Figure 3

Analysis of MLV export by fluorescence microscopy. (A) Map of the reporter MS2- RNA. Red arrows correspond to the Cyan3-labelled probe used in FISH. (B) Reporter MS2-RNA was expressed in NIH3T3 or packaging GPE cell lines transfected or not with peGFP-TapΔC and fixed in 4% paraformaldehyde between 8-12 h p.t for FISH analysis using Cy3-MS2 probes as described [24]. Cells were imaged on a 100× NA 1.4 wide-field microscope (Zeiss AxioimagerZ1) equipped with a Coolsnap HQ2 camera. Stacks of images were recorded for Cy3 (MS2 RNA), GFP (TapΔC) and Hoechst (nucleus) fluorescence over the depth of the cell using a Z step of 0.3 μm, and represented as stacked images. Scale bar is 10 μm. All images were processed using the ImageJ software. (C) For quantitative analysis of viral RNA localization, filtered images (by subtraction of the blurred image using a sigma radius of 20) were stacked and automatically thresholded. Values for the integrated density (Mean Fluorescence intensity × Area corresponding to Cy3 signal) in the different regions of interest, nucleus and cytoplasm, were calculated using the Particle Analysis plugin in ImageJ. Data are expressed as the percentage of nuclear signal, with the mean +/− SEM values of 32 cells for each condition.