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BioAssay: AID 602268

Heat Shock Factor-1 (HSF-1) Luciferase Reporter Measured in Cell-Based System Using Plate Reader - 2038-01_Inhibitor_Dose_DryPowder_Activity

Modified NIH3T3, transformed to express firefly luciferase under the control of a HSF-1 response element, will be exposed to small molecules. After 30min exposure to small molecules, proteasome inhibitor MG132 is added to elicit a stress response. After 8hr incubation in the presence of this stressor, the amount of HSF-1 mediated luciferase expression is measured using a luminescence detection reagent. ..more
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 Tested Compounds
 Tested Compounds
All(6)
 
 
Active(5)
 
 
Inactive(1)
 
 
 Tested Substances
 Tested Substances
All(7)
 
 
Active(5)
 
 
Inactive(2)
 
 
AID: 602268
Data Source: Broad Institute (2038-01_Inhibitor_Dose_DryPowder_Activity)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Probe Production Network
Deposit Date: 2012-02-17
Hold-until Date: 2013-02-16
Modify Date: 2013-02-16

Data Table ( Complete ):           View Active Data    View All Data
Target
BioActive Compounds: 5
Related Experiments
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AIDNameTypeComment
2118Identifying Small Molecules To Probe the Role of Heat Shock Factor 1 in CancerSummarydepositor-specified cross reference: Summary assay
2098Luminescence Cell-Based Primary HTS to Identify Inhibitors of Heat Shock Factor 1 (HSF1).Screeningsame project related to Summary assay
2382Luminescence Cell-Based Dose Confimation HTS to Identify Inhibitors of Heat Shock Factor 1 (HSF1)Confirmatorysame project related to Summary assay
493083HSF-1 induced GFP reporter and Doxycycline induced RFP reporter Measured in Cell-Based System Using Plate Reader - 2038-03_Inhibitor_DoseNoFile_CherryPick_Activity_Set3Confirmatorysame project related to Summary assay
493085HSF-1 induced GFP reporter and Doxycycline induced RFP reporter Measured in Cell-Based System Using Plate Reader - 2038-03_Inhibitor_DoseNoFile_CherryPick_Internal-Standard_Set3Confirmatorysame project related to Summary assay
588827HSF-1 induced GFP reporter and Doxycycline induced RFP reporter Measured in Cell-Based System Using Plate Reader - 2038-03_Inhibitor_Dose_CherryPick_Activity_Set4Confirmatorysame project related to Summary assay
588847Luciferase Inhibition Counter Screen Measured in Biochemical System Using Plate Reader - 2038-06_Inhibitor_Dose_CherryPick_ActivityConfirmatorysame project related to Summary assay
602266Quantitative-PCR analysis for transcription inhibition of HSF-1 target gene Hsp90aa Measured in Cell-Based System Using RT-PCR - 2038-08_Inhibitor_Dose_CherryPick_ActivityConfirmatorysame project related to Summary assay
602269HSF-1 induced GFP reporter and Doxycycline induced RFP reporter Measured in Cell-Based System Using Plate Reader - 2038-03_Inhibitor_Dose_DryPowder_ActivityConfirmatorysame project related to Summary assay
Description:
Keywords:
Heat Shock Factor-1 (HSF-1), Stress Response, MG132, NIH3T3, Luminescence

Assay Overview:
Modified NIH3T3, transformed to express firefly luciferase under the control of a HSF-1 response element, will be exposed to small molecules. After 30min exposure to small molecules, proteasome inhibitor MG132 is added to elicit a stress response. After 8hr incubation in the presence of this stressor, the amount of HSF-1 mediated luciferase expression is measured using a luminescence detection reagent.

Expected Outcome:
Identification of HSF-1 inhibitors in those instances where there is a loss of luminescence signal due to the prevention of HSF-1 being able to drive the expression of the luciferase reporter. Potential false positives in this assay included those compounds which act not by specifically inhibiting HSF-1, but by inhibiting luciferase or are broad spectrum inhibitors of transcription/translation.
Protocol
Protocol: HGL HSF-1 luciferase assay:

The NIH3T3-HGL cell line is modified version of NIH3T3 fibroblasts with an integrated eGFP-Firefly luciferase fusion construct under the control of a heat shock response element. The NIH3T3-HGL cell line was generously provided for this study by Dr. Luke Whitesell.

The HGL cell line is propagated in Opti-media (Invitrogen) supplemented with 5% heat inactivated fetal bovine serum (Invitrogen), 1% penicillin/streptomycin/glutamine at 37 degrees C in CO2 incubators (Thermo Scientific) with 5% CO2, 21% O2, and 95% humidity. For High-Throughput Screening assays, cells are grown in T225 flask (BD Falcon) or Hyperflasks (Corning), harvested at more than 80% confluence using Accumax (Innovative Cell Technologies). Cell number is counted using a Nexelcom Bioscience cell counter (Cellometer Auto M10) and viability is measured by mixing cells 1:1 with a 0.4% Trypan Blue solution (Sigma). Only cultures of >94% viability are utilized for experiments.

(Cell plating):

HGL cells are harvested and re-suspended in Opti-MEM with 2.5% Heat inactivated FBS, 1% penicillin/streptomycin/glutamine. HGL cells (from an initial cell suspension of 200,000 cells/ml) are dispensed using a MultiDrop Combi/Standard tube dispensing cassette (Thermo Scientific) in white bottom 384 well assay plates (Corning) at a final density of 4,000 cells per well in final volume of 30 uL. The cells are kept in suspension using a magnetic bar and a stirrer during the dispensing.

The assay plate (cell plate) are incubated for 24 hours at 37 degrees C in the Liconic CO2 incubator (Liconic Instruments) calibrated at 5% CO2, 21% O2, and 95% humidity.

(Compound pinning into assay plate):

Test compounds plates are pinned into duplicate assay plates using the 100nL pin array. In addition to test compounds, Didemnin B (400nM final concentration), was pinned into designated wells within each assay plate as a positive control.

After 30min incubation with the compounds, 50uM MG132 (Enzo, cat# PI-102) in 1X PBS (Invitrogen, cat# 10010) is added in 1uL with the CombinL (Thermo) (2.5uM final conc. MG132) to induce HSF-1. The cells are incubated in the presence of MG132 for a further 8hrs.

(Reading luminescence from assay plates with Envision):
After 8 hr incubation, 20uL of Steady-Glo luciferase (Promega, cat# E2550) is added to each assay plate using a MultiDrop Combi (Thermo). Luminescence is measured in each well (0.1 second/well) using the Envision plate reader (Perkin Elmer)(Corning plate setting).
Comment
PRESENCE OF CONTROLS: Neutral control wells (NC; n=104) and positive control wells (PC; n=20) were included on every plate.

EXPECTED OUTCOME: Active compounds result in decreasing readout signal.

ACTIVE CONCENTRATION LIMIT:
For each sample, the highest valid tested concentration (Max_Concentration) was determined and the active concentration limit (AC_limit) was set to equal (2)(Max_Concentration).

NORMALIZATION:
The raw signals of the plate wells were normalized using the 'Neutral Controls Minus Inhibitors' method in Genedata Assay Analyzer (v7.0.3):
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.

PATTERN CORRECTION: No plate pattern correction algorithm from Genedata Condoseo (v.7.0.3) was applied.

MEASUREMENT USED TO DETERMINE ACTIVE CONCENTRATION (AC): AC50
AC values were calculated using the curve fitting strategies in Genedata Screener Condoseo (7.0.3).
AC values were calculated up to the active concentration limit described for each sample.
pAC was set to equal -1*log10(AC)

PUBCHEM_ACTIVITY_OUTCOME:
Activity_Outcome = 1 (inactive) when:
a) compound shows activity but in a direction opposite to the expected outcome
in these cases, values describing curve fitting parameters (Sinf, S0, Hill Slope, log_AC50, log_AC50_SE) are set to null
b) curve fit is constant where activity is > -30% and < 30% at all tested concentrations, or
c) AC > AC_limit
Activity_Outcome = 2 (active) when:
AC <= AC_limit
Activity_Outcome = 3 (inconclusive) when:
a) Curve fitting strategy resulted in a constant fit with activity >= -70% but <= -30%, or
b) The fit was deemed not valid due to poor fit quality.

PUBCHEM_ACTIVITY_SCORE:
If PUBCHEM_ACTIVITY_OUTCOME = 1 (inactive) or 3 (inconclusive),
then PUBCHEM_ACTIVITY_SCORE = 0
If PUBCHEM_ACTIVITY_OUTCOME = 2 (active)
then PUBCHEM_ACTIVITY_SCORE = (10)(pAC)
Scores relate to AC in this manner:
120 = 1 pM
90 = 1 nM
60 = 1 uM
30 = 1 mM
0 = 1 M
When the active concentration (AC) is calculated to be greater than the highest valid tested concentration (Max_Concentration), the PUBCHEM_ACTIVITY_SCORE is calculated using Max_Concentration as the basis.
When the active concentration (AC) is calculated to be less than the lowest tested concentration, the PUBCHEM_ACTIVITY_SCORE is calculated using the lowest tested concentration as the basis.

Note:
The individual dose data point columns ('Activity_at_xxuM') reported here represent the median of valid (unmasked) replicate observations at each concentration. These values are the inputs to a curve fitting algorithm.
All other data columns represent values which are derived during the curve fitting algorithm; this may sometimes include automatic further masking of some replicate data points.
Occasionally this results in perceived inconsistencies: for example, between the derived 'Maximal_Activity' and the apparent most active data point.
Categorized Comment - additional comments and annotations
From PubChem:
Assay Format: Cell-based
Assay Cell Type: NIH 3T3
Result Definitions
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TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1AC50_Qualifier'>','=', or '<'String
2AC50_uM*The concentration at which activity reaches 50% of the maximumFloatμM
3pAC50_MEqual to -1*log10(AC50)String
4Hill_SlopeThe slope at AC50Float
5S0_(%)The fitted activity value at zero concentrationFloat%
6Sinf_(%)The fitted activity value at infinite concentrationFloat%
7Num_PointsThe number of data points used to generate the plotInteger
8Max_Activity_(%)The maximum activity value observed, based on mean of replicates per concentrationFloat%
9Max_Activity_Conc_uMThe concentration at which the maximum activity is observedFloatμM
10Max_Concentration_uMMaximum valid test concentrationFloatμM
11Activity_at_0.000046uM_(%) (4.6e-05μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
12Activity_at_0.0001uM_(%) (0.0001μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
13Activity_at_0.000195uM_(%) (0.000195μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
14Activity_at_0.00038uM_(%) (0.00038μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
15Activity_at_0.00075uM_(%) (0.00075μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
16Activity_at_0.0015uM_(%) (0.0015μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
17Activity_at_0.003uM_(%) (0.003μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
18Activity_at_0.006uM_(%) (0.006μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
19Activity_at_0.012uM_(%) (0.012μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
20Activity_at_0.0235uM_(%) (0.0235μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
21Activity_at_0.05uM_(%) (0.05μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
22Activity_at_0.1uM_(%) (0.1μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
23Activity_at_0.195uM_(%) (0.195μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
24Activity_at_0.38uM_(%) (0.38μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
25Activity_at_0.8uM_(%) (0.8μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
26Activity_at_1.6uM_(%) (1.6μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
27Activity_at_3uM_(%) (3μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
28Activity_at_6uM_(%) (6μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
29Activity_at_12uM_(%) (12μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
30Activity_at_26uM_(%) (26μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%

* Activity Concentration. ** Test Concentration.
Additional Information
Grant Number: 1 R03 MH086465-01

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