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idr0019-study.txt
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idr0019-study.txt
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"# Section with generic information about the study including title, description, publication details (if applicable) and contact details"
# Study
Comment[IDR Study Accession] idr0019
Study Title Protein localisation and morphological comparison of normal and cancer cell lines in various media, treated with TNF-alpha or cytoskeleton-modifying small molecules
Study Type high content analysis of cells
Study Type Term Source REF EFO
Study Type Term Accession EFO_0005397
Study Description The aim of the study was to investigate relationships between NF-kappaB nuclear localisation and cell morphology in breast cancer cells.
Study Organism Homo sapiens
Study Organism Term Source REF NCBITaxon
Study Organism Term Accession NCBITaxon_9606
Study Screens Number 1
Study External URL
Study Public Release Date 2017-01-18
Study Version History July 2017: screenA - updated some cell line names to be inline with the names used in EFO. MCF7 -> MCF-7, MCF10A -> MCF 10A, JIMT1 -> JIMT-1, MDA-MB-157 -> MDAMB157, MDA-MB-231 -> MDAMB231, MDA-MB-453 -> MDAMB453, SUM149 -> SUM149PT, SUM159 -> SUM159PT, ZR75.1 -> ZR751.
# Study Publication
Study PubMed ID 26148352
Study Publication Title Cell shape and the microenvironment regulate nuclear translocation of NF-κB in breast epithelial and tumor cells.
Study Author List Sero JE, Sailem HZ, Ardy RC, Almuttaqi H, Zhang T, Bakal C
Study PMC ID PMC4380925
Study DOI http://dx.doi.org/10.15252/msb.20145644
# Study License and Data DOI
Study License CC BY 4.0
Study License URL https://creativecommons.org/licenses/by/4.0/
Study Copyright Sero et al
# Study Contacts
Study Person Last Name Sero
Study Person First Name Julia
Study Person Email Julia.Sero@icr.ac.uk
Study Person Address Institute of Cancer Research, 237 Fulham Rd, London SW3 6JB
Study Person Roles submitter
Term Source Name NCBITaxon EFO Fbbi
Term Source URI http://purl.obolibrary.org/obo/ http://www.ebi.ac.uk/efo/ http://purl.obolibrary.org/obo/
"# Section containing all information relative to each screen in the study including materials used, protocols names and description, phenotype names and description. For multiple assays this section should be repeated."
# Screen; this section should be repeated if a study contains multiple screens
Screen Number 1
Comment[IDR Screen Name] idr0019-sero-nfkappab/screenA
Screen Sample Type cell
Screen Description High Content Analysis was used to quantitatively profile the morphology and NF-kappaB nuclear localization (which serves as a proxy for its activation), in 17 breast cancer and two non_tumor cell lines. Cells were treated with or without TNFa for 1 h or 5 h to capture the first peak and later steady state of NF-kappaB activation.
Screen Size Plates: 7 5D Images: 25,872 Planes: 77,616 Average Image Dimension (XYZCT): 668 x 501 x 1 x 3 x 1 Total Tb: 0.03
Screen Example Image https://idr.openmicroscopy.org/webclient/?show=well-1024987 https://idr.openmicroscopy.org/webclient/img_detail/1859680/ 22_lines_TNF_1h_200145914;O2
Screen Imaging Method spinning disk confocal microscopy
Screen Imaging Method Term Source REF Fbbi
Screen Imaging Method Term Accession FBbi_00000253
Screen Technology Type
Screen Technology Term Source REF
Screen Technology Term Accession
Screen Type
Screen Type Term Source REF
Screen Type Term Accession
ScreenComments
"# 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 idr0019-screenA-library.txt
Library File Format tab-delimited text
Library Type none
Library Type Term Source REF
Library Type Term Accession
Library Manufacturer
Library Version
Library Experimental Conditions cell line media compound dose time
Library Experimental Conditions Term Source REF EFO EFO CHEBI EFO EFO
Library Experimental Conditions Term Accession EFO_0000322 EFO_0000579 CHEBI_37577 EFO_0000428 EFO_0000721
Quality Control Description
# Protocols
Protocol Name growth protocol treatment protocol HCA image acquistion and feature extraction protocol HCA data analysis protocol
Protocol Type growth protocol treatment protocol HCA image acquistion and feature extraction protocol HCA data analysis protocol
Protocol Type Term Source REF EFO EFO EFO EFO
Protocol Type Term Accession EFO_0003789 EFO_0003969 EFO_0007572 EFO_0007573
Protocol Description All cell lines were plated by columns in replicate 384-well plates using a Multidrop liquid handler in DMEM:F12 medium supplemented with 5% heat-inactivated fetal bovine serum (HI-FBS). Approximately 1000 cells per well were seeded in 20 _l of medium and cultured for 72 h. Supplements and treatments were added to each plate, or to top/bottom halves of plates, at the final concentrations and times (h before fixation). Complete MCF10A medium was supplemented with 20 ng/ml EGF, 10 ug/ml insulin, 0.5 mg/ml hydrocortisone, and 100 ng/ml cholera toxin. MCF10A breast epithelial and AU565 breast tumour cells were obtained from ATCC (LCG Standards). MDAMB231, HCC70, HCC1143, HCC1954, MCF7, T47D, BT474, CAMA1, MDAMB453, and hs578T breast tumour and MCF10A non-tumour cells were obtained from the laboratory of Alan Ashworth (Breakthrough Breast Cancer, ICR). SUM149, SUM159, MDAMB157, JIMT1, SKBR3, and ZR75.1 breast tumour cells were given by the laboratory of Jorge Reis-Filho (Breakthrough Breast Cancer, ICR). Non-breast tumour cell lines HeLa (cervical carcinoma) and Hep3B (hepatocarcinoma), and primary human fibroblasts (AG15730) were included for comparison. Human recombinant TNF_ (Life Technologies) was added to a final concentration of 10 ng/ml. Y_27632 (Sigma) was used at 10 uM unless otherwise specified, and nocodazole was used at 0.1 ug/ml unless otherwise specified. Blebbistatin, taxol, and DMSO were obtained from Sigma. H1152 was from Tocris Bioscience. Plasmid transfection with GFPup65/RelA (Addgene; plasmid ID 23255; (Chen et al, 2001)) was performed using Lipofectamine 2000 (Invitrogen), and GFP_positive cells were harvested by FACS 24 h before imaging One hour prior to fixation, a final concentration of 10 uM dihydroethidium (2-hydroethidine, DHE; Invitrogen) was added to all wells to label cell bodies. Cells were fixed with 4% paraformaldehyde for 10 min, washed with PBS, and permeabilised with 0.1% Triton X-100. NF-_B was labelled using anti-p65 rabbit polyclonal antibody (Abcam ab16502; 1:500), followed by Alexa-488 anti-rabbit secondary antibody (Invitrogen, 1:1000). DAPI (1:1000) was used to label nuclei. Image acquisition was performed using an Opera Cell::Explorer_automated spinning disk confocal microscope. The initial screen was performed using a 20x air objective lens (NA = 0.45) (PerkinElmer), and 12-30 fields of view were imaged in each well. Subsequent experiments were imaged using a 20_ water objective lens (NA = 0.6). Cell segmentation was performed using Acapella software (PerkinElmer). Segmentation and feature measurements were performed using Columbus (PerkinElmer) automated analysis software. Nuclei were segmented using DAPI (Channel 1) and cell bodies were detected by DHE (Channel 3). Subcellular regions include: "nucleus", "ring region" (-2 to -7 pixels from nucleus border), "nucleus region" (1 pixel erosion of nucleus), "cytoplasm", and "membrane region" (0 to 5 pixels inward from cell border). The following morphological features were calculated: area, roundness (form factor), length, width, and length/width were determined for nucleus and cell regions, the ratio of nuclear area:cell area ("Nuc/Cell Area") and areas of ring and membrane regions. The extent of cell-cell contact, i.e. "Neighbor Fraction", was calculated as follows: 1 - ((Outer Membrane Region area * 2) / (Inner Membrane Region area * 2)), where the "Outer Membrane Region" is a 1-pixel dilation from the cell edge and the "Inner Membrane Region" is a 1-pixel erosion from the cell edge. Intensities were measured for each channel in each region and regional mean values are reported. The amount of NF-kappaB in the nucleus of each cell was calculated as the log of the ratio of nuclear:ring region intensities (Channel 2). Two measures of edge ruffling ("Ruffliness-Edge" and "Ruffliness-Ridge") were calculated from the "membrane region" texture features SER-Edge and SER-Ridge (scale = 4 px, normalisation by kernel) divided by cell roundness. Nucleoli were visible in the DHE channel and the Spot finder module was used to measured the number, area, and intensity of each nucleolus ("spot"). Texture features were calculated for nuclear DNA using the DAPI channel: Haralick distance = 1 px, "nucleus"; SER (Spot, Hole, Edge, Ridge, Valley, Saddle, Bright, Dark) scale = 0 px, normalisation by region, "nucleus"; and SER-Dark scale = 3 px, normalisation by region, "nucleus region". Border objects were removed and dead or poorly-segmented cells were removed by applying the filter: "Nuc/Cell Area" > 0.02 AND "Ring NFkB Intensity" > 100 Mitotic cells were binned using the following criteria: "Nucleus Roundness" < 0.9 AND "DAPI Nucleus Haralick Sum Variance 1 px" > 0.04 AND "DAPI nucleus Haralick Homogeneity 1 px" < 0.32 AND "Nucleus Region Channel1 SER-Dark 3 px" < 0.001. Interphase cells were further binned using the following criteria: "Nucleus Roundness" > 0.7 AND "DAPI nucleus Haralick Sum Variance 1 px" < 0.1 AND "DAPI nucleus Haralick Homogeneity 1 px" > 0.05. Mean, SD, CV, Median, Max, and Min feature values and counts for all filtered cells, interphase cells only, and mitotic cells only are included for each plate. PCA, clustering, and statistical tests. Principal component analysis was carried out using Cluster 3.0 and MATLAB software based on Z-scores [(value-mean)/SD]. Hierarchi- cal clustering was performed with Cluster 3.0 and visualized using Java Tree View. P values were determined using Student's t-test and ANOVA (Excel and MATLAB). R and R2 values were determined using Excel or MATLAB (Pearson correlation unless otherwise specified).
# Phenotypes
Phenotype Name
Phenotype Description
Phenotype Score Type
Phenotype Term Source REF
Phenotype Term Name
Phenotype Term Accession
Phenotype Term Source REF
Phenotype Term Name
Phenotype Term Accession
# Raw Data Files
Raw Image Data Format TIFF
Raw Image Organization 7 x 384 plates, with approx 12 fields per well.
# Feature Level Data Files (give individual file details unless there is one file per well)
Feature Level Data File Name inline-supplementary-material-11.csv
Feature Level Data File Format comma-delimited text
Feature Level Data File Description This file contains the single cell data used to generate Bayesian network models for 19 cell lines with and without TNF_. It is supplementary dataset 1 from the publication.
Feature Level Data Column Name Treatment condition Cell line Plate row Plate column field of view Object number Cell area Cell_WidthToLength centers_distance Eccentricity form_factor ruffliness Protrusion_area micro_nuclei_count NucbyCytoArea NucbyCytoNFkB logNFkB Nuclear_Roundness nuclei_eccentricity NucleusArea Nuc_WidthToLength ClusterArea NF relative_masscentre_voronoi_area relative_general_voronoi_area lrgprot2_area lrgprot2_compactness lrgprot2_contactlength lrgprot2_HaralickContrast lrgprot2_HaralickCorrelation lrgprot2_HaralickHomogeneity lrgprot2_HaralickSumVariance lrgprot2_membrane_area lrgprot2_prlength lrgprot2_relative_ruffle_area lrgprot2_relative_ruffle_intensity lrgprot2_relative_ruffle_to_membrane lrgprot2_roundness lrgprot2_ruffle_area lrgprot2_servalley lrgprot_area lrgprot_compactness lrgprot_contactlength lrgprot_membrane_area lrgprot_prlength lrgprot_relative_ruffle_area lrgprot_relative_ruffle_intensity lrgprot_relative_ruffle_to_membrane lrgprot_roundness lrgprot_ruffle_area lrgprot_serhole lrgprot_SERridge lrgprot_sersaddle lrgprot_SERspot lrgprot_servalley No_of_protrusions prot_mean_area prot_mean_compactness prot_mean_contactlength prot_mean_prlength prot_mean_ruffle_area prot_relative_area prot_relative_contactlength prot_rel_contactlength_to_NF tall_prot_area tall_prot_compactness tall_prot_contactlength tall_prot_membrane_area tall_prot_prlength tall_prot_relative_ruffle_area tall_prot_relative_ruffle_intensity tall_prot_relative_ruffle_to_membrane tall_prot_roundness tall_prot_ruffle_area nuc_theta p1theta p2theta p1p2_theta p3p2_theta p1p3_theta cluster_no_cells center_cell (n/y) colony_area edge_cell (n/y)
Feature Level Data Column Description control = untreated, tnf1h = 1 h TNFa (10 ng/ml); tnf5h = TNFa 5 h (10 ng/ml) Name of breast tumor or non-tumor cell line Given in pixels, where 1 pixel = 0.64 um half width / full length of cell body Distance between centroid of nucleus and centroid of cell body Eccentricity of best-fit ellipse of cell body roundness of cell body = 3.14159*sqrt(CellArea-BorderLength/2)/BorderLength-0.1) SER Edge texture feature (measures "edge-ness" of pixel intensity distribution) in membrane region divided by form_factor total protrusion area per cell in pixels numebr of micronuclei Nucleus area/cytoplasm area Nuclear intensity/Ring region intensity in NF-kB channel log(NucbyCytoNFkB) roundness of nucleus = 3.14159*sqrt(Nucleusarea-NucleusborderLength/2)/NucleusborderLength-0.1) Eccentricity of best-fit ellipse of nucleus given in pixels helf width ; full length of nucleus Area of cluster in which cell is located (in pixels) Neighbor fraction, i.e. proportion of cell border that is in contact with other cells Voronoi area from cell mass center Voronoi area from cell border Area of second largest protrusion Compactness of pixel intensities in second largest protrusion length of contact between second largest protrusion and cell core length of second largest protrusion (normal to cell border) area of ruffles (high intensity regions) within border of second largest protrusion ruffle intensity/membrane intensity SER Valley feature (see Acapella, PerkinElmer for description of SER features) Area of largest protrusion Compactness of pixel intensities in largest protrusion length of contact between largest protrusion and cell core length of largest protrusion (normal to cell border) area of ruffles (high intensity regions) within border of largest protrusion SER Hole feature (see Acapella, PerkinElmer for description of SER features) ruffle intensity/membrane intensity number of protrusions average area of individual protrusions area of longest protrusion (long axis of protrusion normal to cell border) angle between nucleus body and cell body angle between largest protrusion and cell body angle between second largest protrusion and cell body angle between largest and second largest protrusions angle between second and third largest protrusions angle between largest and third largest protrusions number of cells in cluster is cell in center of cluster? area of colony is cell on edge of colony or cluster?
# Processed Data Files
Processed Data File Name idr0019-screenA-processed.txt
Processed Data File Format tab-delimited text
Processed Data File Description This file lists the morphological clusters the cell lines were grouped into based on hierarchicial clustering of the avearge score of the first eight Principal Components for each cell line.
Processed Data Genome Build For Target Genes
Processed Data Column Name Experimental Condition [Cell Line] Cell Line Morphotype
Processed Data Column Type experimental condition data
Processed Data Column Annotation Level Experimental Condition
Processed Data Column Description The Cell Line studied. The morphological cluster the cell line has been grouped in.
Processed Data Column Source Database
Processed Data Column Source Stem URI
Processed Data Column Link To Library File Experimental Condition [Cell Line]