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BioAssay: AID 624086

TCF/LEF Reporter Assay Measured in Cell-Based System Using Plate Reader - 2133-01_Inhibitor_Dose_DryPowder_Activity

A standard protocol for analyzing TCF/LEF promotor activity using HEK293T cells stabily expressing firefly luciferase driven by TCF/LEF promotor. ..more
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 Tested Compounds
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Active(23)
 
 
Inactive(67)
 
 
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 Tested Substances
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Active(25)
 
 
Inactive(67)
 
 
 Related BioAssays
 Related BioAssays
AID: 624086
Data Source: Broad Institute (2133-01_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 ):           View Active Data    View All Data
BioActive Compounds: 23
Related Experiments
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AIDNameTypeComment
2119Summary of Probe Development Efforts to Identify Inhibitors of Glycogen Synthase Kinase-3 beta (GSK-3b)Summarydepositor-specified cross reference: summary assay
2097Cell-Free Homogeneous Primary HTS to Identify Inhibitors of GSK3beta ActivityScreeningsame project related to Summary assay
434947Luminescence Cell-Free Homogeneous Counter Screen to Identify Inhibitors of ADP-glo ReagentsConfirmatorysame project related to Summary assay
434954Luminescence Cell-Free Homogeneous Dose Retest to Identify Inhibitors of Glycogen Synthase Kinase-3 beta ActivityConfirmatorysame project related to Summary assay
623978Inhibition of Human GSK3 alpha Activity Measured in Biochemical System Using Microfluidics - 2046-11_Inhibitor_Dose_DryPowder_Activity_Set2Confirmatorysame project related to Summary assay
623979Inhibition of Human CDK5 Activity Measured in Biochemical System Using Microfluidics - 2046-12_Inhibitor_Dose_DryPowder_Activity_Set2Confirmatorysame project related to Summary assay
623989Inhibition of Human GSK3 beta Activity Measured in Biochemical System Using Microfluidics - 2046-10_Inhibitor_Dose_DryPowder_Activity_Set2Confirmatorysame project related to Summary assay
623998GSK3beta Measured in Biochemical System Using Plate Reader - 2046-01_Inhibitor_Dose_DryPowder_ActivityConfirmatorysame project related to Summary assay
623999CDK5 Measured in Biochemical System Using Plate Reader - 2046-05_Inhibitor_Dose_DryPowder_ActivityConfirmatorysame project related to Summary assay
624027GSK3beta Assay at Varied ATP concentrations Measured in Biochemical System Using Plate Reader - 2046-04_Inhibitor_Dose_DryPowder_ActivityConfirmatorysame project related to Summary assay
624057Human p-Tau and Total Tau ELISA Measured in Cell-Based System Using Plate Reader - 2133-07_Inhibitor_Dose_DryPowder_ActivityConfirmatorysame project related to Summary assay
624076Kinome profiling single dose point Measured in Biochemical System Using Plate Reader - 2133-09_Inhibitor_SinglePoint_DryPowder_ActivityOthersame project related to Summary assay
624088PathHunter bCatenin (U2-OS) Measured in Cell-Based System Using Plate Reader - 2133-06_Inhibitor_Dose_DryPowder_ActivityConfirmatorysame project related to Summary assay
624091Dose responses of selected compounds against GSK3b Measured in Biochemical System Using Plate Reader - 2133-10_Inhibitor_Dose_DryPowder_ActivityOthersame project related to Summary assay
624108GSK3beta Surface Plasmon Resonance (SPR) Assay Measured in Biochemical System Using Microfluidics - 2133-08_Inhibitor_Dose_DryPowder_ActivityOthersame project related to Summary assay
Description:
Keywords:
HEK293T cells, TCF/LEF

Assay Overview:
A standard protocol for analyzing TCF/LEF promotor activity using HEK293T cells stabily expressing firefly luciferase driven by TCF/LEF promotor.
Inhibition of GSK3-b is known to stabilize beta catenin (b-catenin), an integral component of the canonical wnt pathway involved in morphogenic signaling and cellular growth. When the kinase activity of GSK3-b is inhibited, b-catenin is stabilized and may then translocate to the nucleus where it interacts with members of the T-cell factor/ lymphoid enhancer-binding factor (TCF/LEF) family of transcription factors, inducing transcription. The overall objective is to identify compounds which activate TCF/ LEF transcription through putative GSK3-b inhibition. This assay measures TCF/LEF
transcription activation through use of a firefly luciferase reporter. Potent GSK3-b-selective compounds were previously identified by SPR assay. Selected compounds were applied to reporter cells at various doses and TCF/LEF activation through firefly expression was quantified. Positive controls (CHIR99021 at 10 uM) and negative controls (DMSO) were included in each plate to verify positive responses versus background signal.

Expected Outcome:
activation of TCF/LEF promotor activity
Protocol
TCF/LEF Reporter Gene Assay: An In Vitro Endpoint Assay Using a Firefly Reporter Gene on the TCF/LEF Promotor

Outline:
A standard protocol for analyzing TCF/LEF gene activity in established cell lines stably expressing the TCF/LEF reporter
Reagents:
1. Dual-Glo Luciferase Assay Reagent from Promega (Cat #E2940)
a. Luciferase Buffer
b. Luciferase Substrate
c. Stop & Glo Luciferase Buffer
d. Stop & Glo Luciferase SubstrateTCF/LEF Reporter Gene Assay: An In Vitro Endpoint Assay Using a Firefly Reporter Gene on the TCF/LEF Promotor

Outline:
A standard protocol for analyzing TCF/LEF gene activity in established cell lines stably expressing the TCF/LEF reporter
Reagents:
1. Dual-Glo Luciferase Assay Reagent from Promega (Cat #E2940)
a. Luciferase Buffer
b. Luciferase Substrate
c. Stop & Glo Luciferase Buffer
d. Stop & Glo Luciferase Substrate
Plate Type:
Corning 384 well white, TC treated culture plates (Cat #3707)
Protocol:
1. Culture cells in 384 well plates to appropriate density (6000 cells / well).
a. Total plating volume should be 40 ul.
2. 24 hours after plating, pin cells with 100 nl compounds.
3. 24 hours after incubation with compounds (48 hours after plating), prepare assay reagents by mixing Luciferase Assay solution with Luciferase Subtrate and Stop & Glo solution to Stop & Glo substrate
a. Extra reagents can be aliquotted and frozen for future use and are stable for at least one freeze/thaw cycle.
4. Add a volume of luciferase reagent equal to (1/4) the volume of media to each well
a. For example, in a 384-well plate with 40 u in each well, add 10 u. This gives a final volume of 50 u.
5. Spin the plate down and allow 10 minutes for signal stabilization.
6. Read luciferase signal on the Perkin Elmer Envision
a. The following read parameters are used:
i. Distance between plate & detector (mm) = 0.2
ii. Measurement time(s) = 0.1
iii. Glow (CT2) correction fator = 0
7. Once the initial data is secure, add a volume of Stop & Glo equal to each well equal to the volume of Luciferase reagent added above
a. If 10 u Luciferase Reagent was added, 10 u Stop & Glo should also be added
8. Spin the plate down and allow 10 minutes for signal stabilization.
9. Read the plate on the envision using the same parameters detailed in step four above.
10. Both reagents remain stable for >2 hours. After that, signal may begin to drop off


Plate Type:
Corning 384 well white, TC treated culture plates (Cat #3707)
Protocol:
1. Culture cells in 384 well plates to appropriate density (6000 cells / well).
a. Total plating volume should be 40 ul.
2. 24 hours after plating, pin cells with 100 nl compounds.
3. 24 hours after incubation with compounds (48 hours after plating), prepare assay reagents by mixing Luciferase Assay solution with Luciferase Subtrate and Stop & Glo solution to Stop & Glo substrate
a. Extra reagents can be aliquotted and frozen for future use and are stable for at least one freeze/thaw cycle.
4. Add a volume of luciferase reagent equal to (1/4) the volume of media to each well
a. For example, in a 384-well plate with 40 u in each well, add 10 u. This gives a final volume of 50 u.
5. Spin the plate down and allow 10 minutes for signal stabilization.
6. Read luciferase signal on the Perkin Elmer Envision
a. The following read parameters are used:
i. Distance between plate & detector (mm) = 0.2
ii. Measurement time(s) = 0.1
iii. Glow (CT2) correction fator = 0
7. Once the initial data is secure, add a volume of Stop & Glo equal to each well equal to the volume of Luciferase reagent added above
a. If 10 u Luciferase Reagent was added, 10 u Stop & Glo should also be added
8. Spin the plate down and allow 10 minutes for signal stabilization.
9. Read the plate on the envision using the same parameters detailed in step four above.
10. Both reagents remain stable for >2 hours. After that, signal may begin to drop off
Comment
The total Tau absorbance value from the tTau Elisa assay was divided by the average absorbance value of DMSO wells from the total Tau Elisa assay within the same plate. This ratio was multiplied by 100 to determine a percent of cell survival for each compound at each concentration tested. The percent of cell survival that is reported for each compound reflects the value achieved at a concentration 10 times that of the calculated AC50 for that compound. AC50 values were calculated using the Smart Fit strategy of Genedata Screener Condoseo (7.0.3). If a reported value is greater than 100% cell survival, the starting cell density for that well was originally higher than that in the DMSO wells (averaged).


PubChem Activity Score and Outcome

PUBCHEM_ACTIVITY_SCORE:

Inactive compounds = 0
Active compounds = -10*Log(AC50)

PUBCHEM_ACTIVITY_OUTCOME:

Activity_Outcome = 1 (inactive)
AC50 > highest tested concentration

Activity_Outcome = 2 (active)
AC50 <= highest tested concentration
Categorized Comment - additional comments and annotations
From PubChem:
Assay Format: Cell-based
Assay Cell Type: HEK 293T
Result Definitions
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TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1AC50_uM *The concentration at which activity reaches 50% of the maximumFloatμM
2Activity_at_12nM_(%) The average measured activity of all accepted replicates at the specified concentrationFloat%
3Activity_at_23.5nM_(%) The average measured activity of all accepted replicates at the specified concentrationFloat%
4Activity_at_0.05uM_(%) The average measured activity of all accepted replicates at the specified concentrationFloat%
5Activity_at_0.1uM_(%) The average measured activity of all accepted replicates at the specified concentrationFloat%
6Activity_at_0.195uM_(%) The average measured activity of all accepted replicates at the specified concentrationFloat%
7Activity_at_0.38uM_(%) The average measured activity of all accepted replicates at the specified concentrationFloat%
8Activity_at_0.8uM_(%) The average measured activity of all accepted replicates at the specified concentrationFloat%
9Activity_at_1.6uM_(%) The average measured activity of all accepted replicates at the specified concentrationFloat%
10Activity_at_3uM_(%) The average measured activity of all accepted replicates at the specified concentrationFloat%
11Activity_at_6uM_(%) The average measured activity of all accepted replicates at the specified concentrationFloat%
12Activity_at_12uM_(%) The average measured activity of all accepted replicates at the specified concentrationFloat%
13Activity_at_26uM_(%) 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)
Data Table ( Complete ):     View Active Data    View All Data
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