Translation monitoring and controls. (A) Methodology for labeling and microscopy schematic maps of the two plasmids, scFv-GFP and MCP-RFP, encoding fluorescent proteins that bind the ST and MS2 tags, respectively, both fused to an NLS. (B) Representative images of the GFP/RFP cell line, expressing the scFv-GFP (green) and MCP-RFP (red) proteins, acquired on a widefield microscope. DAPI staining is in cyan, and scale bar is 10 µm. (C) Spot detection with Imaris for nascent Gag (green) and RNA (red). Stack images of a GFP/RFP cell expressing MLV-WT are projected in 3D using the 3D viewer tool in Imaris (on the left). DAPI staining is in light blue. The image on the right shows the spots detected for nascent Gag (green) and RNA (red). Spots outside of the cell were manually removed and spots in the nucleus were excluded using a surface created with DAPI channel. Scale bars are 10 µm. (D) Analysis of the dependence of the red/green spots colocalization on the total number of RNA molecules in the cell. The slope (0.0025) of the simple linear regression function and the low value of the coefficient of determination (R2 = 0.04) indicate a very weak dependence of the percentage of colocalization on the abundance of the red dots in the cells (n = 38 cells). (E) Inhibition of WT FL RNA translation. Comparison between the effects of puromycin and cycloheximide (CHX), which have distinct mechanisms of action. Colocalized red–green dots were quantified, and the proportion of translating FL RNA per cell was calculated for n ≥ 33 cells. The graph shows the mean ± SEM, ns = nonsignificant and ****P ≤ 0.0001 (Mann–Whitney test).
