Fig. 2
Gag transits through the nucleolus during PFV infection. a Schematic representation of the experimental strategy used to study PFV Gag trafficking through the nucleolus in U373MG cells stably expressing the Gag-TRAP-GFP protein (Gag1-200-RevNoLS-GFP). b U373MG cell lines stably expressing GFP, RevNoLS-GFP, Gag1-200-GFP or Gag-TRAP-GFP were infected with replication competent PFV. After 72 h, the localization of Gag (red staining) was analyzed in fixed cells using a rabbit polyclonal antibody specific of the C-terminal half of Gag (aa 382–648). Images were acquired as described in Fig. 1b. c Virions released in the supernatant 72 h after infection were titrated on FAG indicator cells and the percentage of infected (GFP-positive) cells was measured by flow cytometry. The infectivity of virions produced by GFP-expressing U373MG cells was used for normalization. Results from 4 independent experiments performed in three replicates each are expressed as the mean ± SD (standard deviation). Significance compared to GFP was calculated using a one-way ANOVA statistical test with a Bonferroni Multiple comparison post-test (*p < 0.05; **p < 0.01). d The subcellular localization of Gag was studied in PFV infected U373MG cells treated or not with LMB (10 nM, 6 h) and/or exposed to hypoxia (2% O2, 4 h). At 48 h post-infection, cells were fixed and stained with a mouse polyclonal antibody against full-length Gag (green) and rabbit polyclonal anti-nucleolin antibody (ab 22,758, Abcam, 1:800). Two hundreds cells were counted for each sample. Nuclei were stained with DAPI (blue). Images were acquired as described in Fig. 1b. Scale bar represents 10 µm