Figure S1.
Imaging rRNAs via tagging various regions in rRNA. (A) Representative SIM images of nucleoli in living HeLa cells. FC: GFP-RPA43, DFC: HaloTag-FBL, GC: BFP-NPM1. Scale bars, 2 μm and 250 nm (right, enlarged image). Insert magnification: 6×. (B) Quantification of the number of cells per clone in cell lines without or with overexpression of three nucleolar markers (RPA43, FBL, and NPM1). Each dot represents a single clone (n ≥ 50). (C) Measurement of 45S pre-rRNA abundance by qPCR. n = 3 biological replicates. Results in B and C are shown as mean ± SEM. Two-tailed unpaired t test was analyzed for B and C. n.s., not significant. (D) Schematic diagram of human 47S pre-rRNA. TSS, transcription start site. rRNAs can be labeled by integrating MS2 sequence into various regions in rDNA, including 5′-ETS, 18S, ITS1, 5.8S, ITS2, 28S, and 3′-ETS. (E) Representative images to show the colocalization of various MS2-tagged rRNAs with standard nucleolar markers in HeLa cells. FC: RPA43, DFC: FBL, GC: NPM1. All the images are maximum-intensity projections from z stacks. Scale bars, 5 µm. Insert magnification: 3×. Arrows point to visible MS2-tagged rRNAs. (F) Quantification of rRNA accumulation indicated by stdMCP-tdTomato signal intensity in the absence or presence of CX-5461. Each dot represents a single cell (n = 100 for all samples). Green line indicates mean ± SEM. (G and H) Left: live-cell imaging snapshots of U2OS (G) or RPE-1 (H) cells showing accumulation of MS2-tagged 3′-ETS rRNA indicated by stdMCP-tdTomato in the absence or presence of ActD. BFP-NPM1 was imaged to reveal the nucleoli. All images are maximum-intensity projections from z stacks. Scale bars, 5 µm. Right: quantitative analysis of three representative cells showing the dynamic change of 3′-ETS rRNA defined by the total intensity of stdMCP-tdTomato spots in the absence or presence of ActD.

Imaging rRNAs via tagging various regions in rRNA. (A) Representative SIM images of nucleoli in living HeLa cells. FC: GFP-RPA43, DFC: HaloTag-FBL, GC: BFP-NPM1. Scale bars, 2 μm and 250 nm (right, enlarged image). Insert magnification: 6×. (B) Quantification of the number of cells per clone in cell lines without or with overexpression of three nucleolar markers (RPA43, FBL, and NPM1). Each dot represents a single clone (n ≥ 50). (C) Measurement of 45S pre-rRNA abundance by qPCR. n = 3 biological replicates. Results in B and C are shown as mean ± SEM. Two-tailed unpaired t test was analyzed for B and C. n.s., not significant. (D) Schematic diagram of human 47S pre-rRNA. TSS, transcription start site. rRNAs can be labeled by integrating MS2 sequence into various regions in rDNA, including 5′-ETS, 18S, ITS1, 5.8S, ITS2, 28S, and 3′-ETS. (E) Representative images to show the colocalization of various MS2-tagged rRNAs with standard nucleolar markers in HeLa cells. FC: RPA43, DFC: FBL, GC: NPM1. All the images are maximum-intensity projections from z stacks. Scale bars, 5 µm. Insert magnification: 3×. Arrows point to visible MS2-tagged rRNAs. (F) Quantification of rRNA accumulation indicated by stdMCP-tdTomato signal intensity in the absence or presence of CX-5461. Each dot represents a single cell (n = 100 for all samples). Green line indicates mean ± SEM. (G and H) Left: live-cell imaging snapshots of U2OS (G) or RPE-1 (H) cells showing accumulation of MS2-tagged 3′-ETS rRNA indicated by stdMCP-tdTomato in the absence or presence of ActD. BFP-NPM1 was imaged to reveal the nucleoli. All images are maximum-intensity projections from z stacks. Scale bars, 5 µm. Right: quantitative analysis of three representative cells showing the dynamic change of 3′-ETS rRNA defined by the total intensity of stdMCP-tdTomato spots in the absence or presence of ActD.

This Feature Is Available To Subscribers Only

or Create an Account

Close Modal
Close Modal