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idr0139-study.txt
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idr0139-study.txt
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# FILL IN AS MUCH INFORMATION AS YOU CAN. HINTS HAVE BEEN PUT IN SOME FIELDS AFTER THE HASH # SYMBOL. REPLACE THE HINT WITH TEXT WHERE APPROPRIATE.
# STUDY DESCRIPTION SECTION
# Section with generic information about the study including title, description, publication details (if applicable) and contact details
Comment[IDR Study Accession] idr0139
Study Title Nuclear fascin regulates cancer cell survival
Study Type high content screen
Study Type Term Source REF EFO
Study Type Term Accession EFO_0007550
Study Description Fascin is an important regulator of F-actin bundling leading to enhanced filopodia assembly. Fascin is also overexpressed in most solid tumours where it supports invasion through control of F-actin structures at the periphery and nuclear envelope. Recently fascin has been identified in the nucleus of a broad range of cell types but the contributions of nuclear fascin to cancer cell behaviour remain unknown. Here we demonstrate that fascin bundles F-actin within the nucleus to support chromatin organisation and efficient DNA damage response. Fascin associates directly with phosphorylated Histone H3 leading to regulated levels of nuclear fascin to support these phenotypes. Forcing nuclear fascin accumulation through the expression of nuclear-targeted fascin-specific nanobodies or inhibition of Histone H3 kinases results in enhanced and sustained nuclear F-actin bundling leading to reduced invasion, viability and nuclear fascin-specific/driven apoptosis. These findings represent an additional important route through which fascin can support tumorigenesis and provide insight into potential pathways for targeted fascin-dependent cancer cell killing.
Study Key Words Fascin actin nucleus nuclear F-actin HeLa cancer
Study Organism Homo sapiens
Study Organism Term Source REF NCBITaxon
Study Organism Term Accession 9606
Study Screens Number 3
Study External URL
Study BioImage Archive Accession
Study Public Release Date 2023-08-17
# Study Publication
Study PubMed ID 36039640
Study Publication Title Nuclear fascin regulates cancer cell survival
Study Author List Lawson CD, Peel S, Jayo A, Corrigan A, Iyer P, Baxter Dalrymple M, Marsh RJ, Cox S, Van Audenhove I, Gettemans J, Parsons M
Study PMC ID PMC9427113
Study DOI https://doi.org/10.7554/eLife.79283
# Study Contacts
Study Person Last Name Parsons Lawson
Study Person First Name Maddy Campbell
Study Person Email [email protected] [email protected]
Study Person Address Randall Centre for Cell and Molecular Biophysics, KingÕs College London, GuyÕs Campus, London, SE1 1UL, United Kingdom Randall Centre for Cell and Molecular Biophysics, KingÕs College London, GuyÕs Campus, London, SE1 1UL, United Kingdom
Study Person ORCID 0000-0002-2021-8379 0000-0001-5349-0638
Study Person Roles submitter submitter
# Study License and Data DOI
Study License CC BY 4.0
Study License URL https://creativecommons.org/licenses/by/4.0/
Study Copyright Lawson et al
Study Data Publisher University of Dundee
Study Data DOI https://doi.org/10.17867/10000190
Term Source Name NCBITaxon EFO CMPO Fbbi
Term Source File http://purl.obolibrary.org/obo/ http://www.ebi.ac.uk/efo/ http://www.ebi.ac.uk/cmpo/ http://purl.obolibrary.org/obo/
# SCREEN SECTION
# Screen Section containing all information relative to each screen in the study including materials used, protocols names and description, phenotype names and description.
# For multiple screens this section should be repeated. Copy and paste the whole section below and fill out for the next screen.
Screen Number 1
Comment[IDR Screen Name] idr0139-lawson-fascin/screenA
Screen Data DOI https://doi.org/10.17867/10000190a
Screen Sample Type cell
Screen Description To explore the pathways controlling nuclear fascin, we performed unbiased, imaging-based high-content phenotypic screening using fascin KD cells expressing mScarlet-fascin and the nuclear F-actin probe iRFP-nAC. Cells were treated with a library of ~13,000 annotated compounds, targeting multiple signalling pathways, at a single concentration (5 µm) or neutral or positive (leptomycin B) controls for 24 h, followed by fixation and imaging using high content spinning disk confocal microscopy. Resulting data were analysed using automated pipelines to define levels of nuclear fascin normalised to neutral and positive controls. The mean RZÕ of the neutral and positive controls in this screen was 0.5017, indicating excellent assay robustness. This approach identified compounds with a range of activities including those that increased or decreased nuclear fascin relative to the neutral controls. We chose to focus on those that increased nuclear fascin and identified hits based on a minimum threshold value of 30% of the neutral controls, equivalent to 3x the standard deviation of the neutral controls. Following quality control checks (e.g., to exclude images with very few cells), 231 compounds were identified as hits.
Screen Size Plates: 384 5D Images: 61,600 Planes: 246,400 Average Image Dimension (XYZCT): 996 x 996 x 1 x 4 x 1 Total Tb:
Screen Example Images
Screen Imaging Method spinning disk confocal microscopy
Screen Imaging Method Term Source REF Fbbi
Screen Imaging Method Term Accession FBbi_00000253
Screen Technology Type compound screen
Screen Technology Type Term Source REF EFO
Screen Technology Type Term Accession EFO_0007553
Screen Type primary screen
Screen Type Term Source REF EFO
Screen Type Term Accession EFO_0007556
Screen Comments
# Library section. The library file should be supplied separately and it should contain the reagents description including, at the absolute minimum: reagent ID, sequences and position in the layout (= plate + position in the plate)
Library File Name idr0139-screenA-annotation
Library File Format tab-delimited text
Library Type compound library
Library Type Term Source REF EFO
Library Type Term Accession EFO_0007569
Library Manufacturer AstraZeneca
Library Version
Library Experimental Conditions
Library Experimental Conditions Term Source REF EFO
Library Experimental Conditions Term Accession
Quality Control Description Wells with <5 imaged cells were excluded. Fluorescent compounds were automatically detected and excluded based on nuclear/cytoplasmic intensity/size. Wells with nuclear fascin >30% were manually reviewed to validate the presence of nuclear fascin within the population. Wells with very few cells or excessive numbers of dead (small/rounded) cells were excluded.
# Protocols
Protocol Name growth protocol treatment protocol HCS library protocol HCS image acquisition and feature extraction protocol HCS data analysis protocol
Protocol Type growth protocol treatment protocol HCS library protocol HCS image acquisition and feature extraction protocol HCS data analysis protocol
Protocol Type Term Source REF EFO EFO EFO EFO EFO
Protocol Type Term Accession EFO_0003789 EFO_0003969 EFO_0007571 EFO_0007572 EFO_0007573
Protocol Description Fascin was stably knocked down in HeLa cells, then mScarlet-fascin was stably re-expressed at physiological levels. mScarlet-fascin/fascin KD HeLa cells were cultured at 37¡C, 5% CO2 in high-glucose DMEM supplemented with 10% FBS, 1% glutamine and 1% penicillin/streptomycin. Cells were selected and maintained using puromycin, and sub-cultured using 0.05% trypsin-EDTA in PBS. In this assay, mScarlet-fascin/fascin KD HeLa cells were transfected with the nuclear actin probe iRFP-nAC, then plated in CellCarrier Ultra 384 well plates (PerkinElmer) at a concentration of 4,400 cells/well. 24 h after transfection, 10 nM leptomycin b (positive control), vehicle control or 5 µM library compounds were added using an Echo liquid handler (Labcyte/Beckman Coulter). Cells were incubated for a further 24 h then fixed in 4% paraformaldehyde. Library of biologically annotated compounds. Cells were stained with DAPI and Alexa Fluor 488 Phalloidin then imaged using a 20x objective on a CellVoyager CV8000 High-Content Screening System (Yokogawa). Images were analysed using Columbus (PerkinElmer) and Genedata Screener (Genedata) software. Images were analysed using automated pipelines to define levels of nuclear fascin normalised to neutral and positive controls. The nuclear and cytoplasmic compartments were determined using Columbus software (PerkinElmer) and the intensity of mScarlet-fascin in each comparment used to determine the % of nuclear-localized fascin per cell. The mean % nuclear fascin/cell/well was then calculated and values normalised relative to neutral controls (median set to 0) and positive controls (median set to 100). Hits were identified based on a normalised % nuclear fascin/cell/well of >30% of the neutral controls (equivalent to 3x the standard deviation of the neutral controls).
# Phenotypes
Phenotype Name Nuclear fascin localization
Phenotype Description The mean % nuclear fascin/cell/well was calculated and values normalised relative to neutral controls (median set to 0) and positive controls (median set to 100). Hits were identified based on a normalised % nuclear fascin/cell/well of >30% of the neutral controls (equivalent to 3x the standard deviation of the neutral controls).
Phenotype Score Type Automatic
Phenotype Term Source REF GO
Phenotype Term Name regulation of protein localization to nucleus
Phenotype Term Accession GO_1900180
# Raw Data Files
Raw Image Data Format TIFF
Raw Image Organization 50x 384 well plates, 4 fields per well, 4 channels per field
# Feature Level Data Files
Feature Level Data File Name
Feature Level Data File Description
Feature Level Data File Format
Feature Level Data Column Name
Feature Level Data Column Description
# Processed Data Files
Processed Data File Name idr0139-screenA-annotation
Processed Data File Format tab-delimited text
Processed Data File Description Primary screen - nuclear fascin
Processed Data Column Name Plate Well Reagent Identifier Gene Identifier Gene Symbol Score In Validation Screen Has Phenotype Phenotype 1
Processed Data Column Type Plate Well Reagent Identifier Gene Identifier Gene Symbol Data Phenotype Phenotype Phenotype
Processed Data Column Annotation Level well well well well
Processed Data Column Description Nuclear fascin - normalised mean nuclear fascin/cell/well Selected for compound response screen Nuclear fascin >30% Nuclear fascin >30%
Processed Data Column Link To Library File Plate_Well combination
Screen Number 2
Comment[IDR Screen Name] idr0139-lawson-fascin/screenB
Screen Data DOI https://doi.org/10.17867/10000190b
Screen Sample Type cell
Screen Description Compound response screen. 231 compounds identified as hits in primary screen were added to fascin KD cells expressing mScarlet-fascin and the nuclear F-actin probe iRFP-nAC, at 12 different concentrations. Assay conditions were otherwise identical to the primary screen. The mean RZÕ of the neutral and positive controls in this screen was 0.5607, indicating excellent assay robustness. Nuclear fascin and nuclear F-actin were plotted as a function of compound concentration to identify AC50 values for each compound. This compound response analysis identified 96 compounds that were active against nuclear fascin, of which 43 also increased nuclear F-actin.
Screen Size Plates: 384 5D Images: 17,280 Planes: 69,120 Average Image Dimension (XYZCT): 996 x 996 x 1 x 4 x 1 Total Tb:
Screen Example Images
Screen Imaging Method spinning disk confocal microscopy
Screen Imaging Method Term Source REF Fbbi
Screen Imaging Method Term Accession FBbi_00000253
Screen Technology Type compound screen
Screen Technology Type Term Source REF EFO
Screen Technology Type Term Accession EFO_0007553
Screen Type secondary screen
Screen Type Term Source REF EFO
Screen Type Term Accession EFO_0007557
Screen Comments
# Library section. The library file should be supplied separately and it should contain the reagents description including, at the absolute minimum: reagent ID, sequences and position in the layout (= plate + position in the plate)
Library File Name idr0139-screenB-annotation
Library File Format tab-delimited text
Library Type compound library
Library Type Term Source REF EFO
Library Type Term Accession EFO_0007569
Library Manufacturer AstraZeneca
Library Version
Library Experimental Conditions
Library Experimental Conditions Term Source REF EFO
Library Experimental Conditions Term Accession
Quality Control Description Wells with <5 imaged cells were excluded. Fluorescent compounds were automatically detected and excluded based on nuclear/cytoplasmic intensity/size. Wells with nuclear fascin >30% were manually reviewed to validate the presence of nuclear fascin within the population. Wells with very few cells or excessive numbers of dead (small/rounded) cells were excluded.
# Protocols
Protocol Name growth protocol treatment protocol HCS library protocol HCS image acquisition and feature extraction protocol HCS data analysis protocol
Protocol Type growth protocol treatment protocol HCS library protocol HCS image acquisition and feature extraction protocol HCS data analysis protocol
Protocol Type Term Source REF EFO EFO EFO EFO EFO
Protocol Type Term Accession EFO_0003789 EFO_0003969 EFO_0007571 EFO_0007572 EFO_0007573
Protocol Description Fascin was stably knocked down in HeLa cells, then mScarlet-fascin was stably re-expressed at physiological levels. mScarlet-fascin/fascin KD HeLa cells were cultured at 37¡C, 5% CO2 in high-glucose DMEM supplemented with 10% FBS, 1% glutamine and 1% penicillin/streptomycin. Cells were selected and maintained using puromycin, and sub-cultured using 0.05% trypsin-EDTA in PBS. In this assay, mScarlet-fascin/fascin KD HeLa cells were transfected with the nuclear actin probe iRFP-nAC, then plated in CellCarrier Ultra 384 well plates (PerkinElmer) at a concentration of 4,400 cells/well. 24 h after transfection, 10 nM leptomycin b (positive control), vehicle control or 0.00003-30 µM library compounds were added using an Echo liquid handler (Labcyte/Beckman Coulter). Cells were incubated for a further 24 h then fixed in 4% paraformaldehyde. Library of biologically annotated compounds identified as hits in primary screen Cells were stained with DAPI and Alexa Fluor 488 Phalloidin then imaged using a 20x objective on a CellVoyager CV8000 High-Content Screening System (Yokogawa). Images were analysed using Columbus (PerkinElmer) and Genedata Screener (Genedata) software. Images were analysed using automated pipelines to define levels of nuclear fascin normalised to neutral and positive controls. The nuclear and cytoplasmic compartments were determined using Columbus software (PerkinElmer) and the intensity of mScarlet-fascin in each comparment used to determine the % of nuclear-localized fascin per cell. The mean % nuclear fascin/cell/well was then calculated and values normalised relative to neutral controls (median set to 0) and positive controls (median set to 100). Nuclear fascin was plotted as a function of compound concentration to identify AC50 values for each compound.
# Phenotypes
Phenotype Name Nuclear fascin localization Nuclear F-actin
Phenotype Description The mean % nuclear fascin/cell/well was calculated and values normalised relative to neutral controls (median set to 0) and positive controls (median set to 100) Mean nuclear F-actin organisation
Phenotype Score Type Automatic Automatic
Phenotype Term Source REF GO CMPO
Phenotype Term Name regulation of protein localization to nucleus actin filament phenotype
Phenotype Term Accession GO_1900180 CMPO_0000104
# Raw Data Files
Raw Image Data Format TIFF
Raw Image Organization 9x 384 well plates, 5 fields per well, 4 channels per field
# Feature Level Data Files
Feature Level Data File Name
Feature Level Data File Description
Feature Level Data File Format
Feature Level Data Column Name
Feature Level Data Column Description
# Processed Data Files
Processed Data File Name idr0139-screenB-annotation
Processed Data File Format tab-delimited text
Processed Data File Description Compound response - nuclear fascin and F-actin
Processed Data Column Name Plate Well Reagent Identifier Gene Identifier Gene Symbol Score - Nuclear fascin Score - Nuclear F-actin In Validation Screen Has Phenotype Phenoptye Annotation Level Nuclear fascin Nuclear F-actin
Processed Data Column Type Plate Well Reagent Identifier Gene Identifier Gene Symbol Data Data Phenotype Phenotype Phenotype Phenotype Phenotype
Processed Data Column Annotation Level well well compound response across 12 wells compound response across 12 wells compound response across 12 wells compound response across 12 wells compound response across 12 wells
Processed Data Column Description Nuclear fascin - normalised mean nuclear fascin/cell/well Nuclear F-actin - normalised mean nuclear F-actin/cell/well Selected for apoptosis screen Valid compound response for nuclear fascin and/or F-actin Phenotype annotation level Active for nuclear fascin Active for nuclear F-actin
Processed Data Column Link To Library File Plate_Well combination
Screen Number 3
Comment[IDR Screen Name] idr0139-lawson-fascin/screenC
Screen Data DOI https://doi.org/10.17867/10000190c
Screen Sample Type cell
Screen Description To further refine the identified compounds and targets that enhanced nuclear fascin and F-actin in terms of functional importance, we performed an additional screen to detect apoptosis using a representative panel of 52 compounds that inhibited various pathways but all targeted fascin to the nucleus. Fascin KD HeLa cells expressing mScarlet or mScarlet-fascin were used for this analysis to identify fascin-dependent responses to compounds. Cells were treated with all compounds at a range of concentrations and imaged using automated high-content microscopy in the presence of a fluorescence-based caspase reporter (that fluoresced at 488nm only upon onset of apoptosis) over 72 h. Resulting data were quantified to produce apoptosis AC50 values for all compounds in both control and fascin KD cells. From these data, we identified 15 compounds that promoted apoptosis at lower concentrations in mScarlet-fascin expressing cells than in fascin KD cells.
Screen Size Plates: 384 5D Images: 27,264 Planes: 81,792 Average Image Dimension (XYZCT): Phase contrast images: 1392 x 1040 x 1 x 1 x 18; Fluorescent images: 1392 x 1040 x 1 x 2 x 18 Total Tb:
Screen Example Images
Screen Imaging Method time lapse microscopy
Screen Imaging Method Term Source REF Fbbi
Screen Imaging Method Term Accession FBbi_00000249
Screen Technology Type compound screen
Screen Technology Type Term Source REF EFO
Screen Technology Type Term Accession EFO_0007553
Screen Type secondary screen
Screen Type Term Source REF EFO
Screen Type Term Accession EFO_0007557
Screen Comments Time labels (image sequence no./time): 1) 4h, 2) 8h, 3) 12h, 4) 16h, 5) 20h, 6) 24h, 7) 28h, 8) 32h, 9) 36h, 10) 40h, 11) 44h, 12) 48h, 13) 52h, 14) 56h, 15) 60h, 16) 64h, 17) 68h, 18) 72h. Image sequences from plate 1093670859 are missing 7) 28h timepoint due to software error during acquisition.
# Library section. The library file should be supplied separately and it should contain the reagents description including, at the absolute minimum: reagent ID, sequences and position in the layout (= plate + position in the plate)
Library File Name idr0139-screenC-annotation
Library File Format tab-delimited text
Library Type compound library
Library Type Term Source REF EFO
Library Type Term Accession EFO_0007569
Library Manufacturer AstraZeneca
Library Version
Library Experimental Conditions Plates 1093670866 and 1093670873 - Fascin KD HeLa cells expressing mScarlet-fascin (WT); Plates 1093670842 and 1093670859 - Fascin KD HeLa cells expressing mScarlet (KD)
Library Experimental Conditions Term Source REF EFO
Library Experimental Conditions Term Accession
Quality Control Description Positive/negative controls
# Protocols
Protocol Name growth protocol treatment protocol HCS library protocol HCS image acquisition and feature extraction protocol HCS data analysis protocol
Protocol Type growth protocol treatment protocol HCS library protocol HCS image acquisition and feature extraction protocol HCS data analysis protocol
Protocol Type Term Source REF EFO EFO EFO EFO EFO
Protocol Type Term Accession EFO_0003789 EFO_0003969 EFO_0007571 EFO_0007572 EFO_0007573
Protocol Description Fascin was stably knocked down in HeLa cells, then mScarlet (KD) or mScarlet-fascin (WT) was stably expressed. Cells were cultured at 37¡C, 5% CO2 in high-glucose DMEM supplemented with 10% FBS, 1% glutamine and 1% penicillin/streptomycin. Cells were selected and maintained using puromycin, and sub-cultured using 0.05% trypsin-EDTA in PBS. In this assay, cells were plated in CellCarrier Ultra 384 well plates (PerkinElmer) at a concentration of 1,000 cells/well plus 2 mM CellEvent Caspase 3/7 Green detection reagent (ThermoFisher). 24 h after plating, 1 µM staurosporine (positive control), vehicle control or 0.00003-30 µM compounds were added using an Echo liquid handler (Labcyte/Beckman Coulter). Cells were imaged live every 4 h for a further 72 h using an Incucyte Zoom (Sartorius). Biologically annotated compounds selected following compound response screen. Cells were incubated in an IncuCyte Zoom (Sartorius) at 37¡C and imaged at 10x every 4 h for 72 h (first image taken 4 h after adding drugs). Channels = Phase contrast (cells), red (mScarlet) and green (CellEvent apoptosis marker). Data were analysed using IncuCyte Zoom (Sartorius) and Genedata Screener (Genedata) software. Images were acquired every 4 h and apoptosis levels calculated for each condition as the area under the curve over the total period of the experiment.
# Phenotypes
Phenotype Name Apoptosis
Phenotype Description Apoptosis levels calculated for each condition as the area under the curve over the total period of the experiment relative to the total number of cells.
Phenotype Score Type Automatic
Phenotype Term Source REF CMPO
Phenotype Term Name cell apoptosis phenotype
Phenotype Term Accession CMPO_0000220
# Raw Data Files
Raw Image Data Format TIFF
Raw Image Organization Phase contrast images: 4x 384 well plates, 1 field per well, 1 channel per field, 18 timepoints; Fluorescent images: 4x 384 well plates, 1 field per well, 2 channels per field, 18 timepoints.
# Feature Level Data Files
Feature Level Data File Name
Feature Level Data File Description
Feature Level Data File Format
Feature Level Data Column Name
Feature Level Data Column Description
# Processed Data Files
Processed Data File Name idr0139-screenC-annotation
Processed Data File Format tab-delimited text
Processed Data File Description Apoptosis data
Processed Data Column Name Plate Well Reagent Identifier Gene Identifier Gene Symbol Score
Processed Data Column Type Plate Well Reagent Identifier Gene Identifier Gene Symbol Data
Processed Data Column Annotation Level well
Processed Data Column Description Apoptosis levels expressed as ratio of total cell confluence
Processed Data Column Link To Library File Plate_Well combination