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. 2015 Dec 17;90(1):206–221. doi: 10.1128/JVI.01447-15

FIG 9.

FIG 9

Rab7+ late endosomes are not required for langerin-mediated IAV infection. CHO-ctrl, Lec2-ctrl, and Lec2-Lg cells transfected with expression vectors encoding Rab7 or DN Rab7 T22N were incubated at 37°C for 24 h to allow for protein expression. After incubation, cells were infected with 107 PFU BJx109 at 37°C for 1 h, washed, and incubated a further 6 to 8 h before they were fixed and stained for viral NP. (A) Representative images from confocal microscopy show RFP-labeled Rab7 and Rab7 T22N expression in all cell lines (red, left panels), newly synthesized viral NP (green, middle panels), and a merged image (right panels), including staining for double-stranded nuclei acid using DAPI (blue). White arrows indicate examples of cells showing colocalization of Rab7/Rab7 T22N and viral NP. Note that cells are present in the middle panel for Lec2-ctrl cells but did not stain for viral NP. (B) Following transfection and IAV infection, cells were detached using 0.75 mM EDTA in PBS, fixed, and stained for viral NP prior to analysis via flow cytometry. Representative dot plots showing RFP-Rab7/RFP-Rab7 T22N expression (vertical axis) and NP expression (horizontal axis) of CHO-ctrl, Lec2-ctrl, and Lec2-Lg cells are shown. Gates have been included to identify all Rab7/Rab7 T22N+ cells (Q1 and Q4) as well as NP+ cells (Q2 and Q3). Arrows indicate cells with high expression levels of Rab7 or Rab7 T22N. (C) Analysis of transfected and untransfected of CHO-ctrl, Lec2-ctrl, and Lec2-Lg cells shows the percentage of IAV-infected cells in the RFP− fraction of cells transfected with constructs encoding Rab7 (white bars) or DN Rab7 T22N (black bars) (panel i) and the percentage of IAV-infected cells in the RFP+ fraction of cells transfected with constructs encoding Rab7 (white bars) or DN Rab7 T22N (black bars) (panel ii). Data represent the mean (± 1 SD) of triplicate samples and are representative of 2 independent experiments. Statistical significance was assessed using one-way ANOVA with Tukey's post hoc analysis. *, P < 0.05; n.s., not significant.