Journal: eLife

Article Title: High-content synaptic phenotyping in human cellular models reveals a role for BET proteins in synapse assembly

doi: 10.7554/eLife.80168

Figure Lengend Snippet: ( a ) Schematic of stable integration of a doxycycline-responsive NGN2 (iNGN2) cassette into human pluripotent stem cells (hPSCs). TALENs were used to insert iNGN2 into the AAVS1 safe harbor locus of the PPP1R12C gene. ( b ) Summary of human-induced neuron (hN) protocol. hPSCs are differentiated into neural progenitor like cells using extrinsic inhibition of BMP, SMAD, and Wnt in combination with doxycycline-driven transient ectopic NGN2 expression. Zeocin is used as a selective agent. Neural progenitor-like cells were then incubated with neurotrophins in addition to the anti-mitotic agent floxuridine. hNs are then maintained in neurobasal medium and neurotrophins. ( c ) Flowchart of the high-content synaptic screening platform. One day after automated seeding of hNs and hpAs (using a liquid handler, day 5 of hN differentiation), a random plate is selected for assessing plating consistency (Columbus script). On day 18, treatments (shown here as small molecules at 3 µM) are administered in triplicate for 72 hr using a liquid handler; two plates are used as sentinel or reference. On day 21, co-cultures are then stained for synaptic markers using the liquid handler and high-content images are acquired using the Opera Phenix (Perkin Elmer). Data are then processed through CellProfiler and Screener to quantify the number and area of SYNAPSIN1 puncta on MAP2-expressing neurites, the area of MAP2-positive neurites, the number of DAPI-positive nuclei, and the density of SYNAPSIN1 puncta on MAP2-expressing neurites (Z-score). ( d ) Representative images of hNs co-cultured with hpAs and stained for SYNAPSIN1 (red) and MAP2 (green). Cells are counterstained with DAPI (blue). Scale: 100 pixels. Insets: arrows show SYNAPSIN1 puncta localized on MAP2-positive neurites. ( e, f ) Acceptance criteria for plating consistency. Plating consistency for each batch is determined using a Columbus script by quantifying detected hN nuclei per well ( e ) as well as the intra-plate covariance ( f ) of the randomly selected plate on day 6 after hN differentiation. Circles represented the number of neuronal nuclei detected per well from the plates randomly selected on day 6 (one day after seeding the co-culture). Thresholds for inclusion (red dashed lines) are set as above 4000 detected hN nuclei per well and below 12,000 detected hN nuclei per well ( e ) and a covariance below 8% across the plate ( f ). n = 1 x 96 well-plate per Batch which corresponds to 60 wells per 96-well plate per Batch. Error bars are shown as mean +/- SEM. ( g ) Quantification of the density of SYNAPSIN1 puncta on MAP2 neurites after incubation with SYNAPSIN siRNA versus control conditions. n = 6 wells for each condition; ***p<0.0001, ANOVA with Dunnett’s post hoc test. Error bars are shown as SEM.

Article Snippet: antibody , Anti-human SYNAPSIN1 (Rabbit polyclonal) , Millipore , Cat#AB1543 RRID: AB_2200400 , IF (1:1000) WB (1:1000).

Techniques: TALENs, Inhibition, Expressing, Incubation, Staining, Cell Culture, Co-Culture Assay, Control

Journal: eLife

Article Title: High-content synaptic phenotyping in human cellular models reveals a role for BET proteins in synapse assembly

doi: 10.7554/eLife.80168

Figure Lengend Snippet: ( a ) Design of the PCR genotyping strategy for validation of stable integration of the iNGN2 cassette in the AAVS1 safe harbor locus of the H1 genome. ( b ) Cytogenetic analysis performed by Cell Line Genetics on iNGN2-hPSCs demonstrating an apparently normal karyotype 46,XY. ( c ) Example images (left) and quantification (right) of SYNAPSIN1 on MAP2 co-localized with the presynaptic marker synaptophysin (n = 3 wells, 28 fields). ( d ) Example images (left) and quantification (right) of SYNAPSIN1 on MAP2 co-localized with the postsynaptic marker PSD-95 (n = 3 wells, 16 fields, >10 puncta/field threshold).

Article Snippet: antibody , Anti-human SYNAPSIN1 (Rabbit polyclonal) , Millipore , Cat#AB1543 RRID: AB_2200400 , IF (1:1000) WB (1:1000).

Techniques: Biomarker Discovery, Marker

Journal: eLife

Article Title: High-content synaptic phenotyping in human cellular models reveals a role for BET proteins in synapse assembly

doi: 10.7554/eLife.80168

Figure Lengend Snippet: For each acquired field, 5–8 stacks of MAP2, SYNAPSIN1, and DAPI channels are projected in a single plane before segmentation. A colocalization module then assigns the relationship between the identified SYNAPSIN1 puncta contained within or partly touching the MAP2 neurite objects. The area covered by MAP2 neurites and the number of SYNAPSIN1 puncta on MAP2 neurites from each field are then exported to a *.csv spreadsheet.

Article Snippet: antibody , Anti-human SYNAPSIN1 (Rabbit polyclonal) , Millipore , Cat#AB1543 RRID: AB_2200400 , IF (1:1000) WB (1:1000).

Techniques:

Journal: eLife

Article Title: High-content synaptic phenotyping in human cellular models reveals a role for BET proteins in synapse assembly

doi: 10.7554/eLife.80168

Figure Lengend Snippet: ( a ) Schematic of siRNA delivery. hN + hpA co-cultures were untreated or incubated on day 18 with SYNAPSIN1 or control siRNA (1 µM) for 72 hr, stained and processed through the synaptic assay. Scale bar = 100 pixel. ( b ) Examples of output from CellProfiler showing overlay of SYNAPSIN1 puncta (red) on MAP2-expressing neurites (green) from untreated and SYNAPSIN1 siRNA-treated conditions. ( c ) Histograms showing the MAP2-positive neurite coverage and the number of DAPI-positive nuclei after incubation with SYNAPSIN1 siRNA versus control conditions. n = 6 wells for each condition; *p<0.05, ANOVA with Dunnett’s post hoc test. ( d ) Cumulative distribution of the diameter of SYNAPSIN1 puncta.

Article Snippet: antibody , Anti-human SYNAPSIN1 (Rabbit polyclonal) , Millipore , Cat#AB1543 RRID: AB_2200400 , IF (1:1000) WB (1:1000).

Techniques: Incubation, Control, Staining, Expressing

Journal: eLife

Article Title: High-content synaptic phenotyping in human cellular models reveals a role for BET proteins in synapse assembly

doi: 10.7554/eLife.80168

Figure Lengend Snippet: CellProfiler *.csv files (field level) are uploaded with a Genedata parser and filtered through three quality control steps: (i) for each individual field (cross) the area covered by MAP2 must fall within ± 1 standard deviation (σ) to the intra-batch mean (µ) MAP2 coverage (blue crosses represent accepted fields shown within red dashed lines; gray crosses indicated fields that are rejected), (ii) exclusion of the well if ≤4 fields remain following the first quality control step; (iii) exclusion of conditions with one well remaining following the well-level quality control. Data are then imported into Genedata Screener and pattern correction algorithms are leveraged for calculation of Z-score values of the density of SYNAPSIN1 puncta on MAP2 neurites for each condition tested.

Article Snippet: antibody , Anti-human SYNAPSIN1 (Rabbit polyclonal) , Millipore , Cat#AB1543 RRID: AB_2200400 , IF (1:1000) WB (1:1000).

Techniques: Control, Standard Deviation

Journal: eLife

Article Title: High-content synaptic phenotyping in human cellular models reveals a role for BET proteins in synapse assembly

doi: 10.7554/eLife.80168

Figure Lengend Snippet: ( a ) Number of small molecules per pathway used in the primary screen. ( b, c ) Small molecules were screened at 3 µM in triplicate for 72 hr. 13 small molecules did not meet quality control requirements. 92.94% of the control and treated wells had a Z-score for synaptic density between –2 and +2 as depicted by the red dashed lines ( b ). Values are presented as Z-score of the density of SYNAPSIN1 puncta on MAP2-positive neurites ( b , each circle represents either a DMSO or a small molecule treated well) or the area covered by MAP2-expressing neurites ( c , each circle represents the aggregate value of the triplicate). Small molecules decreasing the area covered by MAP2-positive neurites are indicated by circles filled in red, and one small molecule increasing the area covered by MAP2-positive neurites is indicated by circle filled in green.

Article Snippet: antibody , Anti-human SYNAPSIN1 (Rabbit polyclonal) , Millipore , Cat#AB1543 RRID: AB_2200400 , IF (1:1000) WB (1:1000).

Techniques: Control, Expressing

Journal: eLife

Article Title: High-content synaptic phenotyping in human cellular models reveals a role for BET proteins in synapse assembly

doi: 10.7554/eLife.80168

Figure Lengend Snippet: ( a ) Z-score values for the density of SYNAPSIN1 on MAP2-expressing neurites for hN + hpA co-cultures incubated with small molecules in triplicate at 3 µM for 72 hr starting on day 18 of neuronal differentiation (each circle represents the small molecule level aggregate of the replicates). The effects of each individual small molecule were assessed using our in silico pipelines. Small molecules that increased presynaptic density and were selected for further validation are indicated by colored circles. Those in purple are anticarcinogens that reduced presynaptic density and were not selected for further validation. ( b ) Representative immunofluorescence images and CellProfiler output images (field-level) of human co-cultures treated with either 0.1% DMSO, (+)-JQ1, BI-D1870, I-BET151, Rigosertib, or GSK J4 HCl. Scale bar = 1 µm. Note the increase in SYNAPSIN1 puncta on MAP2-expressing neurites following (+)-JQ1, BI-D1870, and I-BET151 compared to 0.1% DMSO control, and the decrease in SYNPASIN1 puncta following treatment with Rigosertib and GSK J4 HCl.

Article Snippet: antibody , Anti-human SYNAPSIN1 (Rabbit polyclonal) , Millipore , Cat#AB1543 RRID: AB_2200400 , IF (1:1000) WB (1:1000).

Techniques: Expressing, Incubation, In Silico, Biomarker Discovery, Immunofluorescence, Control

Journal: eLife

Article Title: High-content synaptic phenotyping in human cellular models reveals a role for BET proteins in synapse assembly

doi: 10.7554/eLife.80168

Figure Lengend Snippet: Human co-cultures were treated on day 18 with 0.1% DMSO or the 17 selected small molecules at various concentrations for 72 hr, stained and processed through the synaptic assay. Dose–response curves show the density of SYNAPSIN1 puncta on MAP2-expressing neurites (same data in histogram format shown in ). Green dots represent the optimal concentration of the chosen small molecules. EC(50) and curves were calculated and drawn using PRISM software. Data are quantified by percentage of intra-plate control (0.1% DMSO) represented as mean values ± SEM, n = 3 biological replicates, n = 3 technical replicates.

Article Snippet: antibody , Anti-human SYNAPSIN1 (Rabbit polyclonal) , Millipore , Cat#AB1543 RRID: AB_2200400 , IF (1:1000) WB (1:1000).

Techniques: Staining, Expressing, Concentration Assay, Software, Control

Journal: eLife

Article Title: High-content synaptic phenotyping in human cellular models reveals a role for BET proteins in synapse assembly

doi: 10.7554/eLife.80168

Figure Lengend Snippet: Human co-cultures were treated on day 18 with 0.1% DMSO or the 17 selected small molecules at various concentrations for 72 hr, stained and processed through the synaptic assay. Histograms show the density of SYNAPSIN1 puncta on MAP2-expressing neurites (same data in dose–response curve format shown in and for (+)-JQ1, Birabresib, and VS-5584 in ; all data are included together here for comparison). Data are quantified by percentage of intra-plate control (0.1% DMSO) represented as mean values ± SEM, n = 3 biological replicates, n = 3 technical replicates. *p<0.05; **p<0.01, ***p<0.001; one-way ANOVA with Dunnett’s multiple comparisons test.

Article Snippet: antibody , Anti-human SYNAPSIN1 (Rabbit polyclonal) , Millipore , Cat#AB1543 RRID: AB_2200400 , IF (1:1000) WB (1:1000).

Techniques: Staining, Expressing, Comparison, Control

Journal: eLife

Article Title: High-content synaptic phenotyping in human cellular models reveals a role for BET proteins in synapse assembly

doi: 10.7554/eLife.80168

Figure Lengend Snippet: hNs were generated using an independent cell line (iNgn2-DS2U human iPSCs), co-cultured with hpAs, treated on day 18 with 0.1% DMSO or selected small molecules at the optimal concentrations for 72 hr, stained and processed through the synaptic assay. Histograms show the density of SYNAPSIN1 puncta on MAP2-positive neurons (left), the area covered by MAP2-expressing neurites (middle), and the number of DAPI-positive nuclei (right). Data are quantified by percentage of intra-plate control (0.1% DMSO) represented as mean values ± SEM, n = 2 biological replicates, n = 8 technical replicates. *p<0.05, **p<0.01, ***p<0.001; one-way ANOVA with Dunnett’s multiple comparisons test. Scale bar = 100 pixel.

Article Snippet: antibody , Anti-human SYNAPSIN1 (Rabbit polyclonal) , Millipore , Cat#AB1543 RRID: AB_2200400 , IF (1:1000) WB (1:1000).

Techniques: Generated, Cell Culture, Staining, Expressing, Control

Journal: eLife

Article Title: High-content synaptic phenotyping in human cellular models reveals a role for BET proteins in synapse assembly

doi: 10.7554/eLife.80168

Figure Lengend Snippet: ( a ) Workflow for small molecule validation in the absence of hpAs. Scale bar = 100 pixel. ( b–d ) In the absence of hpAs, none of the selected small molecules affected SYNAPSIN1 density of hNs, in contrast to the co-culture condition analyzed in parallel. The area covered by MAP2-positive neurites and cell viability was also not impacted as compared to intra-plate DMSO controls. Error bars are shown as mean +/- SEM. ( e ) Schematic of the preparation of the immunoblot samples. ( f ) Representative images of immunoblots and quantification of SYNAPSIN1 normalized to GAPDH and presented as a percentage of DMSO control in both hN alone and hN + hpA co-culture conditions. Note that SYNAPSIN1 protein expression levels were not impacted in any condition. n = 3 biological replicates ( f ). Error bars are shown as mean +/- SEM. Figure 4—source data 1. Source of western blots for SYNAPSIN1 as well as GAPDH loading control following small molecule treatment versus DMSO control.

Article Snippet: antibody , Anti-human SYNAPSIN1 (Rabbit polyclonal) , Millipore , Cat#AB1543 RRID: AB_2200400 , IF (1:1000) WB (1:1000).

Techniques: Biomarker Discovery, Co-Culture Assay, Western Blot, Control, Expressing

Journal: eLife

Article Title: High-content synaptic phenotyping in human cellular models reveals a role for BET proteins in synapse assembly

doi: 10.7554/eLife.80168

Figure Lengend Snippet: ( a, b ) Representative immunofluorescence and CellProfiler output images (field-level) of hN monocultures ( a ) and hN + hpA co-cultures ( b ). Scale bar = 1 µm. ( c–e ) Measurements comparing the density of SYNAPSIN1 puncta on MAP2-positive neurites ( c ), the area covered by individual SYNAPSIN1 puncta ( d ) and the area covered by MAP2-positive neurites ( e ) between hN monocultures and hN + hpA co-cultures. Data are represented as mean values ± SEM, n = 3 biological replicates, n = 8 technical replicates ( c–e ); n > 1000 fields and n > 50,000 SYNAPSIN1 puncta for each condition ( c–e ). ***p<0.001; Kolmogorov–Smirnov unpaired t -test. ( f, g ) Power calculations for hN + hPA co-culture ( f ) and hN monoculture ( g ) validation experiments at the well level for the hypothesis ‘greater than.’ Cohen’s d and the number of wells analyzed for each experiment following 0.5 uM (+)-JQ1, 3 uM I-BET151, and 1 uM Birabresib treatment are indicated by the dotted lines.

Article Snippet: antibody , Anti-human SYNAPSIN1 (Rabbit polyclonal) , Millipore , Cat#AB1543 RRID: AB_2200400 , IF (1:1000) WB (1:1000).

Techniques: Immunofluorescence, Co-Culture Assay, Biomarker Discovery

Journal: eLife

Article Title: High-content synaptic phenotyping in human cellular models reveals a role for BET proteins in synapse assembly

doi: 10.7554/eLife.80168

Figure Lengend Snippet:

Article Snippet: antibody , Anti-human SYNAPSIN1 (Rabbit polyclonal) , Millipore , Cat#AB1543 RRID: AB_2200400 , IF (1:1000) WB (1:1000).

Techniques: Transfection, Construct, Control, Recombinant, Plasmid Preparation, Sequencing, Lysis, Drug discovery, Software

Journal: Scientific Reports

Article Title: Encapsulation of human limbus-derived stromal/mesenchymal stem cells for biological preservation and transportation in extreme Indian conditions for clinical use

doi: 10.1038/s41598-019-53315-x

Figure Lengend Snippet: Phenotypic expression of the biomarkers. Immunostaining of the encapsulated hLSMCs under transit for 3 days: Alginate encapsulated hLSMCs of both groups, stored/under transit for 3 days have shown the expression of Pax6 + , stem-cell biomarkers (ABCG2 + , p63-α + ) and the mesenchymal biomarkers (VIM + , CD90 + , CD105 + and CD45 − ) with respect to the control cells. Blue: DAPI, nuclear stain. Scale: 100 µM.

Article Snippet: The antibody panel was composed of (a) ABCG2 (1:100, 18841, Santa Cruz Biotechnology, USA), Pax6 (1:300, 901301, BioLegend, USA), p63-α (1:100, 4892S, Cell Signalling technology, USA) and Col-III (1:100, ab7778, Abcam, UK), as positive markers of the human limbal stem cell phenotype; HLA-DR (1:100, ab55152, Abcam, UK), and CD45 (1:100, 13197, Cell Signalling Technology, USA) as negative marker for mesenchymal origin, (b) CD73 (1:100, 13160, Cell Signalling Technology, USA), CD105 (1:100, 376381, Santa Cruz Biotechnology, USA), and VIM (1:100, 6260, Santa Cruz Biotechnology, USA) as positive markers of the mesenchymal phenotype.

Techniques: Expressing, Immunostaining, Control, Staining

Journal: Scientific Reports

Article Title: Encapsulation of human limbus-derived stromal/mesenchymal stem cells for biological preservation and transportation in extreme Indian conditions for clinical use

doi: 10.1038/s41598-019-53315-x

Figure Lengend Snippet: Quantification of the gene expression using real-time PCR. Immunostaining of the encapsulated hLSMCs under transit for 5 days: Alginate encapsulated hLSMCs stored at 4 °C did not show expression of the stem-cell (ABCG2 − ). The RT group cells have showed similar phenotype as the control group (ABCG2 + , Pax6 + p63-α + , VIM + , CD90 + , CD105 + , CD45 − , HLADR + , Col-III + , and CD73 + ). Blue: DAPI, nuclear stain. Scale : 100 µM.

Article Snippet: The antibody panel was composed of (a) ABCG2 (1:100, 18841, Santa Cruz Biotechnology, USA), Pax6 (1:300, 901301, BioLegend, USA), p63-α (1:100, 4892S, Cell Signalling technology, USA) and Col-III (1:100, ab7778, Abcam, UK), as positive markers of the human limbal stem cell phenotype; HLA-DR (1:100, ab55152, Abcam, UK), and CD45 (1:100, 13197, Cell Signalling Technology, USA) as negative marker for mesenchymal origin, (b) CD73 (1:100, 13160, Cell Signalling Technology, USA), CD105 (1:100, 376381, Santa Cruz Biotechnology, USA), and VIM (1:100, 6260, Santa Cruz Biotechnology, USA) as positive markers of the mesenchymal phenotype.

Techniques: Gene Expression, Real-time Polymerase Chain Reaction, Immunostaining, Expressing, Control, Staining

Journal: Scientific Reports

Article Title: Encapsulation of human limbus-derived stromal/mesenchymal stem cells for biological preservation and transportation in extreme Indian conditions for clinical use

doi: 10.1038/s41598-019-53315-x

Figure Lengend Snippet: Tabular format denoting the number of cells showing positive expression of the phenotypic biomarkers.

Article Snippet: The antibody panel was composed of (a) ABCG2 (1:100, 18841, Santa Cruz Biotechnology, USA), Pax6 (1:300, 901301, BioLegend, USA), p63-α (1:100, 4892S, Cell Signalling technology, USA) and Col-III (1:100, ab7778, Abcam, UK), as positive markers of the human limbal stem cell phenotype; HLA-DR (1:100, ab55152, Abcam, UK), and CD45 (1:100, 13197, Cell Signalling Technology, USA) as negative marker for mesenchymal origin, (b) CD73 (1:100, 13160, Cell Signalling Technology, USA), CD105 (1:100, 376381, Santa Cruz Biotechnology, USA), and VIM (1:100, 6260, Santa Cruz Biotechnology, USA) as positive markers of the mesenchymal phenotype.

Techniques: Expressing, Control, Biomarker Discovery

Journal: bioRxiv

Article Title: Cryo-EM structures of α-synuclein filaments from Parkinson’s disease and dementia with Lewy bodies

doi: 10.1101/2022.07.12.499706

Figure Lengend Snippet: Sections from brain regions contralateral to those used for cryo-EM structure determination were stained with monoclonal antibody Syn1 (1:1,000). (a), Cingulate cortex from PD; (b), Cingulate cortex from PDD1; (c), Cingulate cortex from PDD2; (d), Frontal cortex from DLB1; (e), Frontal cortex from DLB2; (f), Cingulate cortex from DLB3. Scale bars: a-c, f, 100 μm; d,e, 50 μm.

Article Snippet: Before blocking, membranes were fixed with 1% paraformaldehyde for 30 min. Primary antibodies were: Syn303 [a mouse monoclonal antibody that recognizes residues 1-5 of human α-synuclein ( )] (BioLegend) at 1:4,000, Syn1 [a mouse monoclonal antibody that recognizes residues 91-99 from the NAC region of human α-synuclein ( )] (BD Biosciences) at 1:4,000 and PER4 at 1:4,000.

Techniques: Cryo-EM Sample Prep, Staining

Journal: bioRxiv

Article Title: Cryo-EM structures of α-synuclein filaments from Parkinson’s disease and dementia with Lewy bodies

doi: 10.1101/2022.07.12.499706

Figure Lengend Snippet: PER4 was used at 1:50 in (a-c). (a), PD (Cingulate cortex); (b), PDD1 (Cingulate cortex); (c), DLB3 (Cingulate cortex); Syn303, Syn1 and PER4 were used at 1:4,000 in (d-f). The brain regions used for cryo-EM were also used for immunoblotting. The arrow points to the position of monomeric α-synuclein.

Article Snippet: Before blocking, membranes were fixed with 1% paraformaldehyde for 30 min. Primary antibodies were: Syn303 [a mouse monoclonal antibody that recognizes residues 1-5 of human α-synuclein ( )] (BioLegend) at 1:4,000, Syn1 [a mouse monoclonal antibody that recognizes residues 91-99 from the NAC region of human α-synuclein ( )] (BD Biosciences) at 1:4,000 and PER4 at 1:4,000.

Techniques: Cryo-EM Sample Prep, Western Blot