Fluorescent Cell-Based Secondary Screen to Identify Activators of the Hypoxia Factor Pathway
Assay Overview: High content cell imaging assay on human osteosarcoma U2OS cells over-expression GFP-HIF1alpha to identify compounds inducing hypoxia inducible factor (HIF) expression and nuclear translocation. The small molecules inducing an increased nuclear fluorescent mediated by GFP-HIF1alpha will be considered as active. ..more
BioActive Compounds: 419
Keywords: nuclear translocation and GFP
Assay Overview: High content cell imaging assay on human osteosarcoma U2OS cells over-expression GFP-HIF1alpha to identify compounds inducing hypoxia inducible factor (HIF) expression and nuclear translocation. The small molecules inducing an increased nuclear fluorescent mediated by GFP-HIF1alpha 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 HIF expression and nuclear transcription. Compounds increasing the presence of GFP-HIF1alpha in the nucleus measured by fluorescent microscopy (average nuclear intensity) will be considered as actives.
U2OS HIF1a-GFP High Content Imaging Screen assay:
The U2OS-human HIF1a-GFP cell line (Thermo Scientific, Cat No.066_02) is propagated in DMEM media (Invitrogen, SKU No.11995) supplemented with 10% heat inactivated fetal bovine serum (Invitrogen, Cat. No. 16140089), 1% penicillin/streptomycin/glutamine (Invitrogen, 10378-016) and 0.5 mg/ml Geneticin (Invitrogen, 10131-027) at 37C in carbon dioxide incubators (Thermo Scientific) with 5% carbon dioxide, 21% oxygen, and 95% humidity. For High-Throughput Screening assays, cells are grown in T175 flask (BD Falcon, Ref No. 353112) or Hyperflasks (Corning, Cat No. 10010), harvested at more than 80% confluence using 7 or 75 ml Trypsin-EDTA 0.25% (Cellgro, Cat. No. 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 (Invitrogen, SKU No.11995) as mentioned above (for normal cell propagation) or DMEM medium without phenol (LONZA; Cat. No 12-917F) with 10% heat inactivated fetal bovine serum (Invitrogen, Cat. No. 16140089), 1% penicillin/streptomycin/glutamine (Invitrogen, 10378-016) without Geneticin (for compound screening). Cell number is counted using a Nexelcom Bioscience cell counter (Cellometer(r) 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 automation system (Cy-Bi Well) and microscope (ImageXpress, Molecular Devices) units.
Day 1 (Cell plating):
1. U2OS-HIF1a-GFP cells are harvested and resuspended in DMEM without phenol (LONZA, 12-917F) with 10% heat inactivated fetal bovine serum (Invitrogen, Cat. No. 16140089), 1% penicillin/streptomycin/glutamine (Invitrogen,10378-016) without Geneticin. U2OS HIF1a-GFP cells (from an initial cell suspension of 60,000 cells/ml) are dispensed using a MultiDrop Combi/Standard tube dispensing cassette (Thermo Scientific) in 384-well black with clear bottom tissue treated barcoded assay plates (Corning, Cat.No. 8816BC) at a final density of 2,000 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 37C in the Liconic carbon dioxide incubators (Automation system)(Liconic Instruments) calibrated at 5% carbon dioxide, 21% oxygen, and 95% humidity.
Day 2 (Cherry pick compound pinning into assay plate):
3. The MLPCN Cherry pick compounds plates are transferred from the Compound Management Walkup refrigerator to the CyBi-Well compound transfer unit. First, the base plate and the dose response plate are pinned once using 384 well pin tool (100 nl) and transferred to assay plate. The Cherry pick MLPCN test compounds plates ((1202 compounds), 8 concentrations, dilution factor of 3, starting concentration at 33 microM) are double pinned (2x100 nl) along with the in-plate positive control (32 wells, 200 uM Desferrioxamine DFX, Sigma-Aldrich Cat No.D9533, BRD-K09821361-066-08-4) and transferred into one assay plate. Finally, a second round of the base plate and the dose response plate pinning is achieved at the end of the run. Each compound plate is pinned twice in 2 different assay plates (duplicate).
4. After the pinning has occurred, the dose response, base and MLPCN cherry pick plates are returned into Compound Management Walkup refrigerator. The assay plates treated with compounds are moving back to Liconic carbon dioxide incubator to be incubated for an additional 24 hours.
Day 3 (Plate processing and reading assay plates on microscope):
5. The assay plates are washed twice with PBS (50 ul) and fixed with 2% formaldehyde in water for 20 minutes at room temperature. The assay plates are washed twice again and resuspended in 50 ul PBS with 40 ug/ml DAPI (Invitrogen, D3571). The assay plates are physically transferred to the microscope (ImageXpress, Molecular Devices) where the reading is occurring. Pictures are taken at magnification of 10X and images are processed using MetaXpress software. The average nuclear intensity was used as readout for this assay.
Dose Data Analysis
Neutral control wells (NC) and positive controls wells (PC) were included on every plate.
Active compounds result in increased readout signal.
Normalization and Pattern Correction
The raw signal was normalized using the 'Stimulators Minus Neutral Controls' method in Genedata Assay Analyzer (v7.0.3):
The median raw signal of the intraplate PC wells is set to a normalized activity value of 100.
The median raw signal of the intraplate NC wells is set to a normalized activity of 0.
The raw signal of each compound well is then scaled to these two reference points to obtain a normalized activity value.
Plate patterns were corrected using the 'Runwise Pattern (Multiplicative)' algorithm in Genedata Assay Analyzer (v7.0.3).
EC50 values were calculated using the curve fitting strategies in Genedata
Screener Condoseo (7.0.3). EC50 values were extrapolated up to 1 log
over the highest tested concentration.
PubChem Activity Score and Outcome
Inactive compounds = 0
Active compounds = -10*Log(EC50)
Activity_Outcome = 1 (inactive)
EC50 > 1 log over the highest tested concentration
Activity_Outcome = 2 (active)
EC50 <= 1 log over the highest tested concentration
Activity_Outcome = 3 (inconclusive)
Curve fitting strategy resulted in a constant fit with activity >30% but <70%
The fit was not valid due to poor fit quality.
* Activity Concentration. ** Test Concentration.
Data Table (Concise)