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BioAssay: AID 1907

Luminescence-based confirmation cell-based assay for cytotoxic compounds using the IEC-6 intestinal epithelial cell line.

Name: Luminescence-based confirmation cell-based assay for cytotoxic compounds using the IEC-6 intestinal epithelial cell line. ..more
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 Tested Compounds
 Tested Compounds
All(1923)
 
 
Active(1221)
 
 
Inactive(702)
 
 
 Tested Substances
 Tested Substances
All(1923)
 
 
Active(1221)
 
 
Inactive(702)
 
 
 Related BioAssays
 Related BioAssays
AID: 1907
Data Source: The Scripps Research Institute Molecular Screening Center (IEC6CYTOX_INH_LUMI_1536_3X%INH CRUN)
BioAssay Type: Primary, Primary Screening, Single Concentration Activity Observed
Depositor Category: NIH Molecular Libraries Probe Production Network
BioAssay Version:
Deposit Date: 2009-08-21
Modify Date: 2010-06-16

Data Table ( Complete ):           View Active Data    View All Data
BioActive Compounds: 1221
Related Experiments
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AIDNameTypeComment
1700Primary cell-based high throughput screening assay to identify inhibitors of kruppel-like factor 5 (KLF5)Screeningdepositor-specified cross reference: Primary screen for KLF5 inhibitors.
1825Luminescence-based counterscreen assay for KLF5 inhibitors: cell-based high throughput screening assay to identify cytotoxic compounds using the IEC-6 intestinal epithelial cell line.Screeningdepositor-specified cross reference: Counterscreen for KLF5 inhibitors.
1858Summary of probe development efforts to identify inhibitors of kruppel-like factor 5 (KLF5)Summarydepositor-specified cross reference: Summary AID.
1972Luminescence-based dose response cell-based high throughput screening assay for cytotoxic compounds using the IEC-6 intestinal epithelial cell line.Confirmatorydepositor-specified cross reference
588725Late stage counterscreen results from the probe development effort to identify inhibitors of kruppel-like factor 5 (KLF5): Radioactivity-based biochemical assay to identify modulators of a panel of 48 kinasesOtherdepositor-specified cross reference
588729Late stage counterscreen results from the probe development effort to identify inhibitors of kruppel-like factor 5 (KLF5): Absorbance-based cell-based assay to identify modulators of cancer cell viabilityConfirmatorydepositor-specified cross reference
1834Luminescence-based confirmation cell-based high throughput screening assay to identify inhibitors of kruppel-like factor 5 (KLF5)Screeningsame project related to Summary assay
1905Luminescence-based counterscreen assay for KLF5 inhibitors: cell-based high throughput screening assay to identify cytotoxic compounds using the IEC-6 intestinal epithelial cell line in triplicate.Screeningsame project related to Summary assay
1973Luminescence-based dose response cell-based high throughput screening assay for inhibitors of kruppel-like factor 5 (KLF5).Confirmatorysame project related to Summary assay
1975Luminescence-based counterscreen assay for KLF5 inhibitors: dose response cell-based high throughput screening assay to identify cytotoxic compounds using the IEC-6 intestinal epithelial cell line.Confirmatorysame project related to Summary assay
2749Late stage counterscreen results from the probe development effort to identify inhibitors of kruppel-like factor 5 (KLF5): luminescence-based cell-based dose response assay for cytotoxic compounds using the IEC-6 intestinal epithelial cell lineConfirmatorysame project related to Summary assay
2750Late stage results from the probe development effort to identify inhibitors of kruppel-like factor 5 (KLF5): luminescence-based cell-based dose response assay for inhibitors of KLF5Confirmatorysame project related to Summary assay
434956Late stage counterscreen results from the probe development effort to identify inhibitors of kruppel-like factor 5 (KLF5): luminescence-based cell-based dose response assay for cytotoxic compounds using the IEC-6 intestinal epithelial cell line (Round 1)Confirmatorysame project related to Summary assay
434957Late stage results from the probe development effort to identify inhibitors of kruppel-like factor 5 (KLF5): luminescence-based cell-based dose response assay for inhibitors of KLF5 (Round 1)Confirmatorysame project related to Summary assay
485336Late stage assay provider counterscreen results from the probe development effort to identify inhibitors of kruppel-like factor 5 (KLF5): chemiluminescence-based western blot assay for inhibitors of KLF5 protein levelsConfirmatorysame project related to Summary assay
485338Late stage assay provider counterscreen results from the probe development effort to identify inhibitors of kruppel-like factor 5 (KLF5): luminescence-based cell-based dose response assay for cytotoxic compounds using the DLD-1 cell line (Round 1)Confirmatorysame project related to Summary assay
588539Late stage assay provider counterscreen results for the probe development effort to identify inhibitors of kruppel-like factor 5 (KLF5): Cell cycle analysis of the DLD-1 cell lineOthersame project related to Summary assay
588610Late stage assay provider counterscreen results from the probe development effort to identify inhibitors of kruppel-like factor 5 (KLF5): chemiluminescence-based western blot assay for inhibitors of Epidermal Growth Factor Receptor (EGFR) protein levels in DLD1 cellsOthersame project related to Summary assay
588612Late stage assay provider counterscreen results from the probe development effort to identify inhibitors of kruppel-like factor 5 (KLF5): chemiluminescence-based western blot assay for inhibitors of Phosphorylated Mitogen-Activated Protein Kinase 1 (P-MAPK1; P-ERK1/2) protein levels in DLD1 cellsOthersame project related to Summary assay
588613Late stage assay provider counterscreen results from the probe development effort to identify inhibitors of kruppel-like factor 5 (KLF5): chemiluminescence-based western blot assay for inhibitors of Phosphorylated Epidermal Growth Factor Receptor (EGFR) protein levels in DLD1 cells.Othersame project related to Summary assay
588614Late stage assay provider counterscreen results from the probe development effort to identify inhibitors of kruppel-like factor 5 (KLF5): chemiluminescence-based western blot assay for inhibitors of Mitogen-Activated Protein Kinase 1 (MAPK1; ERK1/2) protein levels in DLD1 cellsOthersame project related to Summary assay
588615Late stage assay provider counterscreen results from the probe development effort to identify inhibitors of kruppel-like factor 5 (KLF5): chemiluminescence-based western blot assay for inhibitors of P38 protein levels in DLD1 cellsOthersame project related to Summary assay
588616Late stage assay provider counterscreen results from the probe development effort to identify inhibitors of kruppel-like factor 5 (KLF5): chemiluminescence-based western blot assay for inhibitors of phospho-P38 protein levels in DLD1 cellsOthersame project related to Summary assay
588617Late stage assay provider counterscreen results from the probe development effort to identify inhibitors of kruppel-like factor 5 (KLF5): chemiluminescence-based western blot assay for inhibitors of KLF5 protein levels in DLD1 cellsOthersame project related to Summary assay
588618Late stage assay provider counterscreen results from the probe development effort to identify inhibitors of kruppel-like factor 5 (KLF5): chemiluminescence-based western blot assay for inhibitors of Early Growth Response 1 (EGR1) protein levels in DLD1 cellsOthersame project related to Summary assay
588640Late stage assay provider counterscreen results from the probe development effort to identify inhibitors of kruppel-like factor 5 (KLF5): chemiluminescence-based western blot assay for inhibitors of P38 protein levels in HCT116 human colorectal carcinoma cellsOthersame project related to Summary assay
588642Late stage assay provider counterscreen results from the probe development effort to identify inhibitors of kruppel-like factor 5 (KLF5): chemiluminescence-based western blot assay for inhibitors of Mitogen-Activated Protein Kinase 1 (MAPK1; ERK1/2) protein levels in HCT116 human colorectal carcinoma cellsOthersame project related to Summary assay
588643Late stage assay provider counterscreen results from the probe development effort to identify inhibitors of kruppel-like factor 5 (KLF5): chemiluminescence-based western blot assay for inhibitors of Phosphorylated Mitogen-Activated Protein Kinase 1 (P-MAPK1; P-ERK1/2) protein levels in HCT116 human colorectal carcinoma cellsOthersame project related to Summary assay
588644Late stage assay provider counterscreen results from the probe development effort to identify inhibitors of kruppel-like factor 5 (KLF5): chemiluminescence-based western blot assay for inhibitors of Early Growth Response 1 (EGR1) protein levels in HCT116 human colorectal carcinoma cellsOthersame project related to Summary assay
588646Late stage assay provider counterscreen results from the probe development effort to identify inhibitors of kruppel-like factor 5 (KLF5): chemiluminescence-based western blot assay for inhibitors of phosphorylated P38 protein levels in HCT116 human colorectal carcinoma cellsOthersame project related to Summary assay
588648Late stage assay provider counterscreen results from the probe development effort to identify inhibitors of kruppel-like factor 5 (KLF5): chemiluminescence-based western blot assay for inhibitors of Epidermal Growth Factor Receptor (EGFR) protein levels in HCT116 human colorectal carcinoma cellsOthersame project related to Summary assay
588650Late stage assay provider counterscreen results from the probe development effort to identify inhibitors of kruppel-like factor 5 (KLF5): chemiluminescence-based western blot assay for inhibitors of Phosphorylated Epidermal Growth Factor Receptor (EGFR) protein levels in HCT116 human colorectal carcinoma cellsOthersame project related to Summary assay
588652Late stage assay provider counterscreen results from the probe development effort to identify inhibitors of kruppel-like factor 5 (KLF5): chemiluminescence-based western blot assay for inhibitors of KLF5 protein levels in HCT116 human colorectal carcinoma cellsOthersame project related to Summary assay
588653Late stage assay provider counterscreen results from the probe development effort to identify inhibitors of kruppel-like factor 5 (KLF5): chemiluminescence-based western blot assay for inhibitors of Phosphorylated Epidermal Growth Factor Receptor (EGFR) protein levels in HT29 human colorectal carcinoma cellsOthersame project related to Summary assay
588654Late stage assay provider counterscreen results from the probe development effort to identify inhibitors of kruppel-like factor 5 (KLF5): chemiluminescence-based western blot assay for inhibitors of Epidermal Growth Factor Receptor (EGFR) protein levels in HT29 human colorectal carcinoma cellsOthersame project related to Summary assay
588655Late stage assay provider counterscreen results from the probe development effort to identify inhibitors of kruppel-like factor 5 (KLF5): chemiluminescence-based western blot assay for inhibitors of Phosphorylated Mitogen-Activated Protein Kinase 1 (P-MAPK1; P-ERK1/2) protein levels in HT29 human colorectal carcinoma cellsOthersame project related to Summary assay
588656Late stage assay provider counterscreen results from the probe development effort to identify inhibitors of kruppel-like factor 5 (KLF5): chemiluminescence-based western blot assay for inhibitors of Mitogen-Activated Protein Kinase 1 (MAPK1; ERK1/2) protein levels in HT29 human colorectal carcinoma cellsOthersame project related to Summary assay
588657Late stage assay provider counterscreen results from the probe development effort to identify inhibitors of kruppel-like factor 5 (KLF5): chemiluminescence-based western blot assay for inhibitors of Early Growth Response 1 (EGR1) protein levels in HT29 human colorectal carcinoma cellsOthersame project related to Summary assay
588658Late stage assay provider counterscreen results from the probe development effort to identify inhibitors of kruppel-like factor 5 (KLF5): chemiluminescence-based western blot assay for inhibitors of phosphorylated P38 protein levels in HT29 human colorectal carcinoma cellsOthersame project related to Summary assay
588659Late stage assay provider counterscreen results from the probe development effort to identify inhibitors of kruppel-like factor 5 (KLF5): chemiluminescence-based western blot assay for inhibitors of P38 protein levels in HT29 human colorectal carcinoma cellsOthersame project related to Summary assay
588660Late stage assay provider counterscreen results from the probe development effort to identify inhibitors of kruppel-like factor 5 (KLF5): chemiluminescence-based western blot assay for inhibitors of KLF5 protein levels in HT29 human colorectal carcinoma cellsOthersame project related to Summary assay
588661Late stage assay provider counterscreen results from the probe development effort to identify inhibitors of kruppel-like factor 5 (KLF5): chemiluminescence-based western blot assay for inhibitors of KLF5 protein levels in SW620 human colorectal carcinoma cellsOthersame project related to Summary assay
588662Late stage assay provider counterscreen results from the probe development effort to identify inhibitors of kruppel-like factor 5 (KLF5): chemiluminescence-based western blot assay for inhibitors of P38 protein levels in SW620 human colorectal carcinoma cellsOthersame project related to Summary assay
588663Late stage assay provider counterscreen results from the probe development effort to identify inhibitors of kruppel-like factor 5 (KLF5): chemiluminescence-based western blot assay for inhibitors of phosphorylated P38 protein levels in SW620 human colorectal carcinoma cellsOthersame project related to Summary assay
588665Late stage assay provider counterscreen results from the probe development effort to identify inhibitors of kruppel-like factor 5 (KLF5): chemiluminescence-based western blot assay for inhibitors of Early Growth Response 1 (EGR1) protein levels in SW620 human colorectal carcinoma cellsOthersame project related to Summary assay
588666Late stage assay provider counterscreen results from the probe development effort to identify inhibitors of kruppel-like factor 5 (KLF5): chemiluminescence-based western blot assay for inhibitors of Mitogen-Activated Protein Kinase 1 (MAPK1; ERK1/2) protein levels in SW620 human colorectal carcinoma cellsOthersame project related to Summary assay
588667Late stage assay provider counterscreen results from the probe development effort to identify inhibitors of kruppel-like factor 5 (KLF5): chemiluminescence-based western blot assay for inhibitors of Epidermal Growth Factor Receptor (EGFR) protein levels in SW620 human colorectal carcinoma cellsOthersame project related to Summary assay
588668Late stage assay provider counterscreen results from the probe development effort to identify inhibitors of kruppel-like factor 5 (KLF5): chemiluminescence-based western blot assay for inhibitors of Phosphorylated Mitogen-Activated Protein Kinase 1 (P-MAPK1; P-ERK1/2) protein levels in SW620 human colorectal carcinoma cellsOthersame project related to Summary assay
588677Late stage assay provider counterscreen results from the probe development effort to identify inhibitors of kruppel-like factor 5 (KLF5): chemiluminescence-based western blot assay for inhibitors of Phosphorylated Epidermal Growth Factor Receptor (EGFR) protein levels in SW620 human colorectal carcinoma cellsOthersame project related to Summary assay
Description:
Source (MLPCN Center Name): The Scripps Research Institute Molecular Screening Center
Affiliation: The Scripps Research Institute, TSRI
Assay Provider: Vincent Yang, Emory University
Network: Molecular Library Probe Production Centers Network (MLPCN)
Grant Proposal Number 1-R03-DA026215-01
Grant Proposal PI: Vincent Yang
External Assay ID: IEC6CYTOX_INH_LUMI_1536_3X%INH CRUN

Name: Luminescence-based confirmation cell-based assay for cytotoxic compounds using the IEC-6 intestinal epithelial cell line.

Description:

Transcription factors are essential regulators of transcription that bind DNA to control both the rate and frequency of gene expression (1). Many diseases of cell homeostasis are associated with aberrant transcription factor activity (2). Colon cancer, in particular, is a disease of uncontrolled proliferation of the epithelial cells that line the intestinal crypts. Kruppel-like factor 5 (KLF5) is a zinc finger-containing transcription factor that binds to GC-rich sequences in promoters of numerous genes (3) including cyclin D1 (4), cyclin B1/Cdc2 (4), and integrin-linked kinase (5). KLF5 is highly expressed in rapidly dividing epithelial cells in intestinal crypts (6). This expression pattern of KLF5, along with studies demonstrating that KLF5 mediates the transforming effects of oncogenic H-Ras (7), and that ectopic expression of KLF5 leads to increased cell proliferation and anchorage-independent growth of cultured intestinal epithelial cells (8, 9), suggest that KLF5 may be involved in colon cancer pathogenesis. Therefore, the identification of selective inhibitors of KLF5 may provide useful tools to elucidate the role of KLF5 as a regulator of cellular proliferation and tumor formation in the intestinal epithelium.

References:

1. Ptashne M. Regulation of transcription: from lambda to eukaryotes. Trends Biochem Sci. 2005 Jun;30(6):275-9.
2. Fre S, Vignjevic D, Schoumacher M, Duffy SL, Janssen KP, Robine S, Louvard D. Adv Cancer Res. 2008;100:85-111. Epithelial morphogenesis and intestinal cancer: new insights in signaling mechanisms.
3. Goldstein BG, Chao HH, Yang Y, Yermolina YA, Tobias JW, Katz JP. Am J Physiol Gastrointest Liver Physiol. 2007 Jun;292(6):G1784-92. Overexpression of Kruppel-like factor 5 in esophageal epithelia in vivo leads to increased proliferation in basal but not suprabasal cells.
4. Ghaleb AM, Nandan MO, Chanchevalap S, Dalton WB, Hisamuddin IM, Yang VW. Kruppel-like factors 4 and 5: the yin and yang regulators of cellular proliferation. Cell Res. 2005 Feb;15(2):92-6.
5. Yang Y, Tetreault MP, Yermolina YA, Goldstein BG, Katz JP. Kruppel-like factor 5 controls keratinocyte migration via the integrin-linked kinase. J Biol Chem. 2008 Jul 4;283(27):18812-20.
6. McConnell BB, Ghaleb AM, Nandan MO, Yang VW. The diverse functions of Kruppel-like factors 4 and 5 in epithelial biology and pathobiology. Bioessays. 2007 Jun;29(6):549-57. Erratum in: Bioessays. 2007 Sep;29(9):946.
7. Nandan MO, Yoon HS, Zhao W, Ouko LA, Chanchevalap S, Yang VW. Kruppel-like factor 5 mediates the transforming activity of oncogenic H-Ras. Oncogene. 2004 Apr 22;23(19):3404-13.
8. Chanchevalap S, Nandan MO, Merlin D, Yang VW. FEBS Lett. 2004 Dec 3;578(1-2):99-105. All-trans retinoic acid inhibits proliferation of intestinal epithelial cells by inhibiting expression of the gene encoding Kruppel-like factor 5.
9. Sun R, Chen X, Yang VW. J Biol Chem. 2001 Mar 9;276(10):6897-900. Intestinal-enriched Kruppel-like factor (Kruppel-like factor 5) is a positive regulator of cellular proliferation.

Keywords:

IEC-6, intestinal epithelial cells, cytotoxicity, cell viability, KLF5, BTEB2, kruppel-like factor 5, cancer, confirmation, HTS, high throughput screen, 1536, inhibitor, inhibition, luciferase, luminescence, CellTiter Glo, Scripps, Scripps Florida, The Scripps Research Institute Molecular Screening Center, SRIMSC, Molecular Libraries Probe Production Centers Network, MLPCN.
Protocol
Assay Overview:
The purpose of this assay is to confirm IEC-6 cell cytotoxicity of compounds tested in a previous set of experiments entitled, "Luminescence-based counterscreen assay for KLF5 inhibitors: cell-based high throughput screening assay to identify cytotoxic compounds using the IEC-6 intestinal epithelial cell line," (PubChem AID 1825). In this assay, non-transformed rat IEC-6 epithelial cells are incubated with tests compounds, followed by determination of cell viability. The assay utilizes the CellTiter-Glo luminescent reagent (Promega) to measure intracellular ATP in viable cells. Luciferase present in the reagent catalyzes the oxidation of beetle luciferin to oxyluciferin and light in the presence of cellular ATP. Well luminescence is directly proportional to ATP levels and cell viability. As designed, compounds that reduce cell viability will reduce ATP levels, luciferin oxidation and light production, resulting in decreased well luminescence. Test compounds were assayed in triplicate at a final nominal concentration of 5 micromolar.
Protocol Summary:
The parental IEC-6 cell line was routinely cultured in T-175 sq cm flasks at 37 degrees C and 95% relative humidity (RH). The growth media consisted of RPMI -1640 supplemented with 10% v/v certified fetal bovine
serum, 2 micrograms/ml human recombinant insulin, and 1X antibiotic mix (penicillin, streptomycin, and neomycin).
Prior to the start of the assay 1250 cells in a 5 microliter volume of growth media were dispensed into each well of 1536-well tissue culture-treated microtiter plates. The assay was started immediately by dispensing 20 nL of test compound in DMSO (0.4 % final DMSO concentration), DMSO alone, or doxorubicin (150 micromolar final concentration) to the appropriate wells. Next, the plates were incubated for 48 hours at 37 degrees C (5% CO2, 95% RH). After equilibrating the plates to room temperature for 30 minutes, the assay was stopped by dispensing 5 microliters of CellTiter-Glo reagent to each well, followed by incubation at room temperature for 15 minutes. Well luminescence was measured on the ViewLux plate reader.
The percent inhibition for each compound was calculated as follows:
% Inhibition = ( 1 - ( ( Test_Compound - Median_High_Control ) / ( Median_Low_Control - Median__High_Control ) ) ) * 100
Where:
Test_Compound is defined as wells containing test compound.
Low_Control is defined as the wells containing DMSO.
High_Control is defined as wells containing doxorubicin.
Any compound that exhibited a percent inhibition greater than the hit cutoff calculated for the original screen was declared active. The reported PubChem Activity Score has been normalized to 100% observed primary inhibition. Negative % inhibition values are reported as activity score zero.
The activity score range for active compounds is 100-21, for inactive 20-0.
List of Reagents:
IEC-6 cell line (provided by Assay Provider)
DMEM medium (Invitrogen, part 11995-065)
100X Penicillin-Streptomycin-Neomycin mix (Invitrogen, part 15640-055)
Human, recombinant insulin (Invitrogen, part 12585-014)
Trypsin-EDTA solution (Invitrogen, part 25200-056)
Fetal Bovine Serum (Invitrogen, part 16000-044)
Cell Titer Glo (Promega, part G75729)
Doxorubicin (Sigma Chemical, part D1515)
T-175 tissue culture flasks (Corning, part 431080)
1536-well plates (Greiner, part 789173)
Comment
Due to the increasing size of the MLPCN compound library, this assay may have been run as two or more separate campaigns, each campaign testing a unique set of compounds. In this case the results of each separate campaign were assigned "Active/Inactive" status based upon that campaign's specific compound activity cutoff value. All data reported were normalized on a per-plate basis. In this assay doxorubicin had an IC50 of approximately 100 nM. Possible artifacts of this assay can include, but are not limited to: dust or lint located in or on wells of the microtiter plate, compounds that non-specifically modulate luciferase activity, and compounds that quench or emit luminescence within the well. All test compound concentrations reported are nominal; the specific concentration for a particular test compound may vary based upon the actual sample provided by the MLSMR. The MLSMR was not able to provide all compounds selected for testing in this AID.
Categorized Comment - additional comments and annotations
From PubChem:
Assay Format: Cell-based
Assay Cell Type: IEC-6
Result Definitions
TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1Inhibition (5μM**)Normalized percent inhibition of the confirmation screen at a compound concentration of 5 micromolar.Float%
2Standard DeviationStandard deviation of the confirmation assay derived from the normalized percent inhibition of the triplicate data for each compound.Float

** Test Concentration.
Additional Information
Grant Number: 1-R03-DA026215-01

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