Figure 4.
Figure 4. Inhibitors of RNA polymerase I and splicing had little influence on the chromatin dynamics. (A) Verification of RNA polymerase I inhibition in RPE-1 cells with CX5461 by EU incorporation. (B) The box plots show RNAPII-Ser5P signal intensity in control and CX5461-treated RPE-1 cells. The median intensities of Ser5P are 71.8 in control (n = 30 cells) and 67.3 in CX5461 (n = 30 cells). N.S. (P = 0.066) by the Wilcoxon rank sum test. (C) MSD plots (±SD among cells) of single nucleosomes in RPE-1 cells treated with polymerase I (RNAPI) inhibitor (CX5461, green), solvent (DMSO, gray), or none (control, black). For each condition, n = 37–39 cells. Note that the effect of RNAPI inhibition on the chromatin dynamics is very small. N.S. (P = 0.40) by the Kolmogorov–Smirnov test for untreated control versus CX5461. (D) MSD plots (±SD among cells) of single nucleosomes in RPE-1 cells treated with splicing inhibitor, Pladienolide B (Pla-B, yellow) or DMSO (gray). For each condition, n = 20 cells. N.S. (P = 0.34) by the Kolmogorov–Smirnov test. (E) Verification of splicing inhibition in RPE-1 cells treated with Pla-B by quantitative RT-PCR. Relative amounts of spliced (Ex 2–3 mRNA) and nonspliced (Int 2 premRNA) CDK6 RNA products in RPE-1 cells treated with Pla-B (yellow) or DMSO (gray) are shown. Schematic representation of primer positions for Ex 2–3 mRNA (pink arrows) and Int 2 pre-mRNA (orange arrows) are also shown at the bottom. Averaged relative amounts of the products were shown with SD (n = 3). N.S. (P = 0.38) and ***, P < 0.0001 (P = 1.7 × 10−6) by Student’s t test. (F) Left: MSD plots (±SD among cells) of single nucleosomes on the interior and peripheral layers of the RPE-1 nuclei treated with RNAPII inhibitor, α-AM (pink), solvent (MQ, light blue), or none (control, black). For each condition, n = 15 cells. Note that on the nuclear periphery, the chromatin dynamics were not significantly affected by α-AM treatment. N.S. (P = 0.075) for control periphery versus α-AM periphery and ***, P < 0.0001 for control periphery versus control interior (P = 3.9 × 10−7) by the Kolmogorov–Smirnov test. There was no significant difference between the MSD of α-AM–treated interior in Fig. 4 F and that of α-AM–treated in Fig. 3 A (P = 0.13). Right: Schematic representation for nuclear interior (top) and periphery (bottom) imaging.

Inhibitors of RNA polymerase I and splicing had little influence on the chromatin dynamics. (A) Verification of RNA polymerase I inhibition in RPE-1 cells with CX5461 by EU incorporation. (B) The box plots show RNAPII-Ser5P signal intensity in control and CX5461-treated RPE-1 cells. The median intensities of Ser5P are 71.8 in control (n = 30 cells) and 67.3 in CX5461 (n = 30 cells). N.S. (P = 0.066) by the Wilcoxon rank sum test. (C) MSD plots (±SD among cells) of single nucleosomes in RPE-1 cells treated with polymerase I (RNAPI) inhibitor (CX5461, green), solvent (DMSO, gray), or none (control, black). For each condition, n = 37–39 cells. Note that the effect of RNAPI inhibition on the chromatin dynamics is very small. N.S. (P = 0.40) by the Kolmogorov–Smirnov test for untreated control versus CX5461. (D) MSD plots (±SD among cells) of single nucleosomes in RPE-1 cells treated with splicing inhibitor, Pladienolide B (Pla-B, yellow) or DMSO (gray). For each condition, n = 20 cells. N.S. (P = 0.34) by the Kolmogorov–Smirnov test. (E) Verification of splicing inhibition in RPE-1 cells treated with Pla-B by quantitative RT-PCR. Relative amounts of spliced (Ex 2–3 mRNA) and nonspliced (Int 2 premRNA) CDK6 RNA products in RPE-1 cells treated with Pla-B (yellow) or DMSO (gray) are shown. Schematic representation of primer positions for Ex 2–3 mRNA (pink arrows) and Int 2 pre-mRNA (orange arrows) are also shown at the bottom. Averaged relative amounts of the products were shown with SD (n = 3). N.S. (P = 0.38) and ***, P < 0.0001 (P = 1.7 × 10−6) by Student’s t test. (F) Left: MSD plots (±SD among cells) of single nucleosomes on the interior and peripheral layers of the RPE-1 nuclei treated with RNAPII inhibitor, α-AM (pink), solvent (MQ, light blue), or none (control, black). For each condition, n = 15 cells. Note that on the nuclear periphery, the chromatin dynamics were not significantly affected by α-AM treatment. N.S. (P = 0.075) for control periphery versus α-AM periphery and ***, P < 0.0001 for control periphery versus control interior (P = 3.9 × 10−7) by the Kolmogorov–Smirnov test. There was no significant difference between the MSD of α-AM–treated interior in Fig. 4 F and that of α-AM–treated in Fig. 3 A (P = 0.13). Right: Schematic representation for nuclear interior (top) and periphery (bottom) imaging.

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