Figure S5.
Detection of MS2-tagged rRNAs in ribosomes and DNA breaks or apoptosis in LiveArt clonal cells. (A) Representative images to show the colocalization of γH2AX immunofluorescence with MS2-tagged rRNAs indicated by stdMCP-tdTomato in LiveArt clonal cells (3′-ETS-1, ITS1, and 5.8S). 5.8S rRNA tagging by transient transfection (excessive rDNA editing) was shown as a control (second row), in which γH2AX are present in the nucleolus. All images are maximum intensity projections from z stacks. Scale bars, 5 µm. Arrows point to visible MS2-tagged rRNAs. (B) Line scan of the relative fluorescence of the signal indicated by the dotted lines in A. (C) Percentage of nucleoli harboring enriched γH2AX signal under different labeling conditions in A. n = 2 biological replicates. Black line indicates the mean value. (D) Schematic illustration of the workflow to detect MS2-tagged rRNAs in ribosomes using PCR. (E) Western blot to detect nucleolar protein NPM1 or ribosomal protein RPL22 for demonstrating the success to isolate ribosomal fractions with high purity. (F) Primer designs and the corresponding PCR products to show whether MS2 cassette was present in the template DNA. (G) Representative images to show the colocalization of CC3 immunofluorescence with cell nucleus indicated by Hoechst 33342 in wild-type cells (stdMCP-tdTomato stable cell line without rRNA tagging) and LiveArt clonal cells (3′-ETS-1, ITS1, and 5.8S). ITS1 clone treated with RITA to induce apoptosis was shown as a positive control (second row), in which CC3 signal was condensed. All images are maximum-intensity projections from z stacks. Scale bars, 10 µm. Arrows point to condensed CC3 signal. (H) The percentage of CC3 positive rate under different conditions in G. n = 3 biological replicates. Data are shown as mean ± SEM. Source data are available for this figure: SourceData FS5.

Detection of MS2-tagged rRNAs in ribosomes and DNA breaks or apoptosis in LiveArt clonal cells. (A) Representative images to show the colocalization of γH2AX immunofluorescence with MS2-tagged rRNAs indicated by stdMCP-tdTomato in LiveArt clonal cells (3′-ETS-1, ITS1, and 5.8S). 5.8S rRNA tagging by transient transfection (excessive rDNA editing) was shown as a control (second row), in which γH2AX are present in the nucleolus. All images are maximum intensity projections from z stacks. Scale bars, 5 µm. Arrows point to visible MS2-tagged rRNAs. (B) Line scan of the relative fluorescence of the signal indicated by the dotted lines in A. (C) Percentage of nucleoli harboring enriched γH2AX signal under different labeling conditions in A. n = 2 biological replicates. Black line indicates the mean value. (D) Schematic illustration of the workflow to detect MS2-tagged rRNAs in ribosomes using PCR. (E) Western blot to detect nucleolar protein NPM1 or ribosomal protein RPL22 for demonstrating the success to isolate ribosomal fractions with high purity. (F) Primer designs and the corresponding PCR products to show whether MS2 cassette was present in the template DNA. (G) Representative images to show the colocalization of CC3 immunofluorescence with cell nucleus indicated by Hoechst 33342 in wild-type cells (stdMCP-tdTomato stable cell line without rRNA tagging) and LiveArt clonal cells (3′-ETS-1, ITS1, and 5.8S). ITS1 clone treated with RITA to induce apoptosis was shown as a positive control (second row), in which CC3 signal was condensed. All images are maximum-intensity projections from z stacks. Scale bars, 10 µm. Arrows point to condensed CC3 signal. (H) The percentage of CC3 positive rate under different conditions in G. n = 3 biological replicates. Data are shown as mean ± SEM. Source data are available for this figure: SourceData FS5.

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