Hypoxia inducible factor pathway Iron Measured in Cell-Based System Using Plate Reader - 2030-06_Activator_Dose_DryPowder_Toxicity
Keywords: Hypoxia, Hypoxia Inducible Factor (HIF), Hypoxia Responsive Element (HRE), luciferase, transcriptional activation, tissue regeneration, ischemia ..more
BioActive Compounds: 29
Keywords: Hypoxia, Hypoxia Inducible Factor (HIF), Hypoxia Responsive Element (HRE), luciferase, transcriptional activation, tissue regeneration, ischemia
Assay Overview: Luciferase assay (Steady-Glo, Promega).
Primary screen using human osteosarcoma U2OS cells stably over-expressing a plasmid containing 3 copies of the Hypoxia Responsive Element linked to luciferase gene (U2OS HRE-luciferase cells) to identify small molecules inducing an increased luciferase activity in these cells. The small molecules inducing expression of luciferase under HRE promoter will be measured by the conversion of luciferin to oxyluciferin and light (luminescence) mediated by the luciferase.
Expected Outcome: Most known HIF inducers are metal chelators. In this experiment, we are testing the ability of these HIF inducer compounds to chelate metal(s) (zinc or iron). For compounds that are metal chelators, the dose response curve generated by these compounds should shift to the right in the medium exposed to a metal (zinc or iron) versus the medium without metal. Compounds where their absolute values (abscissa values, ABSCNNs) are crossing the linear portion of the majority of the compound dose response curves (EC50) is shifting by 10 fold or more to the right and show a reduction of the maximum activity (Sinf) of at least 2 fold will be considered iron and/or zinc chelators. Compounds showing no toxicity (no IC50) in a cell viability test (Celltiter-Glo, Promega) but shifting to the right the ABSCNN value (10 fold) in the gene reporter luciferase assay will be considered as iron and/or zinc chelators.
U2OS HRE-luciferase assay:
The U2OS-HRE-luc cell line was originally obtained from Dr. Margaret Ashcroft's lab and kindly provided by Dr. Shawn Gilbert.
The U2OS-HRE-luc cell line is propagated in DMEM media (Invitrogen, SKU# 11995) supplemented with 10% heat inactivated fetal bovine serum (Invitrogen, Cat# 16140089), 1% penicillin/streptomycin/glutamine (Invitrogen, 10378-016) and 0.1 mg/ml Hygromycin B (Invitrogen, 10687-01) at 37degrees 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 or Invitrogen cat# 31053) with 10% Heat inactivated fetal bovine serum (Invitrogen, Cat# 16140089), 1% penicillin/streptomycin/glutamine (Invitrogen, 10378-016), sodium pyruvate (only used with DMEM without phenol red from Invitrogen (Cat# 11306-070)) and without hygromycin B (for compound screening). 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-HRE-luc cells are harvested and resuspended in DMEM without phenol red (Cambrex, 12-917F) with 10% Heat inactivated fetal bovine serum (Invitrogen, 16140089), 1% penicillin/streptomycin/glutamine (Invitrogen,10378-016) without hygromycin B. U2OS HRE-luciferase cells (from an initial cell suspension of 120,000 cells/ml) are dispensed using a MultiDrop Combi/Standard tube dispensing cassette (Thermo Scientific) in white bottom 384 well assay plates (Corning, Cat.No. 8867BC) at a final density of 6,000 cells per well in final volume of 50 uL. The cells are kept in suspension using a magnetic bar and a stirrer during the dispensing.
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. Before pinning the compounds, 10 ul of 2.5mM Ferric Nitrate (Sigma, F-1143) was added to each well to a final concentration of 50 uM. The dose response compound plate (8 concentrations, 3 fold dilution, starting concentration 7.5 mM) with the positive control (Desferrioxamine, 80 mM)(32 wells spread across the plate) are pinned twice using 384 well pin tool (100 nl) on pin table (Walkup Cybi Well) and transferred to assay plate. Pins are washed with methanol and DMSO between each pinning.
4. The assay plates treated with compounds are moving back to Liconic CO2 incubator to be incubated for an additional 24 hours.
Day 3 (Reading luminescence from assay plates with Envision):
5. Each assay plate is pulled out of the incubator and cooled down at room temperature for 30 minutes. 30 uL/well (384 well) of Steady-Glo luciferase reagent 0.5X (diluted in H2O) (Promega, E2550) is dispensed using the the MultiDrop Combi dispensing cassette from Thermo Scientific. For the toxicity (cell viability) readout, 30 uL/well (384 well) of Celltiter-Glo luciferase reagent 0.5X (diluted in H2O) (Promega, G7573) is dispensed using the the MultiDrop Combi dispensing cassette from Thermo Scientific. The assay plate returned to room temperature for 30 minutes to allow a complete cellular lysis.
6. Luminescence is measured (0.2 second/well) using the ultra sensitive luminescence detector (384-well aperture, 0.5 mm height) in each well using the Envision plate reader (Perkin Elmer)(Corning plate setting).
7. HRE activation is calculated based on the following equation using mean luminescence (RLU) values:
Fold Activation = (Treatment - Background)/(No treatment - Background)
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 decreasing 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 (10)(Max_Concentration).
The raw signals of the plate wells were normalized using the 'Neutral Controls' method in Genedata Assay Analyzer (v7.0.3):
The median raw signal of the intraplate neutral controls (NC) is set to a normalized activity value of 0.
A normalized activity value of 100 is defined as (2)(NC).
A normalized activity value of -50 is defined as (0.5)(NC).
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): AC50
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)
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 >= -70% but <= -30%, or
b) The fit was deemed not valid due to poor fit quality.
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.
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
* Activity Concentration. ** Test Concentration.
Data Table (Concise)