HIF pathway Measured in Cell-Based System Using RT-PCR - 2030-04_Activator_Dose_DryPowder_Activity
Assay Overview: Real time-PCR assay on human osteosarcoma U2OS cells to identify compounds inducing mRNA expression of VEGF, a downstream target of the HIF pathway. The small molecules inducing a lower cp than the negative control (DMSO) will be considered as active. ..more
BioActive Compounds: 33
Keywords: VEGF, metal chelator
Assay Overview: Real time-PCR assay on human osteosarcoma U2OS cells to identify compounds inducing mRNA expression of VEGF, a downstream target of the HIF pathway. The small molecules inducing a lower cp than the negative control (DMSO) 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 the mRNA expression downstream target HIF pathway such as VEGF. Compounds increasing the mRNA level of human VEGF presence (lower cp value) will be considered as actives.
RT-PCR VEGF protocol
The Reverse transcriptase-Polymerase Reaction Chain (RT-PCR) was carried out using the human osteosarcoma U2OS cell line. The U2OS (ATCC, HTB-96) cell line is propagated in DMEM media (Invitrogen, SKU#11995) supplemented with 10% heat inactivated fetal bovine serum (Denville Scientific Inc, Cat# FB5001-H) and 1% penicillin/streptomycin/glutamine (Invitrogen, 10378-016) at 37 degrees 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) with 10% Heat inactivated fetal bovine serum (Invitrogen, Cat# 16140089), 1% penicillin/streptomycin/glutamine (Invitrogen, 10378-016). 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 cells are harvested and resuspended in DMEM without phenol red (Cambrex, 12-917F) with 10% Heat inactivated fetal bovine serum (Denville Scientific Inc, Cat# FB5001-H) and 1% penicillin/streptomycin/glutamine (Invitrogen, 10378-016). U2OS cells (from an initial cell suspension of 40,000 cells/ml) are dispensed using a MultiDrop Combi/Standard tube dispensing cassette (Thermo Scientific) in white bottom 384 well assay plates (Corning, Cat# 8867BC) at a final density of 2,000 cells per well in final volume of 50uL.
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. The dose response compound plates are pinned twice into one assay plate (duplicate). Pins are washed with methanol and DMSO between each pinning.
4. After the pinning has occured, the assay plates treated with compounds are moved back to Liconic CO2 incubator to be incubated for an additional 24 hours.
Day 3 (RT-PCR):
1. Cell to CT lysis
The medium is aspirated and the cells are washed twice (100 uL with PBS) using the ELX405 Plate washer (Biotek). The assay plates are flipped upside down and centrifuged at 1000 rpm 2 minutes to remove the excess liquid. 10 uL of Lysis solution with DNase I (Ambion, from Cell to CT Lysis Mix) is added to each well using the MultiDrop Combi/Standard tube dispensing cassette (Thermo Scientific). Each assay plate is then vortexed for 2 minutes and incubated for an additional 5 minutes at room temperature. At the end of the incubation, the assay plates are centrifuged (1000 rpm, 2 minutes) and 1 uL of stop solution is added with the Multidrop Combi-nl (Thermo Scientific). The assay plate are incubated for 2 minutes at room temperature and processed for reverse transcription.
2. Reverse transcription (RT) (Ambion, Cell to CT RT Mix#4402958)
2X RT Buffer, 5 uL
20X RT Enzyme Mix, 0.5 uL
Nuclease-Free Water, 2.5 uL
8 uL of RT is dispensed into each well of a RT assay plate (Axygen, PCR-384 RGD C). 2 uL of the lysed cells are transferred into RT assay plate using Vario transfer unit (CyBi Well). The RT assay plates are incubated at 37 degrees C for 1 hour and the reverse transcriptase inactivated by incubating the plates for 1 minute at 95 degrees C (8 uL RT Master Mix/10 uL total reaction volume).
4 uL PCR Master Mix/5uL total volume of reaction
2X Roche Master Mix (Probes Master), 2.5 uL
20X FAM Taqman probe/primers set, 0.125 uL
(human VEGF-A, Applied Biosystems 4331182, Hs00900057_m1 inventoried FAM-MGB)
20X VIC Taqman probe/primers set, 0.125 uL
(human actin-beta, Applied Biosystems 4310881E Vic-TAMRA)
PCR H2O, 0.125 uL
4 uL/well of PCR master mix is dispensed in PCR plate (Roche Light Cycler 480 MultiWell Plate 384, Cat# 04 729 749 001) using the Multidrop Combi-nl (Thermo Scientific).
Then, 1 uL/well of RT DNA is transferred in the 4 uL/well PCR plate. The PCR plates are centrifuged 2 minutes at 1000 rpm.
PCR is performed using ThermoCycler (Roche Light Cycler 480 II) with this setup:
1. 95 degrees C, 10 minutes
2. 95 degrees C, 10 seconds
3. 60 degrees C, 30 seconds
Step 2 and 3 (40 cycles)
PRESENCE OF CONTROLS: Neutral control wells (NC) and positive control wells (PC) were included on every plate.
EXPECTED OUTCOME: Active compounds result in increasing readout signal.
ACTIVE CONCENTRATION LIMIT:
For each sample, the highest valid tested concentration (HVC) was determined and the active concentration limit (AC_limit) was set to equal (10)(HVC).
The raw signals of the plate wells were normalized using the 'Stimulators Minus Neutral Controls' method in Genedata Assay Analyzer (v7.0.3):
The median raw signal of the intraplate neutral control wells was set to a normalized activity value of 0.
The median raw signal of the intraplate positive control wells was set to a normalized activity value of 100.
Experimental wells values were scaled to this range.
PATTERN CORRECTION: The plate pattern correction algorithm 'Runwise Pattern (Multiplicative)' in Genedata (v7.0.3) was applied to the normalized plate data.
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.
Activity_Outcome = 1 (inactive) when:
a) curve fit is constant where activity is > -30% and < 30% at all tested concentrations, or
b) AC > AC_limit, or
c) 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
Activity_Outcome = 2 (active) when:
AC <= AC_limit
Activity_Outcome = 3 (inconclusive) when:
a) Curve fitting strategy resulted in a constant fit with activity >= 30% but <= 70%, or
b) The fit was not valid due to poor fit quality.
PUBCHEM_ACTIVITY_SCORE = 0 when PUBCHEM_ACTIVITY_OUTCOME = 1 (inactive) or 3 (inconclusive)
PUBCHEM_ACTIVITY_SCORE = (-10)[Log(AC)], where AC is in molar, when PUBCHEM_ACTIVITY_OUTCOME = 2 (active)
Scores relate to AC in this manner:
120 = 1 pM
90 = 1 nM
60 = 1 uM
30 = 1 mM
0 = 1 M
PUBCHEM_ACTIVITY_SCORE = 100 when the curve fit is constant and showing full inhibition at all tested concentrations.
Categorized Comment - additional comments and annotations
* Activity Concentration. ** Test Concentration.
Data Table (Concise)