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BioAssay: AID 493230

HIF pathway Measured in Cell-Based System Using RT-PCR - 2030-04_Activator_Dose_DryPowder_Activity_Set3

Assay Overview: Real time-PCR assay on human osteosarcoma U2OS cells to identify compounds inducing mRNA expression of VEGF, a downstream target of the HIF pathway. The small molecules inducing a lower cp than the negative control (DMSO) will be considered as active. ..more
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 Tested Compounds
 Tested Compounds
All(89)
 
 
Active(65)
 
 
Inactive(24)
 
 
 Tested Substances
 Tested Substances
All(90)
 
 
Active(66)
 
 
Inactive(24)
 
 
 Related BioAssays
 Related BioAssays
AID: 493230
Data Source: Broad Institute (2030-04_Activator_Dose_DryPowder_Activity_Set3)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Probe Production Network
Deposit Date: 2011-02-16
Hold-until Date: 2012-02-14
Modify Date: 2012-02-15

Data Table ( Complete ):           View Active Data    View All Data
BioActive Compounds: 65
Related Experiments
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AIDNameTypeComment
1971Broad Institute MLPCN Hypoxia Inducible Factor Activation ProjectSummarydepositor-specified cross reference: Summary assay
1910Luminescence Cell-Based Primary HTS to Identify Transcriptional Activators of Hypoxia-Inducible Factor PathwayScreeningsame project related to Summary assay
2089Luminescence Cell-Based Confirmation at Dose to Identify Transcriptional Activators of Hypoxia-Inducible Factor PathwayConfirmatorysame project related to Summary assay
2486Luminescent Cell-Based Dose Titration Retest Counterscreen to Identify Proteasome InhibitorsConfirmatorysame project related to Summary assay
434951Fluorescent Cell-Based Secondary Screen to Identify Activators of the Hypoxia Factor PathwayConfirmatorysame project related to Summary assay
488804Proteasome Measured in Cell-Based System Using Plate Reader - 2030-02_Activator_Dose_DryPowder_Activity_Set2Confirmatorysame project related to Summary assay
488805Hypoxia inducible factor pathway Measured in Cell-Based System Using Plate Reader - 2030-01_Activator_Dose_DryPowder_Activity_Set2Confirmatorysame project related to Summary assay
488820Hypoxia Inducible pathway Measured in Cell-Based System Using Imaging - 2030-03_Activator_Dose_DryPowder_ActivityConfirmatorysame project related to Summary assay
488843HIF pathway Measured in Cell-Based System Using RT-PCR - 2030-04_Activator_Dose_DryPowder_ActivityConfirmatorysame project related to Summary assay
493180Hypoxia inducible factor pathway Measured in Cell-Based System Using Plate Reader - 2030-01_Activator_Dose_DryPowder_Activity_Set3Confirmatorysame project related to Summary assay
493193Hypoxia inducible factor pathway Measured in Cell-Based System Using Plate Reader - 2030-01_Activator_Dose_DryPowder_Activity_Set4Confirmatorysame project related to Summary assay
493195Hypoxia inducible factor pathway Measured in Cell-Based System Using Plate Reader - 2030-01_Activator_Dose_DryPowder_Activity_Set6Confirmatorysame project related to Summary assay
493213Counterscreen Measured in Cell-Based System Using Plate Reader - 2030-02_Activator_Dose_DryPowder_Activity_Set3Confirmatorysame project related to Summary assay
493225Hypoxia inducible pathway Measured in Cell-Based System Using Imaging - 2030-03_Activator_Dose_DryPowder_Activity_Set2Confirmatorysame project related to Summary assay
493227Hypoxia inducible factor pathway Measured in Cell-Based System Using Plate Reader - 2030-01_Activator_Dose_DryPowder_Activity_Set8Confirmatorysame project related to Summary assay
493228Hypoxia inducible factor pathway Iron Measured in Cell-Based System Using Plate Reader - 2030-06_Activator_Dose_DryPowder_ActivityConfirmatorysame project related to Summary assay
493234Hypoxia inducible factor pathway Zinc Measured in Cell-Based System Using Plate Reader - 2030-05_Activator_Dose_DryPowder_Activity_Set2Confirmatorysame project related to Summary assay
493235Hypoxia inducible factor pathway Measured in Cell-Based System Using Plate Reader - 2030-01_Activator_Dose_DryPowder_Activity_Set7Confirmatorysame project related to Summary assay
493236Counterscreen Measured in Cell-Based System Using Plate Reader - 2030-02_Activator_Dose_DryPowder_Activity_Set4Confirmatorysame project related to Summary assay
493237Hypoxia inducible factor pathway Iron Measured in Cell-Based System Using Plate Reader - 2030-06_Activator_Dose_DryPowder_Activity_Set2Confirmatorysame project related to Summary assay
493238HIF pathway Measured in Cell-Based System Using RT-PCR - 2030-04_Activator_Dose_DryPowder_Activity_Set2Confirmatorysame project related to Summary assay
493239Hypoxia inducible pathway Measured in Cell-Based System Using Imaging - 2030-03_Activator_Dose_DryPowder_Activity_Set3Confirmatorysame project related to Summary assay
493249Hypoxia inducible factor pathway Measured in Cell-Based System Using Plate Reader - 2030-01_Activator_Dose_DryPowder_Toxicity_Set7Confirmatorysame project related to Summary assay
504323Hypoxia inducible factor pathway Zinc Measured in Cell-Based System Using Plate Reader - 2030-05_Activator_Dose_DryPowder_ToxicityConfirmatorysame project related to Summary assay
504324Hypoxia inducible factor pathway Zinc Measured in Cell-Based System Using Plate Reader - 2030-05_Activator_Dose_DryPowder_ActivityConfirmatorysame project related to Summary assay
504331Hypoxia inducible factor pathway Iron Measured in Cell-Based System Using Plate Reader - 2030-06_Activator_Dose_DryPowder_ToxicityConfirmatorysame project related to Summary assay
504588Hypoxia inducible factor pathway Measured in Cell-Based System Using Plate Reader - 2030-01_Activator_Dose_DryPowder_Activity_Set10Confirmatorysame project related to Summary assay
504597Hypoxia inducible factor pathway Measured in Cell-Based System Using Plate Reader - 2030-01_Activator_Dose_DryPowder_Activity_Set9Confirmatorysame project related to Summary assay
Description:
Keywords: VEGF, metal chelator

Assay Overview: Real time-PCR assay on human osteosarcoma U2OS cells to identify compounds inducing mRNA expression of VEGF, a downstream target of the HIF pathway. The small molecules inducing a lower cp than the negative control (DMSO) will be considered as active.

Expected Outcome: Confirmation that compounds inducing transcriptional activation of hypoxia responsive element (primary screen) are also acting through induction of the mRNA expression downstream target HIF pathway such as VEGF. Compounds increasing the mRNA level of human VEGF presence (lower cp value) will be considered as actives.
Protocol
Protocol:
RT-PCR VEGF protocol
The Reverse transcriptase-Polymerase Reaction Chain (RT-PCR) was carried out using the human osteosarcoma U2OS cell line. The U2OS (ATCC, HTB-96) cell line is propagated in DMEM media (Invitrogen, SKU#11995) supplemented with 10% heat inactivated fetal bovine serum (Denville Scientific Inc, Cat# FB5001-H) and 1% penicillin/streptomycin/glutamine (Invitrogen, 10378-016) 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 T175 flask (BD Falcon, Ref# 353112) or Hyperflasks (Corning, Cat# 10010), harvested at more than 80% confluence using 7 or 75 ml Trypsin-EDTA 0.25% (Cellgro, Cat# 25-053-CI) for 5 minutes and then the trypsin is inactivated with 7 or 75 ml of complete medium respectively. Cells should not be trypsinized more than 10 minutes because massive cell death will start to occur. Cells are centrifuged at 1000rpm/5min and resuspended in fresh complete DMEM media with phenol red (Invitrogen, SKU# 11995) as mentioned above (for normal cell propagation) or DMEM medium without phenol red (Cambrex, Cat# 12-917F) with 10% Heat inactivated fetal bovine serum (Invitrogen, Cat# 16140089), 1% penicillin/streptomycin/glutamine (Invitrogen, 10378-016). Cell number is counted using a Nexelcom Bioscience cell counter (Cellometer Auto M10) and viability is measured by mixing one volume of cells with one same volume of Trypan Blue solution (0.4%, dilution 1/2). Only cultures of >94% viability are utilized for HTS.
Compound Screening is carried out on the Broad Institute/Chemical Biology Platform Walkup unit:
Day 1 (Cell plating):
1. U2OS cells are harvested and resuspended in DMEM without phenol red (Cambrex, 12-917F) with 10% Heat inactivated fetal bovine serum (Denville Scientific Inc, Cat# FB5001-H) and 1% penicillin/streptomycin/glutamine (Invitrogen, 10378-016). U2OS cells (from an initial cell suspension of 40,000 cells/ml) are dispensed using a MultiDrop Combi/Standard tube dispensing cassette (Thermo Scientific) in white bottom 384 well assay plates (Corning, Cat# 8867BC) at a final density of 2,000 cells per well in final volume of 50uL.
2. The assay plate (cell plate) are placed in Liconic Instruments cassettes (22 plates/cassette) and incubated for 24 hours at 37 degrees C in the Liconic CO2 incubator (Liconic Instruments) calibrated at 5% CO2, 21% O2, and 95% humidity.
Day 2 (Compound pinning into assay plate):
3. The dose response compound plates are pinned twice into one assay plate (duplicate). Pins are washed with methanol and DMSO between each pinning.
4. After the pinning has occurred, the assay plates treated with compounds are moved back to Liconic CO2 incubator to be incubated for an additional 24 hours.
Day 3 (RT-PCR):
1. Cell to CT lysis
The medium is aspirated and the cells are washed twice (100 uL with PBS) using the ELX405 Plate washer (Biotek). The assay plates are flipped upside down and centrifuged at 1000 rpm 2 minutes to remove the excess liquid. 10 uL of Lysis solution with DNase I (Ambion, from Cell to CT Lysis Mix) is added to each well using the MultiDrop Combi/Standard tube dispensing cassette (Thermo Scientific). Each assay plate is then vortexed for 2 minutes and incubated for an additional 5 minutes at room temperature. At the end of the incubation, the assay plates are centrifuged (1000 rpm, 2 minutes) and 1 uL of stop solution is added with the Multidrop Combi-nl (Thermo Scientific). The assay plate are incubated for 2 minutes at room temperature and processed for reverse transcription.
2. Reverse transcription (RT) (Ambion, Cell to CT RT Mix#4402958)
Buffer:
2X RT Buffer, 5 uL
20X RT Enzyme Mix, 0.5 uL
Nuclease-Free Water, 2.5 uL
Procedure:
8 uL of RT is dispensed into each well of a RT assay plate (Axygen, PCR-384 RGD C). 2 uL of the lysed cells are transferred into RT assay plate using Vario transfer unit (CyBi Well). The RT assay plates are incubated at 37 degrees C for 1 hour and the reverse transcriptase inactivated by incubating the plates for 1 minute at 95 degrees C (8 uL RT Master Mix/10 uL total reaction volume).
3. RT-PCR
4 uL PCR Master Mix/5uL total volume of reaction
2X Roche Master Mix (Probes Master), 2.5 uL
20X FAM Taqman probe/primers set, 0.125 uL
(human VEGF-A, Applied Biosystems 4331182, Hs03929005_m1 inventoried FAM-MGB)
20X VIC Taqman probe/primers set, 0.125 uL
(human actin-beta, Applied Biosystems 4310881E Vic-TAMRA)
PCR H2O, 1.25 uL
4 uL/well of PCR master mix is dispensed in PCR plate (Roche Light Cycler 480 MultiWell Plate 384, Cat# 04 729 749 001) using the Multidrop Combi-nl (Thermo Scientific).
Then, 1 uL/well of RT DNA is transferred in the 4 uL/well PCR plate(Roche, Ref#04 902 343 001). The PCR plates are centrifuged 2 minutes at 1000 rpm.
PCR is performed using ThermoCycler (Roche Light Cycler 480 II) with this setup:
Steps:
1. 95 degrees C, 10 minutes
2. 95 degrees C, 10 seconds
3. 60 degrees C, 30 seconds
Step 2 and 3 (40 cycles)
Comment
PRESENCE OF CONTROLS: Neutral control wells (NC; n=32) and positive control wells (PC; n=32) 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 'Stimulators Minus Neutral Controls' 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): 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: U-2 OS
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_0.018uM_(%) (0.018μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
12Activity_at_0.056uM_(%) (0.056μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
13Activity_at_0.16uM_(%) (0.16μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
14Activity_at_0.5uM_(%) (0.5μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
15Activity_at_1.5uM_(%) (1.5μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
16Activity_at_4.6uM_(%) (4.6μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
17Activity_at_13.5uM_(%) (13.5μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
18Activity_at_38uM_(%) (38μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%

* Activity Concentration. ** Test Concentration.
Additional Information
Grant Number: 1 R03 MH082355-01A2

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