HSF-1 induced GFP reporter and Doxycycline induced RFP reporter Measured in Cell-Based System Using Plate Reader - 2038-03_Inhibitor_DoseNoFile_CherryPick_Internal-Standard_Set3
Assay Overview: Modified NIH3T3, transformed to express GFP under the control of a HSF-1 response element, and RFP under the control of a Doxycycline inducible (Tet-On) promoter will be exposed to small molecules. Upon addition of small molecules, Doxycycline is added to wells to induce RFP expression, and cells are exposed to a 2hr thermal heat-shock at 42oC to elicit a stress response and resulting GFP expression. After 48hr incubation, the amount fluorescence of each of these markers is measured using an Envision plate reader. ..more
BioActive Compounds: 362
Keywords: Heat Shock Factor-1 (HSF-1), Stress Response, NIH3T3, GFP, RFP
Assay Overview: Modified NIH3T3, transformed to express GFP under the control of a HSF-1 response element, and RFP under the control of a Doxycycline inducible (Tet-On) promoter will be exposed to small molecules. Upon addition of small molecules, Doxycycline is added to wells to induce RFP expression, and cells are exposed to a 2hr thermal heat-shock at 42oC to elicit a stress response and resulting GFP expression. After 48hr incubation, the amount fluorescence of each of these markers is measured using an Envision plate reader.
This assay identifies inhibitors of HSF-1 in those instances where there is a selective loss of GFP signal due to the prevention of HSF-1 being able to drive the expression of the GFP reporter. These selective inhibitors of HSF-1 should not interfere with the Doxycycline induced RFP expression. Additionally, this assay also identifies those compounds which act as broad spectrum inhibitors of transcription/translation, which will cause a decrease in expression of both GFP and RFP reporters.
Protocol: R4-3T3 GFP/RFP Dual Reporter assay:
The R4-3T3 cell line is modified version of NIH3T3 fibroblasts with an integrated GFP construct under the control of a heat shock response element, and a RFP construct under the control of a doxycycline inducible promoter. The R4-3T3 cell line was generously provided for this study by Dr. Luke Whitesell.
The R4-3T3 cell line is propagated in Opti-MEM (Invitrogen, cat# 31985-088) supplemented with 2.5% heat inactivated fetal bovine serum (FBS) (Invitrogen, cat# 16140089), 1% penicillin/streptomycin/glutamine (Invitrogen, cat# 10378-016), and 200microg/mL G418 (Gibco, cat# 10131) at 37 degrees C in CO2 incubators (Thermo Scientific) with 5% CO2, 21% O2, and 95% humidity. For High-Throughput Screening (HTS) assays, cells are grown in T225 flask (BD Falcon, cat# 353138) or Hyperflasks (Corning, cat# 10010), harvested at more than 80% confluence using Accumax cell detachment solution (Innovative Cell Technologies, cat# AM105). Cell number is counted using a Cellometer Auto M10 cell counter (Nexelcom Bioscience) 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 General system (GS) automation unit:
Day 1 (Cell plating):
1. R4-3T3 cells are harvested and re-suspended in Opti-MEM with 2.5% Heat inactivated FBS, 1% penicillin/streptomycin/glutamine. R4-3T3 cells (from an initial cell suspension of 1x106 cells/ml) are dispensed using a MultiDrop Combi/Standard tube dispensing cassette (Thermo Scientific) in Black clear-bottom 384 well assay plates (Corning, cat# 3985BC) at a final density of 15,000 cells per well in a volume of 30 uL. The cells are kept in suspension using a magnetic bar and a stirrer during the dispensing.
2. The assay plates (cell plates) are incubated for 24 hours at 37 degrees C in incubator calibrated to 5% CO2, 21% O2, and 95% humidity.
Day 2 (Compound pinning into assay plate):
3. Doxycycline is added to each assay plate at a concentration of 8microg/mL (4X) in 10microL of Opti-MEM with 2.5% Heat inactivated FBS, 1% penicillin/streptomycin/glutamine using a MultiDrop Combi/Standard tube dispensing cassette. After this addition, each assay plate has a final volume of 40microL Opti-MEM with 2microg/mL Doxycycline.
4. MLPCN test compounds as well as the in-plate positive control (160iM Didemnin B (DdB), BRD-K58977490-001-01-0, 32 wells, 400nM final conc.) plates are pinned consecutively one after the other and transferred into one assay plate. Each compound plate is pinned into duplicate assay plates.
5. After addition of compounds, the assay plates are incubated for a 2hr at 42 degrees C to induce HSF-1. After this incubation at elevated temperature, the plates are returned to 37 degrees C to incubate for a further 48hrs.
(Reading fluorescence from assay plates with Envision):
6. After 48 hr incubation, fluorescence is measured in each assay plate using Envision plate reader (Perkin Elmer). GFP is measured by bottom-read with a 485nm excitation filter, 505nm dichroic mirror, and 535nm emission filter. RFP measured by bottom-read with 550nm excitation filter, 555nm dichroic mirror, and 595nm emission filter.
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 Minus Inhibitors' 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 (Additive)' 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.
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.
* Activity Concentration. ** Test Concentration.
Data Table (Concise)