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BioAssay: AID 624088

PathHunter bCatenin (U2-OS) Measured in Cell-Based System Using Plate Reader - 2133-06_Inhibitor_Dose_DryPowder_Activity

B-catenin is a downstream target of GSK3b. Inhibition of GSK3b will prevent phosphorylation of b-catenin, increase its stability, and promote its nuclear translocation to activate downstream genes. The increase of b-catenin nuclear localization reflects GSK3b inhibition, and is an indicator of GSK3b activity or that of the GSK3b inhibitors. ..more
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 Tested Compounds
 Tested Compounds
All(41)
 
 
Active(20)
 
 
Inactive(21)
 
 
 Tested Substances
 Tested Substances
All(43)
 
 
Active(22)
 
 
Inactive(21)
 
 
 Related BioAssays
 Related BioAssays
AID: 624088
Data Source: Broad Institute (2133-06_Inhibitor_Dose_DryPowder_Activity)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Probe Production Network, Assay Provider
Deposit Date: 2012-04-12
Hold-until Date: 2013-04-06
Modify Date: 2013-04-10

Data Table ( Complete ):           Active    All
BioActive Compounds: 20
Depositor Specified Assays
AIDNameTypeComment
2119Summary of Probe Development Efforts to Identify Inhibitors of Glycogen Synthase Kinase-3 beta (GSK-3b)summarysummary assay
Description:
Keywords:
U2-OS cells, a-Catenin, a-Galactosidase

Assay Overview:
B-catenin is a downstream target of GSK3b. Inhibition of GSK3b will prevent phosphorylation of b-catenin, increase its stability, and promote its nuclear translocation to activate downstream genes. The increase of b-catenin nuclear localization reflects GSK3b inhibition, and is an indicator of GSK3b activity or that of the GSK3b inhibitors.


Expected Outcome:
Activation of a-Galactosidase reporter
Protocol
PathHunter B-Catenin Functional Assay: An In Vitro Endpoint Assay to Quantify B-Catenin Translocation
Outline:
A standard protocol using U2-OS cells stably expressing two complimentary a-Galactosidase fragments: one on a-Catenin and the other constitutively expressed in the cell nucleus. When a-Catenin translocates to the nucleus, the complimentary fragments form a complete a-Galactosidase which is then quantified by luciferase assay.
Reagents:


1. PathHunter Detection Kit from PathHunter (Cat #93-0001)
a. Galacton Star Substrate
b. Emerald II Solution
c. PathHunter Cell Assay Buffer
d. Cell plating reagent CP22
Plate Type:
Corning 384 well white, TC treated culture plates (Cat #3707)
Protocol:
1. Seed 10,000 cells in 20 il cell plating reagent CP22
2. Incubate cells overnight @ 37 degrees C
3. Induce cells with appropriate doses of GSK3 inhibitors
4. Incubate for 6 hrs @ 37 degrees C
5. Prepare PathHunter Detection Reagent by combining 1 part Galacton Star Substrate with 5 parts Emerald II Solution and 19 Parts PathHunter Cell Assay Buffer as described in product insert
a. Calculations are made by diving total volume desired for the assay by 25, then multiplying the resulting number (vol. for 1 part) by the appropriate number of parts
b. Reagents are stable for 24 hrs at room temperature
c. PathHunter recommends preparing 30% more reagent to account for loading waste
d. Example for a single 96-well plate (7.5 ml/25 = 0.3 ml per part):
i. Cell Assay Buffer- 5.7 ml (19 Parts)
ii. Emerald II Solution (Substrate Reagent 1)- 1.5 ml (5 Parts)
iii. Galacton Star Substrate (Reagent 2)- 0.3 ml (1 Part)
e. Example for a single 384-well plate (6 ml/25 = 0.24 ml per part):
i. Cell Assay Buffer- 4.56 ml (19 Parts)
ii. Emerald II Solution (Substrate Reagent 1)- 1.2 ml (5 Parts)
iii. Galacton Star Substrate (Reagent 2)- 0.24 ml (1 Part)
6. Add 12 il PathHunter Detection Reagent per well
7. Incubate for 1 hr @ room temperature
8. Read luciferase signal on the Perkin Elmer Envision:
a. Distance between plate & detector (mm) = 0.2
b. Measurement time(s) = 0.1
c. Glow (CT2) correction factor = 0
Comment
In this assay GSK3beta inhibition leads to increased beta-galactosidase (beta-gal) signal. A normalized beta-gal value was calculated using the "Neutral Controls Minus Inhibitors" method of Genedata Assay Analyzer (v7.0.3) utilizing neutral control (DMSO) wells and inhibitor positive control wells were present on each plate. IC50 values were calculated using the Smart Fit strategy of Genedata Screener Condoseo (7.0.3).


PubChem Activity Score and Outcome

PUBCHEM_ACTIVITY_SCORE:

Inactive compounds = 0
Active compounds = -10*Log(IC50)

PUBCHEM_ACTIVITY_OUTCOME:

Activity_Outcome = 1 (inactive)
IC50 > 1 log over the highest tested concentration

Activity_Outcome = 2 (active)
EC50 <= 1 log over the highest tested concentration
Result Definitions
Show more
TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1AC50_uM *The concentration at which activity reaches 50% of the maximumFloatμM
2Activity_at_23.5nM_(%) The average measured activity of all accepted replicates at the specified concentrationFloat%
3Activity_at_0.05uM_(%) The average measured activity of all accepted replicates at the specified concentrationFloat%
4Activity_at_0.1uM_(%) The average measured activity of all accepted replicates at the specified concentrationFloat%
5Activity_at_0.195uM_(%) The average measured activity of all accepted replicates at the specified concentrationFloat%
6Activity_at_0.38uM_(%) The average measured activity of all accepted replicates at the specified concentrationFloat%
7Activity_at_0.8uM_(%) The average measured activity of all accepted replicates at the specified concentrationFloat%
8Activity_at_1.6uM_(%) The average measured activity of all accepted replicates at the specified concentrationFloat%
9Activity_at_3uM_(%) The average measured activity of all accepted replicates at the specified concentrationFloat%
10Activity_at_6uM_(%) The average measured activity of all accepted replicates at the specified concentrationFloat%
11Activity_at_12uM_(%) The average measured activity of all accepted replicates at the specified concentrationFloat%
12Activity_at_26uM_(%) The average measured activity of all accepted replicates at the specified concentrationFloat%
13Activity_at_0.05mM_(%) The average measured activity of all accepted replicates at the specified concentrationFloat%

* Activity Concentration.
Additional Information
Grant Number: 1 RO3 MHD87442-01

Data Table (Concise)
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