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Introduction of human GGAA microsatellites into the yeast genome induces gene silencing. A) Constructs carrying eGFP driven by the minimal pCYC1 promoter with upstream 80-nt random or 10×/20×/40 × (GGAA) inserts. B) Confocal images of W303-1A isogenic derivatives carrying the constructs shown in (A): GFP fluorescence (right) and bright field (left); scale bar, 10 μm. C, D) Quantification by flow <t>cytometry</t> ( n = 10,000 cells per sample). C) Distribution of cell counts as a function of Comp-GFP. D) Mean eGFP fluorescence.
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Introduction of human GGAA microsatellites into the yeast genome induces gene silencing. A) Constructs carrying eGFP driven by the minimal pCYC1 promoter with upstream 80-nt random or 10×/20×/40 × (GGAA) inserts. B) Confocal images of W303-1A isogenic derivatives carrying the constructs shown in (A): GFP fluorescence (right) and bright field (left); scale bar, 10 μm. C, D) Quantification by flow cytometry ( n = 10,000 cells per sample). C) Distribution of cell counts as a function of Comp-GFP. D) Mean eGFP fluorescence.

Journal: PNAS Nexus

Article Title: Ancient and animal-specific regulatory modes of EWS::FLI1 revealed by a minimal yeast model

doi: 10.1093/pnasnexus/pgag329

Figure Lengend Snippet: Introduction of human GGAA microsatellites into the yeast genome induces gene silencing. A) Constructs carrying eGFP driven by the minimal pCYC1 promoter with upstream 80-nt random or 10×/20×/40 × (GGAA) inserts. B) Confocal images of W303-1A isogenic derivatives carrying the constructs shown in (A): GFP fluorescence (right) and bright field (left); scale bar, 10 μm. C, D) Quantification by flow cytometry ( n = 10,000 cells per sample). C) Distribution of cell counts as a function of Comp-GFP. D) Mean eGFP fluorescence.

Article Snippet: GFP fluorescence was assessed by SP8 confocal microscopy and by spectral flow cytometry (Cytek Aurora; 10,000 cells/sample).

Techniques: Yeast, Construct, Confocal, Fluorescence, Flow Cytometry

Transcriptional activation by EWS::FLI1 through recognition of GGAA microsatellites in yeast. A) W303-1A derivatives carrying pCYC1 (top), 20 × (GGAA) (middle), or 40 × (GGAA) (bottom) reporters transformed with empty pYES2, EWS::FLI1, or EWS::FLI1 RRLL . Left, representative confocal images (bright-field, top; GFP fluorescence, bottom); middle, flow-cytometry histograms of cell counts as a function of Comp-GFP; right, mean GFP fluorescence. B) Time-course of 20×(GGAA)_ pCYC1 _eGFP cells transformed with pYES2 carrying EWS::FLI1 RRLL (top) or EWS::FLI1 (bottom). Samples collected at the indicated times were immunoblotted with anti-EWSR1 and anti-GFP monoclonal antibodies; arrowheads indicate EWS::FLI1 and GFP. Ponceau staining is shown as a loading and transfer control. Equivalent results were obtained in two further biological replicates. C) Relative EWS::FLI1 and GFP levels, normalized to the mean EWS::FLI1 signal at 6 h of induction (100%). Error bars, SD. * P < 0.05; ns, not significant; two-way ANOVA.

Journal: PNAS Nexus

Article Title: Ancient and animal-specific regulatory modes of EWS::FLI1 revealed by a minimal yeast model

doi: 10.1093/pnasnexus/pgag329

Figure Lengend Snippet: Transcriptional activation by EWS::FLI1 through recognition of GGAA microsatellites in yeast. A) W303-1A derivatives carrying pCYC1 (top), 20 × (GGAA) (middle), or 40 × (GGAA) (bottom) reporters transformed with empty pYES2, EWS::FLI1, or EWS::FLI1 RRLL . Left, representative confocal images (bright-field, top; GFP fluorescence, bottom); middle, flow-cytometry histograms of cell counts as a function of Comp-GFP; right, mean GFP fluorescence. B) Time-course of 20×(GGAA)_ pCYC1 _eGFP cells transformed with pYES2 carrying EWS::FLI1 RRLL (top) or EWS::FLI1 (bottom). Samples collected at the indicated times were immunoblotted with anti-EWSR1 and anti-GFP monoclonal antibodies; arrowheads indicate EWS::FLI1 and GFP. Ponceau staining is shown as a loading and transfer control. Equivalent results were obtained in two further biological replicates. C) Relative EWS::FLI1 and GFP levels, normalized to the mean EWS::FLI1 signal at 6 h of induction (100%). Error bars, SD. * P < 0.05; ns, not significant; two-way ANOVA.

Article Snippet: GFP fluorescence was assessed by SP8 confocal microscopy and by spectral flow cytometry (Cytek Aurora; 10,000 cells/sample).

Techniques: Activation Assay, Yeast, Transformation Assay, Confocal, Fluorescence, Flow Cytometry, Bioprocessing, Staining, Control