Bookmark and Share
BioAssay: AID 652154

HTS for PAX8 inhibitors using PAX8 luciferase reporter gene assay in RMG-I cells Measured in Cell-Based System Using Plate Reader - 7054-01_Inhibitor_SinglePoint_HTS_Activity

A cellular assay that measures Pax8 activity using a Luciferase-based reporter of a well characterized PAX8 transcriptional target, thyroperoxidase (TPO), was used in the primary assay. A 420 bp fragment of the rat TPO promoter that was sensitive to Pax8 levels in RMG-I ovarian cancer cells was stably introduced to create a reporter cell line (RMG-1:TPO-Luc)with a biologically-relevant context. Cells were treated with compound and controls for 24 hours. Following compound treatment, TPO promoter activity (via luciferase light production) was measured using Promega SteadyGlo. ..more
_
   
 Tested Compounds
 Tested Compounds
All(353947)
 
 
Active(4145)
 
 
Inactive(349421)
 
 
Inconclusive(454)
 
 
 Tested Substances
 Tested Substances
All(356682)
 
 
Active(4162)
 
 
Inactive(352066)
 
 
Inconclusive(454)
 
 
AID: 652154
Data Source: Broad Institute (7054-01_Inhibitor_SinglePoint_HTS_Activity)
BioAssay Type: Primary, Primary Screening, Single Concentration Activity Observed
Depositor Category: NIH Molecular Libraries Probe Production Network
Deposit Date: 2013-03-21

Data Table ( Complete ):           View Active Data    View All Data
Target
BioActive Compounds: 4145
Related Experiments
Show more
AIDNameTypeComment
652157Broad Institute Discovering modulators of PAX8 for targeting ovarian cancer Inhibitor Probe ProjectSummarydepositor-specified cross reference
687027HTS for PAX8 inhibitors using PAX8 luciferase reporter gene assay in RMG-I cells Measured in Cell-Based System Using Plate Reader - 7054-01_Inhibitor_Dose_CherryPick_ActivityConfirmatorysame project related to Summary assay
687029PAX8: PAX8-dependent cytoxicity Measured in Cell-Based System Using Plate Reader - 7054-06_Inhibitor_Dose_CherryPick_ActivityConfirmatorysame project related to Summary assay
687032SA5-Pax8: Cytotoxicity IOSE-T80 Measured in Cell-Based System Using Plate Reader - 7054-07_Inhibitor_Dose_CherryPick_ActivityConfirmatorysame project related to Summary assay
743020Counterscreen:CMV reporter Measured in Cell-Based System Using Plate Reader - 7054-04_Inhibitor_Dose_DryPowder_ActivityConfirmatorysame project related to Summary assay
743021PAX8: non-specific cytotoxicity Measured in Cell-Based System Using Plate Reader - 7054-05_Inhibitor_Dose_DryPowder_ActivityConfirmatorysame project related to Summary assay
743043SA10 PAX8: cytotoxicity IGROV1 Measured in Cell-Based System Using Plate Reader - 7054-12_Inhibitor_Dose_DryPowder_ActivityConfirmatorysame project related to Summary assay
743044SA9-PAX8: cytotoxicity NIHOVCAR3 Measured in Cell-Based System Using Plate Reader - 7054-11_Inhibitor_Dose_DryPowder_ActivityConfirmatorysame project related to Summary assay
743045SA5-Pax8: Cytotoxicity IOSE-T80 Measured in Cell-Based System Using Plate Reader - 7054-07_Inhibitor_Dose_DryPowder_ActivityConfirmatorysame project related to Summary assay
743046SA6-Pax8: cytotoxicity A2780 Measured in Cell-Based System Using Plate Reader - 7054-08_Inhibitor_Dose_DryPowder_ActivityConfirmatorysame project related to Summary assay
743047SA2 PAX8:selectivity PAX5 Measured in Cell-Based System Using Plate Reader - 7054-03_Inhibitor_Dose_DryPowder_ActivityConfirmatorysame project related to Summary assay
743048Pax8 Selectivity: PAx2 Measured in Cell-Based System Using Plate Reader - 7054-02_Inhibitor_Dose_DryPowder_ActivityConfirmatorysame project related to Summary assay
743049PAX8: PAX8-dependent cytoxicity Measured in Cell-Based System Using Plate Reader - 7054-06_Inhibitor_Dose_DryPowder_ActivityConfirmatorysame project related to Summary assay
743144SA12 PAX8: cytotoxicity COV362 Measured in Cell-Based System Using Plate Reader - 7054-16_Inhibitor_Dose_DryPowder_ActivityConfirmatorysame project related to Summary assay
743145SA7-Pax8: cytotoxicity COV434 Measured in Cell-Based System Using Plate Reader - 7054-09_Inhibitor_Dose_DryPowder_ActivityConfirmatorysame project related to Summary assay
743149SA8-Pax8: cytotoxicity OV-90 Measured in Cell-Based System Using Plate Reader - 7054-10_Inhibitor_Dose_DryPowder_ActivityConfirmatorysame project related to Summary assay
743150SA11 PAX8: cytotoxicity OVCAR4 Measured in Cell-Based System Using Plate Reader - 7054-13_Inhibitor_Dose_DryPowder_ActivityConfirmatorysame project related to Summary assay
743197SA13 PAX8: cytotoxicity OVCAR8 Measured in Cell-Based System Using Plate Reader - 7054-15_Inhibitor_Dose_DryPowder_ActivityConfirmatorysame project related to Summary assay
Description:
Keywords: ovarian cancer, Pax8, luciferase, SteadyGlo, RMG-1 cells, thyroperoxidase


Assay Overview:
A cellular assay that measures Pax8 activity using a Luciferase-based reporter of a well characterized PAX8 transcriptional target, thyroperoxidase (TPO), was used in the primary assay. A 420 bp fragment of the rat TPO promoter that was sensitive to Pax8 levels in RMG-I ovarian cancer cells was stably introduced to create a reporter cell line (RMG-1:TPO-Luc)with a biologically-relevant context. Cells were treated with compound and controls for 24 hours. Following compound treatment, TPO promoter activity (via luciferase light production) was measured using Promega SteadyGlo.

Expected Outcome: If a compound inhibits Pax8 function, there will be less TPO expression in RMG-1 cells and this will lead to a decrease in luciferase signal.
Protocol
PAX8 Primary Assay Protocol
Cell maintenance and passaging:
RMG-I-Pax8Luc cells: Stably express a Pax8-Luciferase reporter that contains a portion of the thyroperoxidase (TPO) gene promoter. Cells are grown in phenol red free RPMI-1640 media supplemented with 10% FBS (Sigma, F4135), Hygromycin (75 ug/mL) and 1X Pen/Strep (Gibco 15140-122). Cells are grown in a humidified incubator, 37oC, 5% CO2.
Upon thawing, cells are allowed to recover for at least 1 week before any experimental procedure. When flasks reach ~80% confluent, cells are passaged, at a 1:4 ratio:
1.Remove media.
2.Briefly wash with PBS.
3.Add Trypsin (0.25%,Gibco 25200-056)
4.Place flask in incubator for 2-3 minutes.
5.Stop trypsin by adding serum containg medium. Transfer cells to a 50 or 250 mL conical tube. Centrifuge 5 minutes at 1200 rpm.
6.Re-plate 25% of the cells into a new plate containing fresh medium.
Note: The cells are very sensitive to long incubation with trypsin or to insufficient dilution of trypsin in FBS-containing medium.
Important: Medium needs to be replaced every 2-3 days between passages (Avoid yellow color of medium, highly acidic).

Day 1: Preparing cells for experiment:
Cells are trypsinized as stated above, centrifuged, aspirate trypsin/media, resuspended in fresh media and counted. Cells are diluted in fresh medium to a final concentration of 167,000 cells/ml. Cells are dispensed into barcoded 384 well plates (Corning 8867 white, opaque), 30 ul/well (5000 cells/well) using a Thermo-Fisher Multidrop Combi and a standard Combi cassette. Dispensing is done at medium speed to prevent foaming.

Day 2: Treatment with compounds:
100 nL of compounds or controls are added to the cells by pinning method. The positive control used in the HTS is 2 uM mitoxantrone (BRD-K21680192-001-01-5), a known cytotoxin that will reduce signal of luciferase.

Day3: SteadyGlo Luciferase assay:
Measure luciferase activity 24h after compound pinning. Steady-Glo solution (Promega E2550 for assay development and med. chem experiments; X1006 pre-made solution for the HTS) is added using a Thermo-Fisher Multidrop Combi, 10 ul/well (standard cassette at medium speed). Plates are allowed to incubate for 10 minutes at room-temp. Luciferase levels are read on a Perkin-Elmer EnVision, using the USLum (ultra-sensitive) protocol. (Time= 0.5s/well)
Comment
PRESENCE OF CONTROLS: Neutral control wells (NC) and positive control wells (PC) were included on every plate.

EXPECTED OUTCOME: Active compounds result in decreasing readout signal.

NORMALIZATION:
The raw signals of the plate wells were normalized using the 'Neutral Controls Minus Inhibitors' method in Genedata Assay Analyzer (v10.0.2):
The median raw signal of the intraplate neutral control wells was set to a normalized activity value of 0.
The median raw signal of the intraplate positive control wells was set to a normalized activity value of -100.
Experimental wells values were scaled to this range.
All well activities were then multiplied by -1 to create a positive activity readout value range, to match Pubchem convention.


PATTERN CORRECTION: The plate pattern correction algorithm 'Runwise Pattern (Multiplicative)' in Genedata (v10.0.2) was applied to the normalized plate data.

PUBCHEM_ACTIVITY_SCORE:
This was set as equal to the mean of the normalized and corrected sample replicate activities, rounded to the nearest integer .
The minimum PUBCHEM_ACTIVITY_SCORE required for a compound to be called a hit (the activity threshold, or AT) was set at 60.

PERCENTAGE OF ACTIVE REPLICATES:
For each sample, the percentage of replicates (PCT_ACTIVE_REP) which had activity scores >= AT was determined.
The minimum percentage of replicates required for a compound to be called a hit (PAR_T) was set at 100.

PUBCHEM_ACTIVITY_OUTCOME:
Samples passing BOTH threshold criteria were assigned an outcome of 2 (active):
PUBCHEM_ACTIVITY_SCORE >= AT, and PCT_ACTIVE_REP >= PAR_T

Samples passing NEITHER threshold criteria were assigned an outcome of 1 (inactive):
PUBCHEM_ACTIVITY_SCORE < AT, and PCT_ACTIVE_REP < PAR_T

Samples passing AT only were assigned an outcome of 3 (inconclusive) :
PUBCHEM_ACTIVITY_SCORE >= AT, and PCT_ACTIVE_REP < PAR_T

Samples passing PAR_T only were assigned an outcome of 3 (inconclusive) :
PUBCHEM_ACTIVITY_SCORE < AT, and PCT_ACTIVE_REP >= PAR_T
Categorized Comment - additional comments and annotations
From PubChem:
Assay Format: Cell-based
Assay Cell Type: RMG-I
Result Definitions
TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1REPRODUCIBILITY_COSINE_TRANSFORMA measure of how well the activity reproduced across the two samples. Computed as the absolute value of the cosine between the 'replicate vector' (ScoreA, ScoreB ---as well as ScoreC and/or ScoreD where applicable) and the vector (1, 1) representing perfect reproducibility. NULL will appear in this column if a sample was not run in duplicate or if the data produced by one of the replicates was Invalid# Float
2PCT_ACTIVE_REPLICATESThe percentage of replicates which pass the activity threshold.Float
3REPLICATE_A_ACTIVITY_SCORE_12.62uM_(%) (12.62μM**)The calculated activity for the indicated sampleFloat%
4REPLICATE_B_ACTIVITY_SCORE_12.62uM_(%) (12.62μM**)The calculated activity for the indicated sampleFloat%

** Test Concentration.
Additional Information
Grant Number: 1 R03 MH097485-01A1

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