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    Addgene inc 5 utr cgg 99× fmr1 egfp
    a) Live cell imaging of gfp mRNA (control) and gfp mRNA fused to a single copy of SRB-2, SRB-3 or RhoBAST. Bacteria were transformed with the pET plasmid carrying the gfp gene fused to the aptamer tags at the 3′ UTR. Scale bars, 3 μm. b) Quantification of the average TMR fluorescence at the poles of bacteria expressing the different RNA constructs. Each dot represents a single cell; also indicated are the means ± s.d. (N > 30 cells). Statistical comparison was performed by using a two-tailed t-test. ***, P ≤ 0.001; ****, P ≤ 0.0001. c) Live-cell mRNA imaging of endogenously RhoBAST-tagged ompA , cat and rnaseE in E. coli K12. d) Live-cell imaging of circular RhoBAST aptamers transcribed under the control of the U6 promoter using the Tornado system in HEK293T cells. Cells were cotransfected with another GFP-expressing plasmid as a transfection control. e) Live-cell imaging of GFP mRNA fused to RhoBAST 16 transcribed under the CAG promoter in HEK293T cells. f) Live-cell imaging of the trinucleotide CGG repeat-containing <t>FMR1-GFP</t> mRNA fused to RhoBAST 16 transcribed under the CMV promoter in HeLa cells. g) Live-cell imaging of HEK293T cells transfected with a GFP expressing plasmid (negative control) to show the background fluorescence due to the presence of free TMR-DN. At least three independent experiments were carried out with similar results.
    5 Utr Cgg 99× Fmr1 Egfp, supplied by Addgene inc, used in various techniques. Bioz Stars score: 92/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/5 utr cgg 99× fmr1 egfp/product/Addgene inc
    Average 92 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    5 utr cgg 99× fmr1 egfp - by Bioz Stars, 2024-06
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    86
    Addgene inc wthp acg 99gly gfp plasmid
    The FMRpolyG protein itself causes aggregate formation in several cell lines. (A) Schematic representation of the <t>wtHP-99Gly-GFP</t> and mutHP-90Gly-GFP constructs used in this study. Both contain the part of the FMR1 5′UTR that encodes the entire FMRpolyG protein (shown in blue). The wtHP-99Gly-GFP construct gives rise to both a CGG RNA hairpin and the FMRpolyGlycine protein. In mutHP-90Gly-GFP, the CGGs have been substituted with alternative glycine codons to abolish hairpin formation. (B) Confocal images of GFP-positive aggregates in the indicated cell lines, transfected with wtHP-99Gly-GFP or mutHP-90Gly-GFP and analyzed 24–48 h after transfection. (C,D) Quantification of FMRpolyG-GFP aggregates in HEK293 cells 24 h after transfection, presented as number of aggregates per 100 cells (C) or 100 GFP-positive cells (D) . Untransfected cells and cells transfected with GFP-C1 were used as negative controls. Small aggregates range from 1 to 3 μm 2 in size, while large aggregates are those over 3 μm 2 . Calculations are based on three individual experiments, each including >4,500 cells for each plasmid. Error bars represent standard deviation (SD).
    Wthp Acg 99gly Gfp Plasmid, supplied by Addgene inc, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/wthp acg 99gly gfp plasmid/product/Addgene inc
    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    wthp acg 99gly gfp plasmid - by Bioz Stars, 2024-06
    86/100 stars
      Buy from Supplier

    Image Search Results


    a) Live cell imaging of gfp mRNA (control) and gfp mRNA fused to a single copy of SRB-2, SRB-3 or RhoBAST. Bacteria were transformed with the pET plasmid carrying the gfp gene fused to the aptamer tags at the 3′ UTR. Scale bars, 3 μm. b) Quantification of the average TMR fluorescence at the poles of bacteria expressing the different RNA constructs. Each dot represents a single cell; also indicated are the means ± s.d. (N > 30 cells). Statistical comparison was performed by using a two-tailed t-test. ***, P ≤ 0.001; ****, P ≤ 0.0001. c) Live-cell mRNA imaging of endogenously RhoBAST-tagged ompA , cat and rnaseE in E. coli K12. d) Live-cell imaging of circular RhoBAST aptamers transcribed under the control of the U6 promoter using the Tornado system in HEK293T cells. Cells were cotransfected with another GFP-expressing plasmid as a transfection control. e) Live-cell imaging of GFP mRNA fused to RhoBAST 16 transcribed under the CAG promoter in HEK293T cells. f) Live-cell imaging of the trinucleotide CGG repeat-containing FMR1-GFP mRNA fused to RhoBAST 16 transcribed under the CMV promoter in HeLa cells. g) Live-cell imaging of HEK293T cells transfected with a GFP expressing plasmid (negative control) to show the background fluorescence due to the presence of free TMR-DN. At least three independent experiments were carried out with similar results.

    Journal: bioRxiv

    Article Title: RhoBAST - a rhodamine-binding aptamer for super-resolution RNA imaging

    doi: 10.1101/2020.03.12.988782

    Figure Lengend Snippet: a) Live cell imaging of gfp mRNA (control) and gfp mRNA fused to a single copy of SRB-2, SRB-3 or RhoBAST. Bacteria were transformed with the pET plasmid carrying the gfp gene fused to the aptamer tags at the 3′ UTR. Scale bars, 3 μm. b) Quantification of the average TMR fluorescence at the poles of bacteria expressing the different RNA constructs. Each dot represents a single cell; also indicated are the means ± s.d. (N > 30 cells). Statistical comparison was performed by using a two-tailed t-test. ***, P ≤ 0.001; ****, P ≤ 0.0001. c) Live-cell mRNA imaging of endogenously RhoBAST-tagged ompA , cat and rnaseE in E. coli K12. d) Live-cell imaging of circular RhoBAST aptamers transcribed under the control of the U6 promoter using the Tornado system in HEK293T cells. Cells were cotransfected with another GFP-expressing plasmid as a transfection control. e) Live-cell imaging of GFP mRNA fused to RhoBAST 16 transcribed under the CAG promoter in HEK293T cells. f) Live-cell imaging of the trinucleotide CGG repeat-containing FMR1-GFP mRNA fused to RhoBAST 16 transcribed under the CMV promoter in HeLa cells. g) Live-cell imaging of HEK293T cells transfected with a GFP expressing plasmid (negative control) to show the background fluorescence due to the presence of free TMR-DN. At least three independent experiments were carried out with similar results.

    Article Snippet: Blunt-end repaired RhoBAST 16 was cloned into 5′ UTR CGG 99× FMR1-EGFP (Addgene, plasmid #63091) which was digested with the NotI enzyme, blunted and dephosphorylated, to yield 5′ UTR CGG 99× FMR1-EGFP-RhoBAST 16 .

    Techniques: Live Cell Imaging, Transformation Assay, Plasmid Preparation, Fluorescence, Expressing, Construct, Two Tailed Test, Imaging, Transfection, Negative Control

    The FMRpolyG protein itself causes aggregate formation in several cell lines. (A) Schematic representation of the wtHP-99Gly-GFP and mutHP-90Gly-GFP constructs used in this study. Both contain the part of the FMR1 5′UTR that encodes the entire FMRpolyG protein (shown in blue). The wtHP-99Gly-GFP construct gives rise to both a CGG RNA hairpin and the FMRpolyGlycine protein. In mutHP-90Gly-GFP, the CGGs have been substituted with alternative glycine codons to abolish hairpin formation. (B) Confocal images of GFP-positive aggregates in the indicated cell lines, transfected with wtHP-99Gly-GFP or mutHP-90Gly-GFP and analyzed 24–48 h after transfection. (C,D) Quantification of FMRpolyG-GFP aggregates in HEK293 cells 24 h after transfection, presented as number of aggregates per 100 cells (C) or 100 GFP-positive cells (D) . Untransfected cells and cells transfected with GFP-C1 were used as negative controls. Small aggregates range from 1 to 3 μm 2 in size, while large aggregates are those over 3 μm 2 . Calculations are based on three individual experiments, each including >4,500 cells for each plasmid. Error bars represent standard deviation (SD).

    Journal: Frontiers in Genetics

    Article Title: The FMRpolyGlycine Protein Mediates Aggregate Formation and Toxicity Independent of the CGG mRNA Hairpin in a Cellular Model for FXTAS

    doi: 10.3389/fgene.2019.00249

    Figure Lengend Snippet: The FMRpolyG protein itself causes aggregate formation in several cell lines. (A) Schematic representation of the wtHP-99Gly-GFP and mutHP-90Gly-GFP constructs used in this study. Both contain the part of the FMR1 5′UTR that encodes the entire FMRpolyG protein (shown in blue). The wtHP-99Gly-GFP construct gives rise to both a CGG RNA hairpin and the FMRpolyGlycine protein. In mutHP-90Gly-GFP, the CGGs have been substituted with alternative glycine codons to abolish hairpin formation. (B) Confocal images of GFP-positive aggregates in the indicated cell lines, transfected with wtHP-99Gly-GFP or mutHP-90Gly-GFP and analyzed 24–48 h after transfection. (C,D) Quantification of FMRpolyG-GFP aggregates in HEK293 cells 24 h after transfection, presented as number of aggregates per 100 cells (C) or 100 GFP-positive cells (D) . Untransfected cells and cells transfected with GFP-C1 were used as negative controls. Small aggregates range from 1 to 3 μm 2 in size, while large aggregates are those over 3 μm 2 . Calculations are based on three individual experiments, each including >4,500 cells for each plasmid. Error bars represent standard deviation (SD).

    Article Snippet: The wtHP-99Gly-GFP-plasmid and a wtHP-ACG-99Gly-GFP-plasmid (Addgene plasmid # 63091) were kind gifts from Nicolas Charlet-Berguand (Sellier et al., ).

    Techniques: Construct, Transfection, Plasmid Preparation, Standard Deviation

    Expression of wtHP-99Gly-GFP results in several fold higher protein levels than that of mutHP-90-Gly-GFP expression. (A,B) Merge of flow curves showing the differences in GFP fluorescence intensity in HEK-FlpIn cells expressing no GFP-tagged protein (negative control, purple), mutHP-90Gly-GFP (blue) and wtHP-99Gly-GFP (green). Flow analysis and sorting was performed separately on each cell population 24 h after induction of expression. Note the logarithmic scale on the X-axis. GFP-positive cells expressing wtHP-99Gly-GFP were sorted into gate P3. GFP-positive cells expressing mutHP-90Gly-GFP were sorted into gate P2. (C) The graph demonstrates the large difference in mean fluorescence (GFP) intensity between mutHP-90Gly-GFP and wtHP-99Gly-GFP expressing cells. (D) The graph shows the difference in FMRpolyG-GFP protein levels in HEK-FlpIn cells stably expression mutHP-90Gly-GFP and wtHP-99Gly-GFP. The cells were sorted using flow with the gates illustrated in (A,B) . For mutHP-90Gly-GFP cells expressing GFP-levels above the threshold in gate P2 were included, while wtHP-99Gly-GFP expressing cells included are those above the threshold in gate P3. The graph is based on WB-quantification of sorted cells from the two above mentioned cell populations. (E) Western blot of sorted cells expressing wtHP-99Gly-GFP or mutHP-90Gly-GFP. Protein levels of wtHP-99Gly-GFP expressing cell is set to 1 in each experiment. Data in A-D is based on three independent experiments were cells were subject to flow, sorted and lysed 24 h after expression was induced.

    Journal: Frontiers in Genetics

    Article Title: The FMRpolyGlycine Protein Mediates Aggregate Formation and Toxicity Independent of the CGG mRNA Hairpin in a Cellular Model for FXTAS

    doi: 10.3389/fgene.2019.00249

    Figure Lengend Snippet: Expression of wtHP-99Gly-GFP results in several fold higher protein levels than that of mutHP-90-Gly-GFP expression. (A,B) Merge of flow curves showing the differences in GFP fluorescence intensity in HEK-FlpIn cells expressing no GFP-tagged protein (negative control, purple), mutHP-90Gly-GFP (blue) and wtHP-99Gly-GFP (green). Flow analysis and sorting was performed separately on each cell population 24 h after induction of expression. Note the logarithmic scale on the X-axis. GFP-positive cells expressing wtHP-99Gly-GFP were sorted into gate P3. GFP-positive cells expressing mutHP-90Gly-GFP were sorted into gate P2. (C) The graph demonstrates the large difference in mean fluorescence (GFP) intensity between mutHP-90Gly-GFP and wtHP-99Gly-GFP expressing cells. (D) The graph shows the difference in FMRpolyG-GFP protein levels in HEK-FlpIn cells stably expression mutHP-90Gly-GFP and wtHP-99Gly-GFP. The cells were sorted using flow with the gates illustrated in (A,B) . For mutHP-90Gly-GFP cells expressing GFP-levels above the threshold in gate P2 were included, while wtHP-99Gly-GFP expressing cells included are those above the threshold in gate P3. The graph is based on WB-quantification of sorted cells from the two above mentioned cell populations. (E) Western blot of sorted cells expressing wtHP-99Gly-GFP or mutHP-90Gly-GFP. Protein levels of wtHP-99Gly-GFP expressing cell is set to 1 in each experiment. Data in A-D is based on three independent experiments were cells were subject to flow, sorted and lysed 24 h after expression was induced.

    Article Snippet: The wtHP-99Gly-GFP-plasmid and a wtHP-ACG-99Gly-GFP-plasmid (Addgene plasmid # 63091) were kind gifts from Nicolas Charlet-Berguand (Sellier et al., ).

    Techniques: Expressing, Fluorescence, Negative Control, Stable Transfection, Western Blot

    Both transient transfection and stable expression of wtHP-99Gly-GFP and mutHP-90Gly-GFP results in a similar kinetics of FMRpolyG-GFP aggregate formation. (A) Representative confocal images of transfected HEK293 cells, displaying aggregate formation 24, 48, and 72 h after transfection with either wtHP-99Gly-GFP (upper panel) or mutHP-90Gly-GFP (lower panel). Untransfected cells and cells transfected with GFP-C1 were used as negative controls. (B) Quantification of FMRpolyG-GFP aggregates formed 24, 48, and 72 h after transfection. (C) Confocal images of FMRpolyG-GFP aggregate formation in HEK-FlpIn cells stably expressing wtHP-99Gly-GFP (upper panel) or mutHP-90Gly-GFP (lower panel) from a tetracycline-inducible promoter. Doxycycline was added 24, 48, or 72 h prior to fixation. (D,E) Quantification of FMRpolyG-GFP aggregates formed in experiments shown in (B) . Error bars in (B,D,E) represent SD of averages from a minimum of three independent experiments.

    Journal: Frontiers in Genetics

    Article Title: The FMRpolyGlycine Protein Mediates Aggregate Formation and Toxicity Independent of the CGG mRNA Hairpin in a Cellular Model for FXTAS

    doi: 10.3389/fgene.2019.00249

    Figure Lengend Snippet: Both transient transfection and stable expression of wtHP-99Gly-GFP and mutHP-90Gly-GFP results in a similar kinetics of FMRpolyG-GFP aggregate formation. (A) Representative confocal images of transfected HEK293 cells, displaying aggregate formation 24, 48, and 72 h after transfection with either wtHP-99Gly-GFP (upper panel) or mutHP-90Gly-GFP (lower panel). Untransfected cells and cells transfected with GFP-C1 were used as negative controls. (B) Quantification of FMRpolyG-GFP aggregates formed 24, 48, and 72 h after transfection. (C) Confocal images of FMRpolyG-GFP aggregate formation in HEK-FlpIn cells stably expressing wtHP-99Gly-GFP (upper panel) or mutHP-90Gly-GFP (lower panel) from a tetracycline-inducible promoter. Doxycycline was added 24, 48, or 72 h prior to fixation. (D,E) Quantification of FMRpolyG-GFP aggregates formed in experiments shown in (B) . Error bars in (B,D,E) represent SD of averages from a minimum of three independent experiments.

    Article Snippet: The wtHP-99Gly-GFP-plasmid and a wtHP-ACG-99Gly-GFP-plasmid (Addgene plasmid # 63091) were kind gifts from Nicolas Charlet-Berguand (Sellier et al., ).

    Techniques: Transfection, Expressing, Stable Transfection

    The FMRpolyG protein expression induces formation of intranuclear aggregates. (A) Representative confocal images of intranuclear aggregates in HEK293 cells transfected with wtHP-99Gly-GFP (upper panel) or mutHP-90Gly-GFP (lower panel). Untransfected cells and cells transfected with GFP-C1 were used as negative controls. (B) Quantification of aggregates that are intranuclear 24 h post transfection. The graphs represent the percentage of aggregates that are intranuclear, based on three individual experiments, each including a minimum of 45 aggregates per construct.

    Journal: Frontiers in Genetics

    Article Title: The FMRpolyGlycine Protein Mediates Aggregate Formation and Toxicity Independent of the CGG mRNA Hairpin in a Cellular Model for FXTAS

    doi: 10.3389/fgene.2019.00249

    Figure Lengend Snippet: The FMRpolyG protein expression induces formation of intranuclear aggregates. (A) Representative confocal images of intranuclear aggregates in HEK293 cells transfected with wtHP-99Gly-GFP (upper panel) or mutHP-90Gly-GFP (lower panel). Untransfected cells and cells transfected with GFP-C1 were used as negative controls. (B) Quantification of aggregates that are intranuclear 24 h post transfection. The graphs represent the percentage of aggregates that are intranuclear, based on three individual experiments, each including a minimum of 45 aggregates per construct.

    Article Snippet: The wtHP-99Gly-GFP-plasmid and a wtHP-ACG-99Gly-GFP-plasmid (Addgene plasmid # 63091) were kind gifts from Nicolas Charlet-Berguand (Sellier et al., ).

    Techniques: Expressing, Transfection, Construct

    The FMRpolyG protein reduces cell viability and disrupts the lamin architecture. (A) Effect of FMRpolyG on cell viability. HEK293 cells were transfected with the indicated constructs, and cell death measured in GFP positive cells. Cell transfected with GFP-C1 were used as negative controls. Cells were counted as non-viable based on the incorporation of propidium iodide detected by FACS. For each transfection >50 000 cells were counted per experiment. *** p < 0.001; ** p < 0.01; * p < 0.05. The exact p -values, from left to right, are as follows: 0.0009 and 0.0081. (B) Effect of FMRpolyG on lamin architecture. HEK293 cells were transfected with the indicated constructs and stained for Lamin B1 24 h after transfection. Transfection with GFP-C1 served as negative control. Cells were analyzed by confocal imaging, and the fraction of cells with disrupted lamin architecture counted in GFP positive cells (left) and in cells with aggregates (right). The number of GFP positive cells included in the analysis was >440 for GFP-C1 and wtHP-99Gly-GFP. For wtHP-99Gly-GFP > 170 aggregate bearing cells were counted. Due to the low levels of GFP positive cells among those transfected with mutHP-90Gly-GFP, a total of 80 GFP-positive cells and 70 aggregate bearing cells were included from mutHP-90Gly-GFP transfected cell populations. The exact p -values, from left to right, are as follows: 0.0021 ( ** ), 0.0004 ( *** ), 0.0123 ( * ), and 0.0113 ( * ). (C) Representative confocal images showing normal lamin rings in cells expressing GFP (GFP-C1), but disrupted lamin architecture in aggregate-containing cells after transfection of wtHP-99Gly-GFP or mutHP-90Gly-GFP. (D) Effect of stably expressed FMRpolyG on lamin architecture. Expression of GFP or FMRpolyG-GFP were induced with doxycycline for 72 h, followed by Lamin B1 staining, confocal imaging, and counting of GFP positive cells with disrupted lamin architecture. Cell expressing GFP (from GFP-C1) were used as negative controls. For GFP-C1 and wtHP-99Gly-GFP, a minimum of 830 GFP positive cells were quantified. Due to low expression levels in cells expressing mutHP-90Gly-GFP, 150 GFP-positive cells were included for this construct. For wtHP-99Gly-GFP and mutHP-90Gly-GFP, a minimum of 80 aggregate bearing cells were analyzed per construct. The exact p -values, from left to right, are as follows: 0.0186 ( * ), 0.0007 ( *** ), 0.00001 ( *** ), and 0.0004 ( *** ). (E) Representative confocal images showing normal lamin rings in HEK-FlpIn cells expressing GFP and disrupted lamin structures in aggregate-containing cells expressing FMRpolyG-GFP. All graphs in (A,B,D) are based on quantifications of a minimum of three individual experiments and error bars represent SD.

    Journal: Frontiers in Genetics

    Article Title: The FMRpolyGlycine Protein Mediates Aggregate Formation and Toxicity Independent of the CGG mRNA Hairpin in a Cellular Model for FXTAS

    doi: 10.3389/fgene.2019.00249

    Figure Lengend Snippet: The FMRpolyG protein reduces cell viability and disrupts the lamin architecture. (A) Effect of FMRpolyG on cell viability. HEK293 cells were transfected with the indicated constructs, and cell death measured in GFP positive cells. Cell transfected with GFP-C1 were used as negative controls. Cells were counted as non-viable based on the incorporation of propidium iodide detected by FACS. For each transfection >50 000 cells were counted per experiment. *** p < 0.001; ** p < 0.01; * p < 0.05. The exact p -values, from left to right, are as follows: 0.0009 and 0.0081. (B) Effect of FMRpolyG on lamin architecture. HEK293 cells were transfected with the indicated constructs and stained for Lamin B1 24 h after transfection. Transfection with GFP-C1 served as negative control. Cells were analyzed by confocal imaging, and the fraction of cells with disrupted lamin architecture counted in GFP positive cells (left) and in cells with aggregates (right). The number of GFP positive cells included in the analysis was >440 for GFP-C1 and wtHP-99Gly-GFP. For wtHP-99Gly-GFP > 170 aggregate bearing cells were counted. Due to the low levels of GFP positive cells among those transfected with mutHP-90Gly-GFP, a total of 80 GFP-positive cells and 70 aggregate bearing cells were included from mutHP-90Gly-GFP transfected cell populations. The exact p -values, from left to right, are as follows: 0.0021 ( ** ), 0.0004 ( *** ), 0.0123 ( * ), and 0.0113 ( * ). (C) Representative confocal images showing normal lamin rings in cells expressing GFP (GFP-C1), but disrupted lamin architecture in aggregate-containing cells after transfection of wtHP-99Gly-GFP or mutHP-90Gly-GFP. (D) Effect of stably expressed FMRpolyG on lamin architecture. Expression of GFP or FMRpolyG-GFP were induced with doxycycline for 72 h, followed by Lamin B1 staining, confocal imaging, and counting of GFP positive cells with disrupted lamin architecture. Cell expressing GFP (from GFP-C1) were used as negative controls. For GFP-C1 and wtHP-99Gly-GFP, a minimum of 830 GFP positive cells were quantified. Due to low expression levels in cells expressing mutHP-90Gly-GFP, 150 GFP-positive cells were included for this construct. For wtHP-99Gly-GFP and mutHP-90Gly-GFP, a minimum of 80 aggregate bearing cells were analyzed per construct. The exact p -values, from left to right, are as follows: 0.0186 ( * ), 0.0007 ( *** ), 0.00001 ( *** ), and 0.0004 ( *** ). (E) Representative confocal images showing normal lamin rings in HEK-FlpIn cells expressing GFP and disrupted lamin structures in aggregate-containing cells expressing FMRpolyG-GFP. All graphs in (A,B,D) are based on quantifications of a minimum of three individual experiments and error bars represent SD.

    Article Snippet: The wtHP-99Gly-GFP-plasmid and a wtHP-ACG-99Gly-GFP-plasmid (Addgene plasmid # 63091) were kind gifts from Nicolas Charlet-Berguand (Sellier et al., ).

    Techniques: Transfection, Construct, Staining, Negative Control, Imaging, Expressing, Stable Transfection

    FMRpolyG forms dense filamentous aggregates. Cells expressing GFP-tagged FMRpolyG were fixed and imaged by confocal microscopy to localize aggregate-containing cells, and subsequently processed for transmission electron microscopy. Following ultramicrotomy, cells of interest were relocalized within sections and imaged at both intermediate and high magnifications to analyze the surrounding cellular environment and ultrastructural details of the aggregates, respectively. Transmission electron micrographs of boxed areas in confocal images (A,D,G,J,M) are shown at intermediate (B,E,H,K,N) and high (C,F,I,L,O) magnification. Arrow heads indicate the position of aggregates inside cells. (A–F) wtHP-99Gly-GFP aggregates consist of filaments that can be found in parallel orientation to the plane of sectioning over significant lengths (>100 nm), and that lay in close vicinity to the nucleus and to annulate lamellae in ultrathin sections. Note decreased staining toward the center of a large aggregate (F) . (H–L) mutHP-90Gly-GFP aggregates are similar to wtHP-99Gly-GFP aggregates but appear less densely packed and displayed uniform staining throughout the aggregates in all observed cases. A subset of mutHP-90Gly-GFP aggregates show a highly repetitive pattern of low-contrast spots circularly arranged around a central spot of slightly larger size and higher contrast (L) . (N,O) In comparison to FMRpolyG aggregates, GFP-p62 aggregates appear to consist of thinner filaments that radiate in all directions within the plane of sectioning. Scale bars are 5 μm (A,D,G,J,M) , 2 μm (B,E,H,K,N) , and 500 nm (C,F,I,L,O) . n, nucleus; al, annulate lamellae; pm, plasma membrane; m, mitochondria. Red spheres in (J,M) are fiducial beads used for image registration.

    Journal: Frontiers in Genetics

    Article Title: The FMRpolyGlycine Protein Mediates Aggregate Formation and Toxicity Independent of the CGG mRNA Hairpin in a Cellular Model for FXTAS

    doi: 10.3389/fgene.2019.00249

    Figure Lengend Snippet: FMRpolyG forms dense filamentous aggregates. Cells expressing GFP-tagged FMRpolyG were fixed and imaged by confocal microscopy to localize aggregate-containing cells, and subsequently processed for transmission electron microscopy. Following ultramicrotomy, cells of interest were relocalized within sections and imaged at both intermediate and high magnifications to analyze the surrounding cellular environment and ultrastructural details of the aggregates, respectively. Transmission electron micrographs of boxed areas in confocal images (A,D,G,J,M) are shown at intermediate (B,E,H,K,N) and high (C,F,I,L,O) magnification. Arrow heads indicate the position of aggregates inside cells. (A–F) wtHP-99Gly-GFP aggregates consist of filaments that can be found in parallel orientation to the plane of sectioning over significant lengths (>100 nm), and that lay in close vicinity to the nucleus and to annulate lamellae in ultrathin sections. Note decreased staining toward the center of a large aggregate (F) . (H–L) mutHP-90Gly-GFP aggregates are similar to wtHP-99Gly-GFP aggregates but appear less densely packed and displayed uniform staining throughout the aggregates in all observed cases. A subset of mutHP-90Gly-GFP aggregates show a highly repetitive pattern of low-contrast spots circularly arranged around a central spot of slightly larger size and higher contrast (L) . (N,O) In comparison to FMRpolyG aggregates, GFP-p62 aggregates appear to consist of thinner filaments that radiate in all directions within the plane of sectioning. Scale bars are 5 μm (A,D,G,J,M) , 2 μm (B,E,H,K,N) , and 500 nm (C,F,I,L,O) . n, nucleus; al, annulate lamellae; pm, plasma membrane; m, mitochondria. Red spheres in (J,M) are fiducial beads used for image registration.

    Article Snippet: The wtHP-99Gly-GFP-plasmid and a wtHP-ACG-99Gly-GFP-plasmid (Addgene plasmid # 63091) were kind gifts from Nicolas Charlet-Berguand (Sellier et al., ).

    Techniques: Expressing, Confocal Microscopy, Transmission Assay, Electron Microscopy, Staining

    Proteasomes are recruited to FMRpolyG aggregates. (A) Representative confocal fluorescence microscopy images of HEK293 cells transfected with wtHP-99Gly-GFP (upper panel) or mutHP-90Gly-GFP (lower panel) and immunostained with antibodies to the proteasome, ubiquitin and p62. Fraction of FMRpolyG-GFP aggregates which co-localized with the proteasome (B) , ubiquitin (C) , p62 (D) , or LC3B (E) , after transfection of wtHP-99Gly-GFP (black bars) or mutHP-90Gly-GFP (white bars). Cells were stained for the indicated endogenous proteins. Quantifications were performed using the image analyzing software Volocity, and are based on 3–6 experiments. For (B) the total number of aggregates included in the quantification was >65 per construct. The remaining graphs (C–E) are based on analysis of a total of > 190 GFP-positive aggregates per construct. (F–H) FMRpolyG is mainly degraded by the proteasome. Except for the negative controls (uninduced cells), HEK-FlpIn cells were treated with tetracycline (1 μg/ml) for 48 h to induce accumulation of GFP-p62 (F) or FMRpolyG-GFP (G,H) , respectively. Degradation was then measured by flow cytometry of the entire cell population (>20,000 cells for each condition, per experiment), as a loss in mean GFP intensity after the removal of tetracycline (Tet Off). The experiments were performed as indicated in the absence or presence of Baf-A1 or MG132. All graphs are based on a minimum of three independent experiments. The exact p -values in (F) are 0.67 (for the n.s. difference), and 0.006 (for difference marked by two stars). The exact p -values, from left to right, in (H) are as follows: 0.029 (one star), 0.402 (n.s.), 0.048 (one star), and 0.933 (n.s.). Error bars represent SD.

    Journal: Frontiers in Genetics

    Article Title: The FMRpolyGlycine Protein Mediates Aggregate Formation and Toxicity Independent of the CGG mRNA Hairpin in a Cellular Model for FXTAS

    doi: 10.3389/fgene.2019.00249

    Figure Lengend Snippet: Proteasomes are recruited to FMRpolyG aggregates. (A) Representative confocal fluorescence microscopy images of HEK293 cells transfected with wtHP-99Gly-GFP (upper panel) or mutHP-90Gly-GFP (lower panel) and immunostained with antibodies to the proteasome, ubiquitin and p62. Fraction of FMRpolyG-GFP aggregates which co-localized with the proteasome (B) , ubiquitin (C) , p62 (D) , or LC3B (E) , after transfection of wtHP-99Gly-GFP (black bars) or mutHP-90Gly-GFP (white bars). Cells were stained for the indicated endogenous proteins. Quantifications were performed using the image analyzing software Volocity, and are based on 3–6 experiments. For (B) the total number of aggregates included in the quantification was >65 per construct. The remaining graphs (C–E) are based on analysis of a total of > 190 GFP-positive aggregates per construct. (F–H) FMRpolyG is mainly degraded by the proteasome. Except for the negative controls (uninduced cells), HEK-FlpIn cells were treated with tetracycline (1 μg/ml) for 48 h to induce accumulation of GFP-p62 (F) or FMRpolyG-GFP (G,H) , respectively. Degradation was then measured by flow cytometry of the entire cell population (>20,000 cells for each condition, per experiment), as a loss in mean GFP intensity after the removal of tetracycline (Tet Off). The experiments were performed as indicated in the absence or presence of Baf-A1 or MG132. All graphs are based on a minimum of three independent experiments. The exact p -values in (F) are 0.67 (for the n.s. difference), and 0.006 (for difference marked by two stars). The exact p -values, from left to right, in (H) are as follows: 0.029 (one star), 0.402 (n.s.), 0.048 (one star), and 0.933 (n.s.). Error bars represent SD.

    Article Snippet: The wtHP-99Gly-GFP-plasmid and a wtHP-ACG-99Gly-GFP-plasmid (Addgene plasmid # 63091) were kind gifts from Nicolas Charlet-Berguand (Sellier et al., ).

    Techniques: Fluorescence, Microscopy, Transfection, Staining, Software, Construct, Flow Cytometry