Figure 3.
Functional evaluation of LiveArt clonal cell lines. (A) Representative images illustrating the changes of stdMCP-tdTomato labeled rRNAs when treated with 5-FU in LiveArt clonal cells (3′-ETS-1, ITS1, and 5.8S). All images are maximum-intensity projections from z stacks. Scale bars, 10 µm. Arrows point to visible MS2-tagged rRNAs. (B) Bar graph showing the percentage of cells with visible rRNA indicated by stdMCP-tdTomato in the absence or presence of 5-FU (n ≥ 124). (C) Total intensity of visible stdMCP-tdTomato spots in each cell was measured under different conditions to generate the plot. Each dot represents a single cell (n = 100). Data are displayed as mean ± SEM. Two-tailed paired t test, ***P ≤ 0.001. (D) Analysis of pre-rRNA processing by Northern blotting. stdMCP-tdTomato stable cell line without rRNA tagging was used as the wild-type control (WT, lane 1), which are the parental cells of the three LiveArt clonal cell lines (lanes 2–4). Probe hybridized to ITS1 region was used for detection. (E) Left: Puromycin incorporation into control and LiveArt cells detected by anti-puromycin blot. GAPDH immunoblot is shown as a loading control (bottom). Right: Graphs show the quantifications of puromycin incorporation, indicating mean ± SEM (n = 3 replicates). (F) Left: Representative examples of polysome profiles from WT and LiveArt clonal cells. Right: Polysome abundance analysis of profiles performed in left. The ratios of polysome to monosome are presented as mean ± SEM (n = 3 replicates). (G) Representative images illustrating the formation of clonal cells after growing from a single cell for 5 d. Cell nuclei were stained by Hoechst 33342. All images are from single focal plane. Scale bars, 100 µm. (H) Quantification of the number of cells per clone in different cell lines in E (n = 3 replicates). Each dot represents a single clone (n ≥ 50). Data are displayed as mean ± SEM. n.s., not significant. One-way ANOVA with Tukey’s post hoc was used to test differences between groups. Source data are available for this figure: SourceData F3.

Functional evaluation of LiveArt clonal cell lines. (A) Representative images illustrating the changes of stdMCP-tdTomato labeled rRNAs when treated with 5-FU in LiveArt clonal cells (3′-ETS-1, ITS1, and 5.8S). All images are maximum-intensity projections from z stacks. Scale bars, 10 µm. Arrows point to visible MS2-tagged rRNAs. (B) Bar graph showing the percentage of cells with visible rRNA indicated by stdMCP-tdTomato in the absence or presence of 5-FU (n ≥ 124). (C) Total intensity of visible stdMCP-tdTomato spots in each cell was measured under different conditions to generate the plot. Each dot represents a single cell (n = 100). Data are displayed as mean ± SEM. Two-tailed paired t test, ***P ≤ 0.001. (D) Analysis of pre-rRNA processing by Northern blotting. stdMCP-tdTomato stable cell line without rRNA tagging was used as the wild-type control (WT, lane 1), which are the parental cells of the three LiveArt clonal cell lines (lanes 2–4). Probe hybridized to ITS1 region was used for detection. (E) Left: Puromycin incorporation into control and LiveArt cells detected by anti-puromycin blot. GAPDH immunoblot is shown as a loading control (bottom). Right: Graphs show the quantifications of puromycin incorporation, indicating mean ± SEM (n = 3 replicates). (F) Left: Representative examples of polysome profiles from WT and LiveArt clonal cells. Right: Polysome abundance analysis of profiles performed in left. The ratios of polysome to monosome are presented as mean ± SEM (n = 3 replicates). (G) Representative images illustrating the formation of clonal cells after growing from a single cell for 5 d. Cell nuclei were stained by Hoechst 33342. All images are from single focal plane. Scale bars, 100 µm. (H) Quantification of the number of cells per clone in different cell lines in E (n = 3 replicates). Each dot represents a single clone (n ≥ 50). Data are displayed as mean ± SEM. n.s., not significant. One-way ANOVA with Tukey’s post hoc was used to test differences between groups. Source data are available for this figure: SourceData F3.

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