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Results: 5

1.
Fig. 2.

Fig. 2. From: CAG expansion induces nucleolar stress in polyglutamine diseases.

Expanded CAG RNAs interfered with the formation of rRNA transcription preinitiation complex and promoted hypermethylation of rRNA promoter. (A and B) Chromatin immunoprecipitation of the largest subunit of RNA polymerase I (RPA194) and rRNA promoter (A) and upstream binding factor (UBF) and upstream control element (UCE) (B) in CAG RNA-expressing cells. “+” indicates that antibody was present in the immunoprecipitation reactions and “−” indicates that antibody was not included in the reactions. (C) The effect of DNA methyltransferase inhibitor 5-azacytidine on expanded CAG RNA-induced UCE CpG DNA hypermethylation. Real-time PCR analysis of UCE DNA methylation status is shown. Error bars represent ±SD. All experiments were repeated three times.

Ho Tsoi, et al. Proc Natl Acad Sci U S A. 2012 August 14;109(33):13428-13433.
2.
Fig. 4.

Fig. 4. From: CAG expansion induces nucleolar stress in polyglutamine diseases.

Nucleolin overexpression restored rRNA transcription and suppressed nucleolar stress. (A) Real-time PCR analysis of upstream control element (UCE) DNA methylation status in cells coexpressing CAG RNAs and NCL. Error bars represent ±SD. This experiment was repeated three times. (B and C) Chromatin immunoprecipitation of upstream binding factor (UBF) and UCE in CAG RNA-expressing cells with or without NCL coexpression. Real-time PCR analysis was performed to determine UBF–UCE interaction. Error bars represent ±SD. This experiment was repeated three times. (D) Real-time PCR analysis of pre-45s rRNA expression levels in cells cotransfected with CAG and NCL constructs. Error bars represent ±SD. This experiment was repeated three times. (E) The effect of NCL overexpression on p53 protein stabilization and cleavage of caspase 3 and poly(ADP ribose) polymerase in expanded CAG RNA-expressing cells. “+” indicates NCL overexpression and “−” indicates no NCL overexpression. This experiment was repeated three times, and a representative blot is shown.

Ho Tsoi, et al. Proc Natl Acad Sci U S A. 2012 August 14;109(33):13428-13433.
3.
Fig. 5.

Fig. 5. From: CAG expansion induces nucleolar stress in polyglutamine diseases.

A proposed model for the activation of nucleolar stress signaling by expanded CAG RNAs. The expression of expanded CAG RNA interacts directly with nucleolin (NCL, in light blue). The expanded CAG-specific RNA–protein interaction causes CpG hypermethylation (inverted triangles) of the upstream control element (UCE, in purple) in the rRNA promoter and results in reduction of rRNA transcription. A functional ribosome consists of both rRNAs and ribosomal proteins. A reduced level of rRNAs causes accumulation of free ribosomal proteins (such as RpL5, RpL11, and RpL23, in orange). The interaction between free ribosomal proteins and E3 ubiquitin ligase MDM2 (in blue) leads to mitochondrial accumulation of p53. The interaction between p53 and antiapoptotic proteins (Bcl-xL, in orange) causes oligomerization of proapoptotic proteins (Bak, in gray) on the mitochondrial membrane and results in cytochrome c (in light purple) release. Cytosolic cytochrome c in turn activates the caspase cascade and induces apoptosis.

Ho Tsoi, et al. Proc Natl Acad Sci U S A. 2012 August 14;109(33):13428-13433.
4.
Fig. 3.

Fig. 3. From: CAG expansion induces nucleolar stress in polyglutamine diseases.

Physical interaction between expanded CAG RNAs and nucleolin compromised the binding of nucleolin to upstream control element. (A) Pull-down of MJDCAG RNA by means of NCL immunoprecipitation. “+” indicates that antibody was present in the immunoprecipitation reactions and “−” indicates that no antibody was included in the reactions. This experiment was repeated three times, and a representative gel is shown. (B) Direct physical interaction between NCL and expanded CAG RNA. Purified GST-NCL protein and in vitro transcribed RNAs (CAG78 and CUG78) were used in the binding reactions. Nonfusion GST protein was used as a negative control. Western blotting was performed to confirm the expression of the GST-NCL protein. This experiment was repeated four times, and a representative blot is shown. (C) Real-time PCR analysis of pre-45s rRNA expression levels in NCL siRNA-treated cells. Error bars represent ±SD. This experiment was repeated three times. (D) Real-time PCR analysis of UCE DNA methylation status in NCL siRNA-treated cells. Error bars represent ±SD. This experiment was repeated three times. (E) Coimmunoprecipitation of upstream binding factor (UBF) and upstream control element (UCE) in NCL siRNA-treated cells. Real-time PCR analysis was performed to determine UBF–UCE interaction. Error bars represent ±SD. This experiment was repeated three times. (F) Chromatin immunoprecipitation of NCL with expanded CAG RNAs and UCE in cells. “+” indicates that antibody was present in the immunoprecipitation reactions and “−” indicates that no antibody was included in the reactions. This experiment was repeated three times, and a representative gel is shown.

Ho Tsoi, et al. Proc Natl Acad Sci U S A. 2012 August 14;109(33):13428-13433.
5.
Fig. 1.

Fig. 1. From: CAG expansion induces nucleolar stress in polyglutamine diseases.

Expanded CAG RNAs perturbed rRNA transcription and induced nucleolar stress. (A–C) Real-time PCR analysis of pre-rRNA expression in Drosophila (A and B) and pre-45s rRNA, GAPDH, U6, and tRNAmet expression in cell (C) models expressing CAG constructs. Error bars represent ±SD. This experiment was repeated three times. (D–F) Coimmunoprecipitation of ribosomal proteins [RpL5 (D), RpL11 (E), and RpL23 (F)] and MDM2 E3 ubiquitin ligase in CAG RNA-expressing cells. (G) Effect of expanded CAG RNA expression on p53 protein stability. CHX represents cycloheximide and was used to inhibit de novo protein synthesis. Tubulin was used as a loading control. (H) Effect of expanded CAG RNA expression on mitochondrial accumulation of p53. Tubulin and glutamate dehydrogenase (GDH) were used, respectively, as cytosolic and mitochondrial fractionation controls. (I) Coimmunoprecipitation of Bcl-xL with p53 and Bak. (J) Cytosolic and mitochondrial fractionations of cytochrome c in CAG RNA-expressing cells. Tubulin and GDH were used as cytosolic and mitochondrial fractionation controls, respectively. (K) Effect of expanded CAG RNA expression on cleavage of caspase 3 and poly(ADP ribose) polymerase. Tubulin was used as a loading control. “+” indicates that antibody was present in the immunoprecipitation reactions and “−” indicates that no antibody was included in the reactions. All experiments were repeated three times, and representative blots or gels are shown.

Ho Tsoi, et al. Proc Natl Acad Sci U S A. 2012 August 14;109(33):13428-13433.

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