HSF-1 induced GFP reporter and Doxycycline induced RFP reporter Measured in Cell-Based System Using Plate Reader - 2038-03_Inhibitor_Dose_DryPowder_Activity
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: 5
Keywords: Heat Shock Factor-1 (HSF-1), Stress Response, NIH3T3, GFP, RFP
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-media (Invitrogen) supplemented with 5% heat inactivated fetal bovine serum (Invitrogen), 1% penicillin/streptomycin/glutamine 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 T225 flask (BD Falcon) or Hyperflasks (Corning), harvested at more than 80% confluence using Accumax (Innovative Cell Technologies). Cell number is counted using a Nexelcom Bioscience cell counter (Cellometer Auto M10) and viability is measured by mixing cells 1:1 with a 0.4% Trypan Blue solution (Sigma). Only cultures of >94% viability are utilized for experiments.
Compound Screening is carried out within the Broad Institute/Chemical Biology Platform.
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) 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.
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.
(Compound pinning into assay plate):
Doxycycline is added to each assay plate at a concentration of 8ug/mL (4X) in 10uL 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 40uL Opti-MEM with 2microg/mL Doxycycline.
Test compounds plates are pinned into duplicate assay plates using the 100nL pin array. In addition to test compounds, Didemnin B (400nM final concentration), was pinned into designated wells within each assay plate as a positive control.
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):
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 excititation filter, 505nm dichroic mirror, and 535nm emission filter. RFP measured by bottom-read with 550nm excitiation filter, 555nm dichroic mirror, and 595nm emission filter.
PRESENCE OF CONTROLS: Neutral control wells (NC; n=104) and positive control wells (PC; n=20) were included on every plate.
EXPECTED OUTCOME: Active compounds result in decreasing readout signal.
The compounds were assayed in multiple independent instances using an identical protocol; each instance is called a 'test'. For each test, the following analysis was applied:
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 (2)(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: 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.
Once the data for each test was processed, the test number was appended to all column headers in that test's data set. The individual test results were then aggregated as follows:
1. The final PUBCHEM_ACTIVITY_OUTCOME was set to:
a. '2' (active) when all test outcomes were '2' (active), or
b. '1' (inactive) when all test outcomes were '1' (inactive), or
c. '3' (inconclusive) when the test outcomes were mixed.
2. The final ACTIVE_CONCENTRATION (AC) was set as follows:
a. If the final PUBCHEM_ACTIVITY_OUTCOME = 2, AC was set as the mean of the constituent test active concentrations;
b. If the final PUBCHEM_ACTIVITY_OUTCOME = 1 or 3, AC was left empty.
3. The final PUBCHEM_ACTIVITY_SCORE was calculated based on the aggregated ACTIVE_CONCENTRATION, using the same logic described above for individual test scores.
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)