector control A549 cells were infected with IAV at an MOI of 5 for 60 min, fixed, and then permeabilized at 2 h, 4 h, and 7 h p.i. for immunofluorescence staining with the anti-NP antibody. Nuclei were stained with DAPI. Localization of the viral NP indicative of the vRNP complex (a) and quantitative analysis of nucleocytoplasmic distribution of the NP at 7 h p.i (n = 3–5) (b). Representative images are shown (original magnification ×400, scale bar = 50 μm). c, d Galectin-3-knockdown and vector control A549 cells were infected with IAV at an MOI of 100 for 60 min in the presence of cycloheximide (100 µg/ml), fixed, and then permeabilized at 2 h p.i. for immunofluorescence staining with the anti-NP antibody. Nuclei were stained with DAPI. IAV-infected A549 cells in the presence of bafilomycin A1 (100 nM) were used as a positive control for inhibition of vRNP nuclear import. Localization of the viral NP (c) and quantitative analysis of nuclear localization of the NP at 2 h (n = 4) (d). Representative images are shown (original magnification ×200, scale bar = 50 μm). Due to different infectivity of A549 cells transduced with different shRNAs, NP-positive (green staining) cells were examined for their nuclear/cytoplasmic localization at 7 h p.i. (a, b) or nuclear localization at 2 h p.i. (c, d). The NP observed in both the nucleus and cytoplasm is denoted as Nuclear + cytoplasmic, whereas the NP observed only in the nucleus is denoted as Nuclear