Characterizations of additional 3′-ETS clones. (A) Representative images to show the specific location of MS2-tagged rRNAs labeled by stdMCP-tdTomato in the nucleoli labeled by BFP-NPM1. Scale bar, 5 µm. (B) Line scan of the relative fluorescence of the signal indicated by the dotted lines in A. (C) Quantification of MS2-tagged rRNA accumulation in 3′-ETS clones (3′-ETS-2, 3′-ETS-3) by quantifying the total intensity of stdMCP-tdTomato spots in the absence or presence of ActD. Each dot represents a single cell (n ≥ 100). Data are displayed as mean ± SEM. (D and E) Left: Representative images to show the co-localization of stdMCP-tdTomato (red) with RNA-FISH (green) using FISH probes that could not recognize MS2- tagged rRNAs (D) or could specifically bind to MS2V5_17X in MS2-tagged rRNAs (E) in 3′-ETS clones. Colocalization ratios are indicated on the corresponding images. n ≥ 53 cells. Right: Line scan of the relative fluorescence of the signal indicated by the dotted lines in the images. All images are maximum-intensity projections from z stacks. Nuclei are outlined with white circles. Scale bars, 10 µm (large-field image) and 5 µm (single-cell image).