Figure 2.
Figure 2. Decrease in the amount of active RNAPII and RNA synthesis by RNAPII inhibitors. (A) Scheme for RNAPII regulation by phosphorylation of its CTD repeats, (YSPTSPS) × 52. In the initiation process, RNAPII, in which Ser5 of CTD is phosphorylated, stays around the initiation site (red, RNAPII-Ser5P) on the template DNA. For elongation, with phosphorylation of Ser2 of CTD, the RNAPII complex goes along the template DNA (green, RNAPII-Ser2P). Note that the scheme is highly simplified. (B) Left: Effect of RNAPII inhibitors on RNAPII activity. RNAPII activity in RPE-1 cells was monitored by immunostaining of the active RNAPII marker (Stasevich et al., 2014), Ser5P, of RNAPII CTD. The inhibitors used were α-AM, DRB, and ActD. As solvent controls, DMSO and ultrapure water (MQ) were used. First row, DNA staining with DAPI; second row, immunostaining of Ser5P of RNAPII CTD; third row, merged images. Right: The quantification of RNAPII-Ser5P signal intensity is shown as a box plot. The median intensities of Ser5P: 32.6 (n = 118 cells) in control; 22.4 (n = 121 cells) in DRB; 17.5 (n = 141 cells) in α-AM; 29.0 (n = 130 cells) in ActD. ***, P < 0.0001 by the Wilcoxon rank sum test for control versus DRB (P < 2.2 × 10−16), and for control versus α-AM (P < 2.2 × 10−16). (C) Left: Active RNAPII (Ser5P) distribution in RPE-1 cells with DRB or α-AM or without inhibitors (untreated control) observed by immunostaining. Typical images with deconvolution by DeltaVision Softworx software are shown. RNAPII-Ser5P formed clusters and distributed in the nucleoplasm except for nuclear periphery and nucleoli. Right: The intensity line profiles (bottom) of DAPI (blue) and RNAPII-Ser5P (green) on the dotted line in the merged image (top) show that the nuclear periphery regions (arrows) are quite free from RNAPII-Ser5P signals. (D) Left: Verification of RNA synthesis inhibition by RNAPII inhibitors (α-AM, DRB, and ActD) with EU incorporation into RNA. The incorporated EU was detected with Alexa Fluor 594–labeling by click chemistry. For each condition, n = 45–53 cells. Right: Box plot of EU signal intensity. The median intensities of EU are 13.5 (n = 49 cells) in control, 4.44 (n = 48 cells) in DRB, 1.69 (n = 45 cells) in α-AM, 0.975 (n = 45 cells) in ActD, and 16.1 (n = 53 cells) in DMSO. Note that the inhibitor treatments decreased RNA transcription. ***, P < 0.0001 by the Wilcoxon rank sum test for control versus DRB (P < 2.2 × 10−16), for control versus α-AM (P < 2.2 × 10−16), and for control versus ActD (P < 2.2 × 10−16).

Decrease in the amount of active RNAPII and RNA synthesis by RNAPII inhibitors. (A) Scheme for RNAPII regulation by phosphorylation of its CTD repeats, (YSPTSPS) × 52. In the initiation process, RNAPII, in which Ser5 of CTD is phosphorylated, stays around the initiation site (red, RNAPII-Ser5P) on the template DNA. For elongation, with phosphorylation of Ser2 of CTD, the RNAPII complex goes along the template DNA (green, RNAPII-Ser2P). Note that the scheme is highly simplified. (B) Left: Effect of RNAPII inhibitors on RNAPII activity. RNAPII activity in RPE-1 cells was monitored by immunostaining of the active RNAPII marker (Stasevich et al., 2014), Ser5P, of RNAPII CTD. The inhibitors used were α-AM, DRB, and ActD. As solvent controls, DMSO and ultrapure water (MQ) were used. First row, DNA staining with DAPI; second row, immunostaining of Ser5P of RNAPII CTD; third row, merged images. Right: The quantification of RNAPII-Ser5P signal intensity is shown as a box plot. The median intensities of Ser5P: 32.6 (n = 118 cells) in control; 22.4 (n = 121 cells) in DRB; 17.5 (n = 141 cells) in α-AM; 29.0 (n = 130 cells) in ActD. ***, P < 0.0001 by the Wilcoxon rank sum test for control versus DRB (P < 2.2 × 10−16), and for control versus α-AM (P < 2.2 × 10−16). (C) Left: Active RNAPII (Ser5P) distribution in RPE-1 cells with DRB or α-AM or without inhibitors (untreated control) observed by immunostaining. Typical images with deconvolution by DeltaVision Softworx software are shown. RNAPII-Ser5P formed clusters and distributed in the nucleoplasm except for nuclear periphery and nucleoli. Right: The intensity line profiles (bottom) of DAPI (blue) and RNAPII-Ser5P (green) on the dotted line in the merged image (top) show that the nuclear periphery regions (arrows) are quite free from RNAPII-Ser5P signals. (D) Left: Verification of RNA synthesis inhibition by RNAPII inhibitors (α-AM, DRB, and ActD) with EU incorporation into RNA. The incorporated EU was detected with Alexa Fluor 594–labeling by click chemistry. For each condition, n = 45–53 cells. Right: Box plot of EU signal intensity. The median intensities of EU are 13.5 (n = 49 cells) in control, 4.44 (n = 48 cells) in DRB, 1.69 (n = 45 cells) in α-AM, 0.975 (n = 45 cells) in ActD, and 16.1 (n = 53 cells) in DMSO. Note that the inhibitor treatments decreased RNA transcription. ***, P < 0.0001 by the Wilcoxon rank sum test for control versus DRB (P < 2.2 × 10−16), for control versus α-AM (P < 2.2 × 10−16), and for control versus ActD (P < 2.2 × 10−16).

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