Figure 8.
Investigation of Pol I transcriptional control using LiveArt. (A) Representative images illustrating the changes of 3′-ETS-1 rRNA when UBF, RRN-3, or SRFBP1 was downregulated by shRNA in clonal cells. BFP protein was imaged to indicate shRNA expression. All images are maximum-intensity projections from z stacks. Scale bars, 10 µm. Arrows point to visible MS2-tagged rRNAs. (B) Left: Bar graph showing the proportion of cells with visible stdMCP-tdTomato spots representing 3′-ETS-1 rRNAs under various conditions in A (n ≥ 178). Right: Quantification of 3′-ETS-1 rRNAs accumulation in each cell by measuring total intensity of individual stdMCP-tdTomato spot. Each dot represents a single cell (n = 100). Gray line indicates mean ± SEM. (C and D) Measurement of 45S pre-rRNA abundance (C) or shRNA efficiency (D) by qPCR. n = three biological replicates displayed as mean ± SEM. (E) Left: Bar graph showing the proportion of cells with visible ITS1 rRNA indicated by stdMCP-tdTomato under different conditions (n ≥ 154). Right: Quantification of ITS1 rRNA accumulation in each cell by measuring total intensity of individual stdMCP-tdTomato spot. Each dot represents a single cell (n = 100). Gray line indicates mean ± SEM. (F) qPCR to examine relative expression of 45S-preRNA (left) and SRFBP1 (right) in SRFBP1 down-regulated samples from E. n = 3 technical replicates. (G) Representative traces (red) demonstrating the real-time synthesis of MS2-tagged rRNAs (3′-ETS-1) by measuring the maximum intensity of stdMCP-tdTomato spots in corresponding cells that were infected with negative control (NC) shRNA or shRNAs to specifically down-regulate SRFPB1 and RRN3, respectively. (H) Quantification of burst duration and pause duration by imaging 3′-ETS-1 clone. n ≥ 100. Data are all displayed as mean ± SEM. One-way ANOVA with Tukey’s post hoc was used to test differences between groups. ***P ≤ 0.001, **P ≤ 0.01. n.s., not significant.

Investigation of Pol I transcriptional control using LiveArt. (A) Representative images illustrating the changes of 3′-ETS-1 rRNA when UBF, RRN-3, or SRFBP1 was downregulated by shRNA in clonal cells. BFP protein was imaged to indicate shRNA expression. All images are maximum-intensity projections from z stacks. Scale bars, 10 µm. Arrows point to visible MS2-tagged rRNAs. (B) Left: Bar graph showing the proportion of cells with visible stdMCP-tdTomato spots representing 3′-ETS-1 rRNAs under various conditions in A (n ≥ 178). Right: Quantification of 3′-ETS-1 rRNAs accumulation in each cell by measuring total intensity of individual stdMCP-tdTomato spot. Each dot represents a single cell (n = 100). Gray line indicates mean ± SEM. (C and D) Measurement of 45S pre-rRNA abundance (C) or shRNA efficiency (D) by qPCR. n = three biological replicates displayed as mean ± SEM. (E) Left: Bar graph showing the proportion of cells with visible ITS1 rRNA indicated by stdMCP-tdTomato under different conditions (n ≥ 154). Right: Quantification of ITS1 rRNA accumulation in each cell by measuring total intensity of individual stdMCP-tdTomato spot. Each dot represents a single cell (n = 100). Gray line indicates mean ± SEM. (F) qPCR to examine relative expression of 45S-preRNA (left) and SRFBP1 (right) in SRFBP1 down-regulated samples from E. n = 3 technical replicates. (G) Representative traces (red) demonstrating the real-time synthesis of MS2-tagged rRNAs (3′-ETS-1) by measuring the maximum intensity of stdMCP-tdTomato spots in corresponding cells that were infected with negative control (NC) shRNA or shRNAs to specifically down-regulate SRFPB1 and RRN3, respectively. (H) Quantification of burst duration and pause duration by imaging 3′-ETS-1 clone. n ≥ 100. Data are all displayed as mean ± SEM. One-way ANOVA with Tukey’s post hoc was used to test differences between groups. ***P ≤ 0.001, **P ≤ 0.01. n.s., not significant.

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