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BioAssay: AID 1825

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.

Name: 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. ..more
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AID: 1825
Data Source: The Scripps Research Institute Molecular Screening Center (IEC6CYTOX_INH_LUMI_1536_%INH)
BioAssay Type: Primary, Primary Screening, Single Concentration Activity Observed
Depositor Category: NIH Molecular Libraries Probe Production Network
Deposit Date: 2009-06-18

Data Table ( Complete ):           View Active Data    View All Data
BioActive Compounds: 2261
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 to identify inhibitors of kruppel-like factor 5 (KLF5).
1858Summary of probe development efforts to identify inhibitors of kruppel-like factor 5 (KLF5)Summarydepositor-specified cross reference
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.Screeningdepositor-specified cross reference
1907Luminescence-based confirmation cell-based assay for cytotoxic compounds using the IEC-6 intestinal epithelial cell line.Screeningdepositor-specified cross reference
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
2201Summary of probe development efforts to identify cytotoxic compounds using the IEC-6 intestinal epithelial cell line.Summarydepositor-specified cross reference
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 lineConfirmatorydepositor-specified cross reference
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 KLF5Confirmatorydepositor-specified cross reference
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)Confirmatorydepositor-specified cross reference
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)Confirmatorydepositor-specified cross reference
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 levelsConfirmatorydepositor-specified cross reference
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)Confirmatorydepositor-specified cross reference
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 lineOtherdepositor-specified cross reference
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 cellsOtherdepositor-specified cross reference
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 cellsOtherdepositor-specified cross reference
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.Otherdepositor-specified cross reference
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 cellsOtherdepositor-specified cross reference
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 cellsOtherdepositor-specified cross reference
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 cellsOtherdepositor-specified cross reference
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 cellsOtherdepositor-specified cross reference
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 cellsOtherdepositor-specified cross reference
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 cellsOtherdepositor-specified cross reference
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 cellsOtherdepositor-specified cross reference
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 cellsOtherdepositor-specified cross reference
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 cellsOtherdepositor-specified cross reference
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 cellsOtherdepositor-specified cross reference
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 cellsOtherdepositor-specified cross reference
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 cellsOtherdepositor-specified cross reference
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 cellsOtherdepositor-specified cross reference
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 cellsOtherdepositor-specified cross reference
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 cellsOtherdepositor-specified cross reference
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 cellsOtherdepositor-specified cross reference
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 cellsOtherdepositor-specified cross reference
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 cellsOtherdepositor-specified cross reference
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 cellsOtherdepositor-specified cross reference
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 cellsOtherdepositor-specified cross reference
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 cellsOtherdepositor-specified cross reference
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 cellsOtherdepositor-specified cross reference
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 cellsOtherdepositor-specified cross reference
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 cellsOtherdepositor-specified cross reference
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 cellsOtherdepositor-specified cross reference
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 cellsOtherdepositor-specified cross reference
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 cellsOtherdepositor-specified cross reference
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 cellsOtherdepositor-specified cross reference
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 cellsOtherdepositor-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
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
1834Luminescence-based confirmation cell-based high throughput screening assay to identify inhibitors of kruppel-like factor 5 (KLF5)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
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_%INH

Name: 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.

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. Kr#ppel-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. Epub 2001 Jan 10. 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, primary screen, counterscreen, 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 identify compounds that are cytotoxic to non-transformed IEC-6 rat intestinal epithelial cells. This assay also serves as a counterscreen to a set of previous experiments entitled, "Primary cell-based high throughput screening assay to identify inhibitors of kruppel-like factor 5 (KLF5)," (PubChem AID 1700). In this assay, rat IEC-6 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 singlicate at a final nominal concentration of 5 uM.

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 25 nL of test
compound in DMSO (0.5 % 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 median value of all test compound wells.
High_Control is defined as wells containing doxorubicin.

For this uHTS counterscreen, a mathematical algorithm was used to determine nominally inhibiting compounds. Two values were calculated for each assay plate: (1) the average percent inhibition of test compound wells and (2) three times their standard deviation. The sum of these two values was used as a cutoff parameter for each plate, i.e. any compound that exhibited greater % inhibition than that particular plate's cutoff parameter was declared active. The reported PubChem Activity Score has been normalized to 100% of the highest observed primary inhibition value. Negative % inhibition values are reported as activity score zero.

The inactive compounds of this assay have activity score range of 0 to 20 and active compounds range of activity score is 21 to 100.

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.
Result Definitions
TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1Inhibition (5μM**)Normalized percent inhibition of the counterscreen screen at a compound concentration of 5 micromolar.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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