Bookmark and Share
BioAssay: AID 651964

Late-stage results from the probe development effort to identify transcriptional activators of heat shock protein 70 (Hsp70): fluorescence-based biochemical quantitative polymerase chain reaction assay to assess gene expression changes in downstream target genes

Name: Late-stage results from the probe development effort to identify transcriptional activators of heat shock protein 70 (Hsp70): fluorescence-based biochemical quantitative polymerase chain reaction assay to assess gene expression changes in downstream target genes. ..more
_
   
 Tested Compounds
 Tested Compounds
All(1)
 
 
Active(1)
 
 
 Tested Substances
 Tested Substances
All(1)
 
 
Active(1)
 
 
AID: 651964
Data Source: The Scripps Research Institute Molecular Screening Center (DOWNSTREAM GENES_AG_FLUO_QPCR)
BioAssay Type: Panel
Depositor Category: NIH Molecular Libraries Screening Center Network, Assay Provider
Deposit Date: 2012-12-28

Data Table ( Complete ):           View Active Data    View All Data
BioActive Compound: 1
Related Experiments
Show more
AIDNameTypeComment
1203Primary cell-based high-throughput screening assay to identify transcriptional activators of heat shock protein 70 (Hsp70)Screeningdepositor-specified cross reference: Primary screen (Hsp70 activators in singlicate)
1252Dose response cell-based high-throughput screening assay to identify transcriptional activators of heat shock protein 70 (Hsp70)Confirmatorydepositor-specified cross reference: Dose response (Hsp70 activators in triplicate)
1259Cytotoxicity counterscreen assay for transcriptional activators of heat shock protein 70 (Hsp70)Confirmatorydepositor-specified cross reference: Dose response counterscreen (cytotoxocity in triplicate)
588815Summary of the probe development efforts to identify transcriptional activators of heat shock protein 70 (Hsp70).Summarydepositor-specified cross reference: Summary (Hsp70 activators)
651945Late-stage results from the probe development effort to identify transcriptional activators of heat shock protein 70 (Hsp70): fluorescence-based biochemical quantitative polymerase chain reaction assay to assess Hsp70 gene expression changesOtherdepositor-specified cross reference: Late-stage assay (Hsp70 gene expression changes)
651948Late-stage results from the probe development effort to identify transcriptional activators of heat shock protein 70 (Hsp70): chemiluminescence-based biochemical western blot assay for transcriptional activators of heat shock protein Hsp70Otherdepositor-specified cross reference: Late-stage assay (Hsp70 transcriptional activators)
651950Late stage assay for the probe development effort to identify transcriptional activators of heat shock protein 70 (Hsp70). Luminescence-based-cell-based high-throughput dose response assay for Hsp70 activatorsOtherdepositor-specified cross reference: Late stage dose response (Hsp70 activators in triplicate)
651992Late-stage results from the probe development effort to identify transcriptional activators of heat shock protein 70 (Hsp70): yellow fluorescent protein (YFP) quenching assay to assess changes in trafficking of DeltaF508-CFTROtherdepositor-specified cross reference
651963Late-stage results from the probe development effort to identify transcriptional activators of heat shock protein 70 (Hsp70): fluorescence-based cell-based microscopic assay to assess aggregation of polyQ35 in C. elegans larvaeOthersame project related to Summary assay
Description:
Source (MLSCN Center Name): The Scripps Research Institute Molecular Screening Center
Center Affiliation: The Scripps Research Institute (TSRI)
Assay Provider: Richard Morimoto, Northwestern University
Network: Molecular Library Screening Center Network (MLSCN)
Grant Proposal Number: 5 R21 NS056337-02
Grant Proposal PI: Richard Morimoto

External Assay ID: DOWNSTREAM GENES_AG_FLUO_QPCR

Name: Late-stage results from the probe development effort to identify transcriptional activators of heat shock protein 70 (Hsp70): fluorescence-based biochemical quantitative polymerase chain reaction assay to assess gene expression changes in downstream target genes.

Description:

The human heat shock protein 70 (Hsp70) family is evolutionarily conserved among all organisms from archaebacteria to humans, suggesting an essential role in cell survival (1, 2). Under circumstances of transient cell stress, the heat shock response and activities of molecular chaperones can restore protein homeostasis. In human disease, however, misfolded proteins can accumulate when polyglutamine-expansion proteins are chronically expressed over the life of the cell. Elevated expression of molecular chaperones suppresses protein misfolding/aggregation and toxicity phenotypes in various model systems of Huntington's disease, Alzheimer's disease, Parkinson's disease, and Amyotrophic Lateral Sclerosis (ALS). Mutations in the respective proteins huntingtin, tau, alpha-synuclein, and superoxide dismutase (SOD1), associated with these diseases, result in the appearance of misfolded species that adopt alternate conformations. These observations led to the proposal that a common feature of diverse diseases of protein conformation is the appearance of alternate folded states that self-associate and form toxic species and protein aggregates.

A role for Hsp70 family proteins in controlling these events has been widely studied. Studies with mammalian tissue culture cells, transgenic mice, Drosophila, and C. elegans have established that the heat shock response can be activated in cells expressing aggregation-prone proteins, suggesting a role for molecular chaperones as an adaptive survival response (3, 4). Moreover, a direct relationship with polyglutamine diseases is suggested by the co-localization of several heat shock proteins, including Hdj-1, Hdj-2, Hsp70 and ubiquitin with polyglutamine aggregates in the tissues of affected individuals, transgenic mice and tissue culture cells (5). Finally, overexpression of Hsp70 can suppress the toxicity associated with the accumulation of misfolded proteins (6-8). High throughput screening initiatives aimed at the identification of compounds that enhance the heat shock response, in particular Hsp70, will provide insights into this conserved cellular process and may lead to novel therapeutics for these devastating disorders.

References:

1.Gupta, R.S., and Singh, B. 1994. Phylogenetic analysis of 70 kD heat shock protein sequences suggests a chimeric origin for the eukaryotic cell nucleus. Curr Biol 4:1104-1114.
2.Lindquist, S., and Craig, E.A. 1988. The heat-shock proteins. Annu Rev Genet 22:631-677.
3.Satyal, S.H., Schmidt, E., Kitagawa, K., Sondheimer, N., Lindquist, S., Kramer, J.M., and Morimoto, R.I. 2000. Polyglutamine aggregates alter protein folding homeostasis in Caenorhabditis elegans. Proc Natl Acad Sci U S A 97:5750-5755.
4.Wyttenbach, A., Carmichael, J., Swartz, J., Furlong, R.A., Narain, Y., Rankin, J., and Rubinsztein, D.C. 2000. Effects of heat shock, heat shock protein 40 (HDJ-2), and proteasome inhibition on protein aggregation in cellular models of Huntington's disease. Proc Natl Acad Sci U S A 97:2898-2903.
5.Cummings, C.J., Mancini, M.A., Antalffy, B., DeFranco, D.B., Orr, H.T., and Zoghbi, H.Y. 1998. Chaperone suppression of aggregation and altered subcellular proteasome localization imply protein misfolding in SCA1. Nat Genet 19:148-154.
6.Krobitsch, S., and Lindquist, S. 2000. Aggregation of huntingtin in yeast varies with the length of the polyglutamine expansion and the expression of chaperone proteins. Proc Natl Acad Sci U S A 97:1589-1594.
7.Kazemi-Esfarjani, P., and Benzer, S. 2000. Genetic suppression of polyglutamine toxicity in Drosophila. Science 287:1837-1840.
8.Warrick, J.M., Chan, H.Y., Gray-Board, G.L., Chai, Y., Paulson, H.L., and Bonini, N.M. 1999. Suppression of polyglutamine-mediated neurodegeneration in Drosophila by the molecular chaperone HSP70. Nat Genet 23:425-428.

Keywords:

Hsp70, HSPA1A, HSF1, heat shock transcription factor 1, Glutamate-cysteine ligase, modifier subunit, GCLM, Ddit3 DNA-damage inducible transcript 3, CHOP, Hspa5 heat shock protein 5, BiP, Hmox1 heme oxygenase (decycling) 1, HO-1, chaperone, agonist, activator, QPCR, RTPCR, qRT-PCR, RNA, polymerase chain reaction, PCR, mouse embryo fibroblasts, MEFs, mouse 8-gene multiplex, celastrol, primers, cDNA, mRNA, counterscreen, streptavidin-phycoerythrin, SAPE, fluorescence, late stage, late stage AID, assay provider, powders, Scripps, Scripps Research Institute Molecular Screening Center, Molecular Library Screening Center Network, MLSCN.
Panel Information
Targets
    Data Table(Active)    Data Table(All)Show more
PID§NameSubstancePanel TargetsDescriptionAdditional Information
ActiveInactive
1Hsp701heat shock 70 kDa protein 1A [Mus musculus] [gi:124339829]
Taxonomy id: 10090
Gene id: 193740
2GCLM1glutamate--cysteine ligase regulatory subunit [Mus musculus] [gi:6680019]
Taxonomy id: 10090
Gene id: 14630
3CHOP1DNA damage-inducible transcript 3 protein [Mus musculus] [gi:160707929]
Taxonomy id: 10090
Gene id: 13198
4BiP1Heat shock protein 5 [Mus musculus] [gi:29748016]
Taxonomy id: 10090
Gene id: 14828
5HO-11heme oxygenase 1 [Mus musculus] [gi:6754212]
Taxonomy id: 10090
Gene id: 15368

§ Panel component ID.
Protocol
Assay Overview:
The purpose of this assay is to determine whether powder samples of a compound identified as a transcriptional activator of heat shock protein 70 (Hsp70) modulates the gene expression of Hsp70 involved in the heat shock response and protein homeostasis, as well as the expression of four other stress-responsive proteostasis network pathways in addition to the heat shock response. These are the regulatory subunit of glutamate-cysteine ligase (GCLM), the proapoptotic growth-arrest-inducible and DNA-damage-inducible gene (CHOP), the UPR-inducible heat shock protein 5 gene (BiP), and heme oxidase 1 (HO-1). In this assay, mouse embryonic fibroblasts (MEFs) are incubated with test compound and harvested. Quantitative reverse transcription polymerase chain reaction (qRT-PCR) is performed using mouse 8-gene multiplex probe sets. RNA product is detected by streptavidin-phycoerythrin (SAPE) fluorescence. Gene expression is normalized to TATA-box binding protein (TBP) housekeeping gene and compared to levels in cells treated with DMSO only. As designed, test compounds that induce an increase in target gene expression will result in an increase in amplified RNA product and an increase in well fluorescence. Cells are treated with test compound in a 7-point dilution series from 0.15 to 10 uM).
Protocol Summary:
Mouse embryonic fibroblasts (MEFs) were maintained on Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% v/v fetal bovine serum (FBS), 0.1 mM nonessential amino acids, 100 U/ml penicillin/streptomycin, and 55 uM 2-mercaptoethanol. The cells were seeded at a density of 12,000 cells/well in 96-well plates with an overnight incubation at 37 C in 5% CO2. Cells were treated with test compound and the reference positive controls MG132 (1 uM), geldanamycin (1 uM), tunicamycin (1 uM), and sulphoraphane (1 uM). Untreated and DMSO-treated cells served as negative controls. Cell lysis with 50% [v/v] Panomics Lysis Mixture (Lysis Mixture + 10 ul/ml Proteinase K) was performed 6 hours post-compound treatment. Lysed cells were heated at 50 C to ensure appropriate lysis and the plates were then frozen at -80 C. Cell lysates, thawed at room temperature on the day of the assay, were pooled with mouse 8-gene multiplex probe sets and with 8 different sets of magnetic capture beads in a 100 ul/well volume. Biomek FX was used at every liquid transfer step to reduce variability by human error. The eight plates containing lysate-probe-bead mixtures were incubated at 54 C +/- 1 C on a shaking platform for an overnight incubation in the dark (18-20 hours). The following day the hybridization plates were compressed by transferring the hybridized lysates into a single magnetic capture plate. The plate was kept on a magnet to hold the beads and then washed with Panomics Wash Buffer 2.0 on a BioTek ELx405 select plate washer to remove any unbound sample. This step was followed by serial hybridizations and washings of the branched DNA (bDNA) pre-amplifier (1 hour, 50 C), bDNA amplifier (1 hour, 50 C), label probe (1 hour, 50 C), and streptavidin-phycoerythrin (SAPE, 30 minutes, room temperature). The plate was then washed with SAPE wash buffer to remove unbound SAPE and each well was analyzed with the Luminex FlexMap3D. SAPE fluorescence measured from each bead was proportional to the number of mRNA transcripts captured by the beads.
Fold inductions in gene expression were obtained for each gene per well by normalizing the raw data first to the DMSO control and then to the TATA-box binding protein (TBP) housekeeping gene.
PubChem Activity Outcome and Score:
The following applies to each panel in this assay:
Compounds were considered active in up-regulating stress responsive pathways if they caused a greater than or equal to 2.0-fold induction in gene expression compared to the DMSO levels (set at 1.0) at any compound concentration tested.
Overall Outcome and Score:
The overall outcome was active if the compound was active in at least one panel, inactive otherwise.
The overall score is 0 if the compound was inactive, otherwise the score is taken as the fraction of panels where the compound is active, multiplied by 100.
The PubChem Activity Score range for active compounds is 100-100. There are no inactive compounds.
List of reagents:
Mouse embryo fibroblast (MEF) cells (provided by Assay Provider)
DMEM (Invitrogen)
Mouse 8-gene multiplex probe sets (Luminex Technology)
Luminex FlexMap3D (Luminex Technology)
Comment
This assay was performed by the assay provider with powder samples of purchased test compound.
Categorized Comment - additional comments and annotations
From PubChem:
Assay Format: Biochemical
Result Definitions
Show more
TIDNameDescriptionPID§Panel TargetsHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1Outcome [Hsp70]One of Active, Inactive, or Not Tested1heat shock 70 kDa protein 1A [Mus musculus]Outcome
2Fold Induction at 10 uM [Hsp70] (10μM**)Fold induction of mRNA expression of Target Gene at 10 uM concentration1String
3Standard Deviation at 10 uM [Hsp70]Standard deviation of value of fold induction at 10 uM concentration1String
4Fold Induction at 5 uM [Hsp70] (5μM**)Fold induction of mRNA expression of Target Gene at 5 uM concentration1String
5Standard Deviation at 5 uM [Hsp70]Standard deviation of value of fold induction at 5 uM concentration1String
6Fold Induction at 2.5 uM [Hsp70] (2.5μM**)Fold induction of mRNA expression of Target Gene at 2.5 uM concentration1String
7Standard Deviation at 2.5 uM [Hsp70]Standard deviation of value of fold induction at 2.5 uM concentration1String
8Fold Induction at 1.25 uM [Hsp70] (1.25μM**)Fold induction of mRNA expression of Target Gene at 1.25 uM concentration1String
9Standard Deviation at 1.25 uM [Hsp70]Standard deviation of value of fold induction at 1.25 uM concentration1String
10Fold Induction at 0.6 uM] [Hsp70] (0.6μM**)Fold induction of mRNA expression of Target Gene at 0.6 uM concentration1String
11Standard Deviation at 0.6 uM [Hsp70]Standard deviation of value of fold induction at 0.6 uM concentration1String
12Fold Induction at 0.3 uM [Hsp70] (0.3μM**)Fold induction of mRNA expression of Target Gene at 0.3 uM concentration1String
13Standard Deviation at 0.3 uM [Hsp70]Standard deviation of value of fold induction at 0.3 uM concentration1String
14Fold Induction at 0.15 uM [Hsp70] (0.15μM**)Fold induction of mRNA expression of Target Gene at 0.15 uM concentration1String
15Standard Deviation at 0.15 uM [Hsp70]Standard deviation of value of fold induction at 0.15 uM concentration1String
16Outcome [GCLM]One of Active, Inactive, or Not Tested2glutamate--cysteine ligase regulatory subunit [Mus musculus]Outcome
17Fold Induction at 10 uM [GCLM] (10μM**)Fold induction of mRNA expression of Target Gene at 10 uM concentration2String
18Standard Deviation at 10 uM [GCLM]Standard deviation of value of fold induction at 10 uM concentration2String
19Fold Induction at 5 uM [GCLM] (5μM**)Fold induction of mRNA expression of Target Gene at 5 uM concentration2String
20Standard Deviation at 5 uM [GCLM]Standard deviation of value of fold induction at 5 uM concentration2String
21Fold Induction at 2.5 uM [GCLM] (2.5μM**)Fold induction of mRNA expression of Target Gene at 2.5 uM concentration2String
22Standard Deviation at 2.5 uM [GCLM]Standard deviation of value of fold induction at 2.5 uM concentration2String
23Fold Induction at 1.25 uM [GCLM] (1.25μM**)Fold induction of mRNA expression of Target Gene at 1.25 uM concentration2String
24Standard Deviation at 1.25 uM [GCLM]Standard deviation of value of fold induction at 1.25 uM concentration2String
25Fold Induction at 0.6 uM] [GCLM] (0.6μM**)Fold induction of mRNA expression of Target Gene at 0.6 uM concentration2String
26Standard Deviation at 0.6 uM [GCLM]Standard deviation of value of fold induction at 0.6 uM concentration2String
27Fold Induction at 0.3 uM [GCLM] (0.3μM**)Fold induction of mRNA expression of Target Gene at 0.3 uM concentration2String
28Standard Deviation at 0.3 uM [GCLM]Standard deviation of value of fold induction at 0.3 uM concentration2String
29Fold Induction at 0.15 uM [GCLM] (0.15μM**)Fold induction of mRNA expression of Target Gene at 0.15 uM concentration2String
30Standard Deviation at 0.15 uM [GCLM]Standard deviation of value of fold induction at 0.15 uM concentration2String
31Outcome [CHOP]One of Active, Inactive, or Not Tested3DNA damage-inducible transcript 3 protein [Mus musculus]Outcome
32Fold Induction at 10 uM [CHOP] (10μM**)Fold induction of mRNA expression of Target Gene at 10 uM concentration3String
33Standard Deviation at 10 uM [CHOP]Standard deviation of value of fold induction at 10 uM concentration3String
34Fold Induction at 5 uM [CHOP] (5μM**)Fold induction of mRNA expression of Target Gene at 5 uM concentration3String
35Standard Deviation at 5 uM [CHOP]Standard deviation of value of fold induction at 5 uM concentration3String
36Fold Induction at 2.5 uM [CHOP] (2.5μM**)Fold induction of mRNA expression of Target Gene at 2.5 uM concentration3String
37Standard Deviation at 2.5 uM [CHOP]Standard deviation of value of fold induction at 2.5 uM concentration3String
38Fold Induction at 1.25 uM [CHOP] (1.25μM**)Fold induction of mRNA expression of Target Gene at 1.25 uM concentration3String
39Standard Deviation at 1.25 uM [CHOP]Standard deviation of value of fold induction at 1.25 uM concentration3String
40Fold Induction at 0.6 uM] [CHOP] (0.6μM**)Fold induction of mRNA expression of Target Gene at 0.6 uM concentration3String
41Standard Deviation at 0.6 uM [CHOP]Standard deviation of value of fold induction at 0.6 uM concentration3String
42Fold Induction at 0.3 uM [CHOP] (0.3μM**)Fold induction of mRNA expression of Target Gene at 0.3 uM concentration3String
43Standard Deviation at 0.3 uM [CHOP]Standard deviation of value of fold induction at 0.3 uM concentration3String
44Fold Induction at 0.15 uM [CHOP] (0.15μM**)Fold induction of mRNA expression of Target Gene at 0.15 uM concentration3String
45Standard Deviation at 0.15 uM [CHOP]Standard deviation of value of fold induction at 0.15 uM concentration3String
46Outcome [BiP]One of Active, Inactive, or Not Tested4Heat shock protein 5 [Mus musculus]Outcome
47Fold Induction at 10 uM [BiP] (10μM**)Fold induction of mRNA expression of Target Gene at 10 uM concentration4String
48Standard Deviation at 10 uM [BiP]Standard deviation of value of fold induction at 10 uM concentration4String
49Fold Induction at 5 uM [BiP] (5μM**)Fold induction of mRNA expression of Target Gene at 5 uM concentration4String
50Standard Deviation at 5 uM [BiP]Standard deviation of value of fold induction at 5 uM concentration4String
51Fold Induction at 2.5 uM [BiP] (2.5μM**)Fold induction of mRNA expression of Target Gene at 2.5 uM concentration4String
52Standard Deviation at 2.5 uM [BiP]Standard deviation of value of fold induction at 2.5 uM concentration4String
53Fold Induction at 1.25 uM [BiP] (1.25μM**)Fold induction of mRNA expression of Target Gene at 1.25 uM concentration4String
54Standard Deviation at 1.25 uM [BiP]Standard deviation of value of fold induction at 1.25 uM concentration4String
55Fold Induction at 0.6 uM] [BiP] (0.6μM**)Fold induction of mRNA expression of Target Gene at 0.6 uM concentration4String
56Standard Deviation at 0.6 uM [BiP]Standard deviation of value of fold induction at 0.6 uM concentration4String
57Fold Induction at 0.3 uM [BiP] (0.3μM**)Fold induction of mRNA expression of Target Gene at 0.3 uM concentration4String
58Standard Deviation at 0.3 uM [BiP]Standard deviation of value of fold induction at 0.3 uM concentration4String
59Fold Induction at 0.15 uM [BiP] (0.15μM**)Fold induction of mRNA expression of Target Gene at 0.15 uM concentration4String
60Standard Deviation at 0.15 uM [BiP]Standard deviation of value of fold induction at 0.15 uM concentration4String
61Outcome [HO-1]One of Active, Inactive, or Not Tested5heme oxygenase 1 [Mus musculus]Outcome
62Fold Induction at 10 uM [HO-1] (10μM**)Fold induction of mRNA expression of Target Gene at 10 uM concentration5String
63Standard Deviation at 10 uM [HO-1]Standard deviation of value of fold induction at 10 uM concentration5String
64Fold Induction at 5 uM [HO-1] (5μM**)Fold induction of mRNA expression of Target Gene at 5 uM concentration5String
65Standard Deviation at 5 uM [HO-1]Standard deviation of value of fold induction at 5 uM concentration5String
66Fold Induction at 2.5 uM [HO-1] (2.5μM**)Fold induction of mRNA expression of Target Gene at 2.5 uM concentration5String
67Standard Deviation at 2.5 uM [HO-1]Standard deviation of value of fold induction at 2.5 uM concentration5String
68Fold Induction at 1.25 uM [HO-1] (1.25μM**)Fold induction of mRNA expression of Target Gene at 1.25 uM concentration5String
69Standard Deviation at 1.25 uM [HO-1]Standard deviation of value of fold induction at 1.25 uM concentration5String
70Fold Induction at 0.6 uM] [HO-1] (0.6μM**)Fold induction of mRNA expression of Target Gene at 0.6 uM concentration5String
71Standard Deviation at 0.6 uM [HO-1]Standard deviation of value of fold induction at 0.6 uM concentration5String
72Fold Induction at 0.3 uM [HO-1] (0.3μM**)Fold induction of mRNA expression of Target Gene at 0.3 uM concentration5String
73Standard Deviation at 0.3 uM [HO-1]Standard deviation of value of fold induction at 0.3 uM concentration5String
74Fold Induction at 0.15 uM [HO-1] (0.15μM**)Fold induction of mRNA expression of Target Gene at 0.15 uM concentration5String
75Standard Deviation at 0.15 uM [HO-1]Standard deviation of value of fold induction at 0.15 uM concentration5String

** Test Concentration. § Panel component ID.
Additional Information
Grant Number: 5 R21 NS056337-02

Classification
PageFrom: