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BioAssay: AID 602296

Sustained Induction of HSF-1 Measured in Cell-Based System Using Plate Reader - 2038-07_Activator_Dose_CherryPick_Activity

Confirmation testing of small molecules originally identified in PubChem Bioassay AID 2098 as increasing expression of a firefly luciferase under the control of a HSF-1 response element in modified NIH3T3 cells. This assay sought to address 3 specific aims. ..more
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 Tested Compounds
 Tested Compounds
All(1953)
 
 
Active(194)
 
 
Inactive(1492)
 
 
Inconclusive(267)
 
 
 Tested Substances
 Tested Substances
All(1953)
 
 
Active(194)
 
 
Inactive(1492)
 
 
Inconclusive(267)
 
 
AID: 602296
Data Source: Broad Institute (2038-07_Activator_Dose_CherryPick_Activity)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Probe Production Network
BioAssay Version:
Deposit Date: 2012-03-05
Hold-until Date: 2012-03-09
Modify Date: 2012-03-09

Data Table ( Complete ):           View Active Data    View All Data
Target
BioActive Compounds: 194
Related Experiments
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AIDNameTypeComment
493224Luminescence Cell-Based Primary HTS to Identify Activators of Heat Shock Factor 1 (HSF1)Summarydepositor-specified cross reference: Summary assay
435004Luminescence Cell-Based Dose Retest to Identify Potentiators of Heat Shock Factor 1 (HSF1)Confirmatorysame project related to Summary assay
504408Heat Shock Factor-1 (HSF-1) Measured in Cell-Based System Using Plate Reader - 2038-01_Activator_SinglePoint_HTS_ActivityScreeningsame project related to Summary assay
602273Rescue of Alpha-Synuclein Toxicity in Yeast Measured in Microorganism System Using Plate Reader - 2038-04_Activator_Dose_CherryPick_ActivityConfirmatorysame project related to Summary assay
602279Rescue of Alpha-Synuclein Toxicity in Yeast Measured in Microorganism System Using Plate Reader - 2038-04_Activator_Dose_DryPowder_ActivityConfirmatorysame project related to Summary assay
602280Rescue of BEM3 Toxicity in Yeast Measured in Microorganism System Using Plate Reader - 2038-12_Activator_Dose_DryPowder_ActivityConfirmatorysame project related to Summary assay
624441Fluorescence Cell-Based assay to Identify Reactive Oxygen Species Inducers in U2OS cells Measured in Cell-Based System Using Plate Reader - 2038-14_Activator_Dose_DryPowder_ActivityConfirmatorysame project related to Summary assay
624442Fluorescence Cell-Based assay to Identify Reactive Oxygen Species Inducers in U2OS cells Measured in Cell-Based System Using Plate Reader - 2038-14_Activator_Dose_DryPowder_Activity_Set2Confirmatorysame project related to Summary assay
624444Luminescent Cell-based Secondary Assay to Evaluate the Proteasomal Inhibition Activity of HSF1 Inducers Measured in Cell-Based System Using Plate Reader - 2038-13_Activator_Dose_DryPowder_ActivityConfirmatorysame project related to Summary assay
624445Luminescent Cell-based Secondary Assay to Evaluate the Proteasomal Inhibition Activity of HSF1 Inducers Measured in Cell-Based System Using Plate Reader - 2038-13_Activator_Dose_DryPowder_Activity_Set2Confirmatorysame project related to Summary assay
Description:
Keywords: Heat Shock Factor-1 (HSF-1), Induction, Stress Response, NIH3T3, Luminescence

Assay Overview:

Confirmation testing of small molecules originally identified in PubChem Bioassay AID 2098 as increasing expression of a firefly luciferase under the control of a HSF-1 response element in modified NIH3T3 cells. This assay sought to address 3 specific aims.

i.Are these compounds capable of long-term (48hr) induction of a HSF-1 reporter, compared to the shorter interval (8hr) originally assayed.

ii.Are these compounds capable of inducing a HSF-1 reporter as single agents, since the proteosome inhibitor, MG132, was present in the original protocol.

iii.Are these compounds capable of inducing HSF-1 reporter without inducing cell toxicity.

Cells expressing luciferase under the control of a HSF-1 response element were exposure to test compounds for 48hr. After 48hr incubation, the amount of HSF-1 mediated luciferase expression is measured using a luminescence detection reagent.

Expected Outcome:

Identification of HSF-1 inducers in those instances where there is an increase of luminescence signal due to the enhancement of HSF-1 being able to drive the expression of the luciferase reporter. Compounds which do not, or fail to sustain induction of HSF-1 should show no increase in luciferase activity relative to untreated controls. Compounds which induce cell toxicity should a decrease in luciferase activity relative to sub-toxic doses and/or a decrease relative to basal level expression in untreated controls. Potential false positives in this assay included those compounds which act not by specifically inducing HSF-1, but by stabilizing luciferase or are broad spectrum inducers of transcription/translation.
Protocol
Sustained Induction of HSF-1/Cell Toxicity 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.
Compound Screening was carried out on the Broad Institute/Chemical Biology Platform Walk-Up Instruments:
(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 133,333 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 40 uL. The cells are kept in suspension using a magnetic bar and a stirrer during the dispensing.
The assay plate (cell plate) are placed in Thermo 3110 Incubator and incubated overnight at 37 degrees C, 5% CO2, 21% O2, and 95% humidity.
(Compound Treatment):
Test compounds plates are pinned into duplicate assay plates using the 100nL pin array. In addition to test compounds, Doxorubicin (10uM final concentration), was pinned into designated wells within each assay plate as a positive control.
After pinning, the plates are returned to 37 degrees C and incubated for a further 48 hours.
(Reading luminescence from assay plates with Envision):
After 48 hour incubation, 40uL of Steady-Glo Luciferase (Promega) diluted 1:8 in PBS is added to each assay plate using the MultiDrop Combi/long tubing dispensing cassette (Thermo Scientific). Luminescence is measured in each well (0.1 second/well, Corning plate setting) using an Envision plate reader (Perkin Elmer).
Comment
PRESENCE OF CONTROLS: Neutral control wells (NC; n=36) were included on every plate.
EXPECTED OUTCOME: Active compounds result in increasing 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 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: The plate pattern correction algorithm 'Runwise Pattern (Multiplicative)' in Genedata (v7.0.3) was applied to the normalized plate data.
MEASUREMENT USED TO DETERMINE ACTIVE CONCENTRATION (AC): absACnn, the concentration at which the curve crosses threshold 40.0
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 >= 30% but <= 70%, 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
From ChEMBL:
Assay Type: Functional
Result Definitions
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TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1AbsAC40_Qualifier<, =, or >String
2AbsAC40_uM*The concentration at which the fitted curve passes activity threshold 40.FloatμM
3pAbsAC40_MEqual to -1*log10(AbsAC40).Float
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_0.195uM_(%) (0.195μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
12Activity_at_0.38uM_(%) (0.38μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
13Activity_at_0.8uM_(%) (0.8μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
14Activity_at_1.6uM_(%) (1.6μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
15Activity_at_3uM_(%) (3μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
16Activity_at_6uM_(%) (6μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
17Activity_at_12uM_(%) (12μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
18Activity_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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