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BioAssay: AID 1665

High Throughput Imaging Assay for Beta-Catenin

Cancer is one of the most tragic afflictions in modern society, and often results from alterations in the mitotic process. In normal cells, beta-catenin is found predominantly associated with the plasma membrane. However, in tumor cells, beta -catenin redistributes to the nucleus where it interacts with transcription factors to stimulate the expression of proteins that promote mitosis. The more ..
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 Tested Compounds
 Tested Compounds
All(193544)
 
 
Active(587)
 
 
Inactive(192905)
 
 
Inconclusive(52)
 
 
 Tested Substances
 Tested Substances
All(193658)
 
 
Active(587)
 
 
Inactive(193019)
 
 
Inconclusive(52)
 
 
AID: 1665
Data Source: Burnham Center for Chemical Genomics (BCCG-A186-Beta-Catenin-Imaging-Assay)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Probe Production Network
BioAssay Version:
Deposit Date: 2009-04-07
Modify Date: 2010-12-30

Data Table ( Complete ):           View Active Data    View All Data
Target
BioActive Compounds: 587
Related Experiments
AIDNameTypeComment
1685Summary Assay for Beta-CateninSummarydepositor-specified cross reference
Description:
Data Source: Sanford-Burnham Center for Chemical Genomics (SBCCG)
Source Affiliation: Sanford-Burnham Medical Research Institute (SBMRI, San Diego, CA)
Network: NIH Molecular Libraries Screening Centers Network (MLSCN)
Grant Proposal Number: 1 R03 MH082378-01
Assay Provider: Dr. Patrick M. McDonough, Vala Sciences Inc.

Cancer is one of the most tragic afflictions in modern society, and often results from alterations in the mitotic process. In normal cells, beta-catenin is found predominantly associated with the plasma membrane. However, in tumor cells, beta -catenin redistributes to the nucleus where it interacts with transcription factors to stimulate the expression of proteins that promote mitosis. The pathway is also important in CNS-related disorders ranging including manic depression, and Alzheimer's. The assay involves exposing HeLa cells to test compounds that are likely to alter beta-catenin distribution; the cells are then immunostained for endogenous beta-catenin and imaged using high content robotic microscopy workstations. Specialized image analysis software is used to quantify cellular beta-catenin localization. In preliminary experiments, an inhibitor of glycogen synthase kinase 3-a (GSK-3), an enzyme typically regulated by the Wnt-signal transduction pathway, induced nuclear localization of beta-catenin.

The goal of this screen is to identify chemicals that are likely to regulate GSK-3 or other proteins that control beta-catenin localization. Discovery of such chemicals will contribute to our understanding of the regulation of beta-catenin localization and potentially help lead to development of novel therapeutics, depression, and dementia.

References:
Hayashida, Y., Honda, K., Idogawa, M., Ino, Y., Ono, m., Tsuchida, A., Aoki, T., Hirohashi, S., Yamada, T. 2005. E-cadherin regulates the association between beta-catenin and actinin-4. Cancer Res. 65:8836-8845.

Lee, J., M., Dedhar, S., Kalluri, R., Thompson, E. W. 2005. The epithelial-mesenchymal transition: new insights in signaling, development, and disease. J. Cell. Biol. 172:973-981.

Polakis, P. 2007. The many ways of Wnt in cancer. Current Opionion in Genetics & Development 17:45-51.
Protocol
Assay Materials:
1) 384-well plates, black with clear bottom (Greiner# 781091)
2) Hela cells (ATCC, CCL-2, Human cervical adenocarcinoma)
3) Culture Media: Phenol-red free DMEM with L-glutamine, Pen-strep, and 10% Fetal Bovine Serum.
4) Positive Control Working Solution: GSK-3 inhibitor IX (EMD, 361552, 10mM stock in DMSO) diluted to 100uM in water. Additional DMSO is added to achieve a DMSO concentration of 2%.
5) Negative Control Working Solution: 2% DMSO in water.
6) Fixative Working Solution: 6% Paraformaldehyde (PFA) in PBS.
7) Permeabilization Buffer Working Solution: 0.1% Triton-X (Fisher Scientific, ICN19397190) in PBS.
8) Blocking Buffer Working Solution: 5% Goat Serum (Fisher Scientific, ICN642921), 1% BSA (Sigma, A7888), 0.5% Tween-20 (Sigma, P9416) in PBS.
9) Primary Antibody Working Solution: Rabbit anti-Beta-Catenin Polyclonal antibody (Vala Sciences) diluted 1:200 in blocking buffer.
10) Secondary Antibody Working Solution: AlexaFluor 568 Goat Anti-Rabbit IgG secondary antibody (Invitrogen, A11036) diluted 1:100 in blocking buffer.
11) Nuclear Stain Working Solution: DAPI (Invitrogen, D1306) diluted to 150ng/ml in DAPI buffer (10mM TRIS, 10mM EDTA, 100mM NaCl, pH 7.4).

Primary Screen Protocol:
1) 76ul of cell suspension (79,000 cells/ml in culture medium) was dispensed in each well of the assay plates using a Wellmate bulk dispenser.
2) Incubate plates overnight or approx. 20 hours at 37oC and 5% CO2.
3) 4ul of 100uM compound solution was added to columns 3 through 24 of the assay plates for a final assay compound concentration of 5uM and 0.1% DMSO. Compound addition was done on a Biomek FX with 384-head dispenser (Beckman).
4) 4ul of negative control (2% DMSO) working solution was added to column 2 using the Biomek FX with 384-head dispenser for a final assay concentration of 0.1% DMSO.
5) 4ul of the positive control (100uM GSK-3 inhibitor IX) working solution was added to column 1 of each plate manually for a final assay concentration of 5uM GSK-3 inhibitor IX and 0.1% DMSO.
6) Plates were incubated for 24 hours at 37oC and 5% CO2.
7) Media was aspirated leaving 20ul liquid in each well using a Titertek plate washer.
8) 40ul of fixative working solution was added to each well using a Wellmate bulk dispenser (Matrix) for a final concentration of 4% PFA and plates were incubated for 40 minutes at room temperature.
9) Fixative was aspirated and plates were washed twice with 80ul PBS leaving 20ul liquid in each well using a Titertek plate washer.
10) 40ul of permeabilization buffer working solution was added to each well using the Wellmate bulk dispenser and plates were incubated for 40 minutes at room temperature.
11) Permeabilization buffer was aspirated and plates were washed twice with 50ul PBS leaving 20ul liquid in each well using a Titertek plate washer.
12) 5ul of primary antibody working solution was added to each well using the Wellmate bulk dispenser (Matrix) and plates were incubated for 1 hour at room temperature.
13) Primary antibody was aspirated and plates were washed twice with 50ul PBS leaving 20ul liquid in each well using a Titertek plate washer.
14) 5ul of secondary antibody working solution was added to each well using the Wellmate bulk dispenser (Matrix) and plates were incubated for 1 hour at room temperature.
15) Secondary antibody was aspirated and plates were washed three times with 80ul PBS leaving 20ul liquid in each well using a Titertek plate washer.
16) 40ul of DAPI working solution was added using a Wellmate bulk dispenser for a final DAPI concentration of 100ng/ml and plates were sealed.

Hit Confirmation Protocol:
1) 45ul of cell suspension (133,000 cells/ml in culture medium) was dispensed in each well of the assay plates using a Wellmate bulk dispenser.
2) Incubate plates overnight or approx. 20 hours at 37oC and 5% CO2.
3) Test compounds were prepared from 10mM DMSO stock as follows:
Compounds were diluted in PBS to 100uM for plate set1 on day 1 and 200uM for the repeat plate set on day 2. This initial dilution was then serially diluted (1:1) with a 1% DMSO solution in PBS in 384-well compound plates.
4) Control compounds were diluted as follows:
DMSO working solution was 1% DMSO in PBS.
GSK3 working solution was 50uM in PBS plus additional DMSO to achieve a 1% DMSO concentration.
5) Control compounds were added to columns 1 and 24 (negative controls) and 2 and 23 (positive controls) on the 384-well compound plate.
6) 5ul of the compounds was then added to the assay plate using the Biomek FX. Final test compound concentrations were 0.02 to 10uM for day 1 and 0.04 to 20uM for day2. Control compound GSK3 was 5uM. DMSO was 0.1%.
7) Primary screen protocols 6 thru 16 were then followed.

HCS System Settings and Image Analysis Protocol:
1) Image acquisition was performed on an IC100 system (Beckman Coulter) with 45 plate capacity BRT loader/stacker and the following settings:
- 20x 0.5 NA Plan Fluor air objective
- Acquisition camera set to 2-by-2 binning for an image size of 672 by 512 pixels
- 2 channels were acquired: Channel 0 = DAPI (nuclei) and Channel 1 = Alexa 568 (Beta-Catenin).
- 6 (2-by-3) fields per well.

2) Image analysis was performed using the CyteSeer software (Vala Sciences) with the "Protein Expression" algorithm where "Nucleus" represents DAPI-stained nuclei and "Protein" represents Beta-Catenin antibody stain.

CyteSeer software settings:
Channel: "Nucleus"
Nucleus Enabled: TRUE
Nucleus Folder Name: channel_0
Nucleus Thresholding Method: Savitsky-Golay
Nucleus Sensitivity: 100.00%
Nucleus Filtering Method: None
Nucleus Filtering Radius: 1
Nucleus Minimum Size: 7

Channel: "Protein"
Protein Enabled: TRUE
Protein Folder Name: channel_1
Protein Thresholding Method: Mode Plus Width Threshold
Protein Sensitivity: 100.00%
Protein Filtering Method: None
Protein Filtering Radius: 1
Protein Minimum Size: 1

Metrics from NUCLEI IMAGES: cell count ("CellCount"), nuclear area ("Area Nm"), total integrated intensity of the nucleus ("TII Ni Nm").
Metrics from CELL (BETA-CATENIN) IMAGES: median pixel intensity of protein image under nuclear mask ("MPI Pi Nm"), median pixel intensity of Protein image under cytoplasm mask ("MPI Pi Cm"). The ratio ("Pi Nm by Pi Cm") representing the ratio of median beta-catenin intensity under the nuclear mask to median beta-catenin intensity under the cytoplasmic mask was then calculated in Matlab by dividing each cell's median pixel intensity of protein (beta-catenin) image under nuclear mask ("MPI Pi Nm") by the median pixel intensity of protein (beta-catenin) image under cytoplasm mask ("MPI Pi Cm").

3) Actives from the primary screen were determined using CBIS software (ChemInnovations) by calculating the % activation of the "Pi Nm by Pi Cm" metric and using a hit criteria of Activity > 50%. Wells with cell counts <50 in the 6 acquired images were flagged as cytotoxic/low cell count. All flagged wells were excluded from hit selection and were assigned an outcome of "inconclusive".

The primary screen actives were then tested for dose response hit confirmation. Compounds with an IC50<10uM were considered "confirmed actives". IC50 values were calculated using CBIS software (ChemInnovations) employing a sigmoidal dose-response equation through non-linear regression.
Comment
Compounds with a %Inhibition > 50% at 5 uM concentration and greater than 50 cellcount are defined as actives in this assay. Wells with cell counts <50 in the 6 acquired images were flagged as cytotoxic/low cell count. All flagged wells were excluded from hit selection and were assigned an outcome of "inconclusive".

The primary screen actives were then tested for dose response hit confirmation betwen 0-10uM and 0-20 uM. Compounds with an average IC50<10uM were considered "confirmed actives".

1) First tier (0-40 range) is reserved for primary screening data. The score is correlated with % displacement in the assay demonstrated by a compound at 5 uM concentration:
a. If primary % inhibition is less than 0%, then the assigned score is 0
b. If primary % inhibition is greater than 100%, then the assigned score is 40
c. If primary % inhibition is between 0% and 100%, then the calculated score is (% Inhibition)*0.4
2) Second tier (41-80 range) is reserved for dose-response confirmation data
a. Inactive compounds of the confirmatory stage are assigned a score value equal 41.
b. The score is linearly correlated with compound potency and, in addition, provides a measure of the likelihood that the compound is not an artifact based on the available information.
Score = 44 + 6*(pIC50 - 3)
where pIC50 is a negative log(10) of the IC50 value expressed in mole/L concentration units. This equation results in score values above 50 for compounds that demonstrate high potency and predictable behavior. Compounds that are inactive in the assay or whose concentration-dependent behavior is likely to be an artifact of that assay will generally have lower score values.

3) Third tier (81-100 range) is reserved for resynthesized true positives and their analogues and is not applicable to this assay.
Categorized Comment - additional comments and annotations
From ChEMBL:
Assay Format: Cell-based
Assay Type: Functional
Assay Cell Type: HeLa
Result Definitions
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TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1IC50_Mean_QualifierThis qualifier is to be used with the next TID, IC50_Mean. If the qualifier is "=", the IC50 result equals the value in that column; If the qualifier is ">", the IC50 result is greater than that value. If the qualifier is "<", the IC50 result is less than that value.String
2IC50_Mean*The mean od the IC50 values determined using sigmoidal dose response equation. FloatμM
3IC50_Qualifier_1This qualifier is to be used with the next TID, IC50_1. If the qualifier is "=", the IC50 result equals the value in that column; If the qualifier is ">", the IC50 result is greater than that value. If the qualifier is "<", the IC50 result is less than that value.String
4IC50_1IC50 value determined using sigmoidal dose response equation. Tested range 0-10 uMFloatμM
5Std.Err(IC50)_1Standard Error of IC50 valueFloatμM
6nH_1Hill coefficient determined using sigmoidal dose response equationFloat
7IC50_Qualifier_2This qualifier is to be used with the next TID, IC50_2. If the qualifier is "=", the IC50 result equals the value in that column; If the qualifier is ">", the IC50 result is greater than that value. If the qualifier is "<", the IC50 result is less than that value.String
8IC50_2IC50 value determined using sigmoidal dose response equation. Tested range 0-20 uMFloatμM
9Std.Err(IC50)_2Standard Error of IC50_2 valueFloatμM
10nH_2Hill coefficient determined using sigmoidal dose response equationFloat
11%Inhibition at 5 uM (5μM**)%Inhibition in primary screeningFloat
12CellCount (5μM**)Number of cellsFloatcells
13AreaNm (5μM**)Nucleus Area in wellFloatum^2
14TIINiNM (5μM**)Total integrated intensity of the nucleus FloatRFU
15MPIPiNm (5μM**)Median pixel intensity of protein image under nuclear mask FloatRFU
16MPIPiCm (5μM**)Median pixel intensity of Protein image under cytoplasm mask FloatRFU
17PiNmbyPiCm (5μM**)The ratio of median beta-catenin intensity under the nuclear mask to median beta-catenin intensity under the cytoplasmic mask Float

* Activity Concentration. ** Test Concentration.
Additional Information
Grant Number: 1 R03 MH082378-01

Data Table (Concise)
Data Table ( Complete ):     View Active Data    View All Data
Classification
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