Figure 7.
Bursting kinetics of rDNA transcription under various conditions. (A) Representative images illustrating the changes of MS2-tagged rRNAs (3′-ETS or 5.8S) when cells were stimulated with FBS for 30 min after serum starvation for 24 h. All images are maximum-intensity projections from z stacks. Scale bars, 10 μm. Total intensity of stdMCP-tdTomato spots in each cell was quantified and plotted under different conditions (n ≥ 100). Data are shown as mean ± SEM. Two-tailed paired t test, **P ≤ 0.01, ***P ≤ 0.001. Arrows point to visible MS2-tagged rRNAs. (B) LiveArt imaging snapshots showing transcriptional bursts of rDNA in three 3′-ETS clones under different conditions. stdMCP-tdTomato signal was imaged for 8 h (4-min interval), but only snapshots between 80 and 200 min are shown. See Video 7 for dynamics. Arrows point to visible MS2-tagged rRNAs. (C) Representative traces (red) illustrating the real-time synthesis of MS2-tagged rRNAs in single cells by measuring the maximum intensity of stdMCP-tdTomato spots at all time points. Gray traces denote the background signal. (D) Histograms of burst and pause durations demonstrating the transcriptional bursting of rDNA in three 3′-ETS clones at normal conditions. n ≥ 100. (E) Quantification of burst amplitude defined by the maximum total intensity of stdMCP-tdTomato spots in a burst. n ≥ 100. (F and G) Quantification of burst duration, pause duration, and burst amplitude by imaging 3′-ETS clone 1 and 2, respectively. n ≥ 100. The negative control (NC) results in F and G are the same dataset as that of 3′-ETS-1 in D. Data are all displayed as mean ± SEM. One-way ANOVA with Tukey’s post hoc was used to test differences between groups. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, n.s., not significant.

Bursting kinetics of rDNA transcription under various conditions. (A) Representative images illustrating the changes of MS2-tagged rRNAs (3′-ETS or 5.8S) when cells were stimulated with FBS for 30 min after serum starvation for 24 h. All images are maximum-intensity projections from z stacks. Scale bars, 10 μm. Total intensity of stdMCP-tdTomato spots in each cell was quantified and plotted under different conditions (n ≥ 100). Data are shown as mean ± SEM. Two-tailed paired t test, **P ≤ 0.01, ***P ≤ 0.001. Arrows point to visible MS2-tagged rRNAs. (B) LiveArt imaging snapshots showing transcriptional bursts of rDNA in three 3′-ETS clones under different conditions. stdMCP-tdTomato signal was imaged for 8 h (4-min interval), but only snapshots between 80 and 200 min are shown. See Video 7 for dynamics. Arrows point to visible MS2-tagged rRNAs. (C) Representative traces (red) illustrating the real-time synthesis of MS2-tagged rRNAs in single cells by measuring the maximum intensity of stdMCP-tdTomato spots at all time points. Gray traces denote the background signal. (D) Histograms of burst and pause durations demonstrating the transcriptional bursting of rDNA in three 3′-ETS clones at normal conditions. n ≥ 100. (E) Quantification of burst amplitude defined by the maximum total intensity of stdMCP-tdTomato spots in a burst. n ≥ 100. (F and G) Quantification of burst duration, pause duration, and burst amplitude by imaging 3′-ETS clone 1 and 2, respectively. n ≥ 100. The negative control (NC) results in F and G are the same dataset as that of 3′-ETS-1 in D. Data are all displayed as mean ± SEM. One-way ANOVA with Tukey’s post hoc was used to test differences between groups. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, n.s., not significant.

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