Sustained Induction of HSF-1 Measured in Cell-Based System Using Plate Reader - 2038-07_Activator_Dose_CherryPick_Activity
Confirmation testing of small molecules originally identified in PubChem Bioassay AID 2098 as increasing expression of a firefly luciferase under the control of a HSF-1 response element in modified NIH3T3 cells. This assay sought to address 3 specific aims. ..more
BioActive Compounds: 194
Keywords: Heat Shock Factor-1 (HSF-1), Induction, Stress Response, NIH3T3, Luminescence
Confirmation testing of small molecules originally identified in PubChem Bioassay AID 2098 as increasing expression of a firefly luciferase under the control of a HSF-1 response element in modified NIH3T3 cells. This assay sought to address 3 specific aims.
i.Are these compounds capable of long-term (48hr) induction of a HSF-1 reporter, compared to the shorter interval (8hr) originally assayed.
ii.Are these compounds capable of inducing a HSF-1 reporter as single agents, since the proteosome inhibitor, MG132, was present in the original protocol.
iii.Are these compounds capable of inducing HSF-1 reporter without inducing cell toxicity.
Cells expressing luciferase under the control of a HSF-1 response element were exposure to test compounds for 48hr. After 48hr incubation, the amount of HSF-1 mediated luciferase expression is measured using a luminescence detection reagent.
Identification of HSF-1 inducers in those instances where there is an increase of luminescence signal due to the enhancement of HSF-1 being able to drive the expression of the luciferase reporter. Compounds which do not, or fail to sustain induction of HSF-1 should show no increase in luciferase activity relative to untreated controls. Compounds which induce cell toxicity should a decrease in luciferase activity relative to sub-toxic doses and/or a decrease relative to basal level expression in untreated controls. Potential false positives in this assay included those compounds which act not by specifically inducing HSF-1, but by stabilizing luciferase or are broad spectrum inducers of transcription/translation.
Sustained Induction of HSF-1/Cell Toxicity Assay:
The NIH3T3-HGL cell line is modified version of NIH3T3 fibroblasts with an integrated eGFP-Firefly luciferase fusion construct under the control of a heat shock response element. The NIH3T3-HGL cell line was generously provided for this study by Dr. Luke Whitesell.
The HGL 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 was carried out on the Broad Institute/Chemical Biology Platform Walk-Up Instruments:
HGL cells are harvested and re-suspended in Opti-MEM with 2.5% Heat inactivated FBS, 1% penicillin/streptomycin/glutamine. HGL cells (from an initial cell suspension of 133,333 cells/ml) are dispensed using a MultiDrop Combi/Standard tube dispensing cassette (Thermo Scientific) in white bottom 384 well assay plates (Corning) at a final density of 4,000 cells per well in final volume of 40 uL. The cells are kept in suspension using a magnetic bar and a stirrer during the dispensing.
The assay plate (cell plate) are placed in Thermo 3110 Incubator and incubated overnight at 37 degrees C, 5% CO2, 21% O2, and 95% humidity.
Test compounds plates are pinned into duplicate assay plates using the 100nL pin array. In addition to test compounds, Doxorubicin (10uM final concentration), was pinned into designated wells within each assay plate as a positive control.
After pinning, the plates are returned to 37 degrees C and incubated for a further 48 hours.
(Reading luminescence from assay plates with Envision):
After 48 hour incubation, 40uL of Steady-Glo Luciferase (Promega) diluted 1:8 in PBS is added to each assay plate using the MultiDrop Combi/long tubing dispensing cassette (Thermo Scientific). Luminescence is measured in each well (0.1 second/well, Corning plate setting) using an Envision plate reader (Perkin Elmer).
PRESENCE OF CONTROLS: Neutral control wells (NC; n=36) 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 (Max_Concentration) was determined and the active concentration limit (AC_limit) was set to equal 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 (Multiplicative)' in Genedata (v7.0.3) was applied to the normalized plate data.
MEASUREMENT USED TO DETERMINE ACTIVE CONCENTRATION (AC): absACnn, the concentration at which the curve crosses threshold 40.0
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 >= 30% but <= 70%, 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
Assay Format: Cell-based
Assay Cell Type: NIH 3T3
Assay Type: Functional
* Activity Concentration. ** Test Concentration.
Data Table (Concise)