Luminescence Cell-Based Dose Retest to Identify Potentiators of Heat Shock Factor 1 (HSF1)
Assay Overview: Confirmation testing of small molecules identified as increasing expression of a firefly luciferase under the control of a HSF-1 response element in modified NIH3T3 cells. For consistency with the primary assay in which these compounds were identified, after 30min exposure to small molecules, proteasome inhibitor MG132 is added to elicit a stress response. After an additional 8hr incubation, the amount of HSF-1 mediated luciferase expression is measured using a luminescence detection reagent. ..more
BioActive Compounds: 514
Keywords: Heat Shock Factor-1 (HSF-1), Stress Response, MG132, NIH3T3, Luminescence
Assay Overview: Confirmation testing of small molecules identified as increasing expression of a firefly luciferase under the control of a HSF-1 response element in modified NIH3T3 cells. For consistency with the primary assay in which these compounds were identified, after 30min exposure to small molecules, proteasome inhibitor MG132 is added to elicit a stress response. After an additional 8hr incubation, the amount of HSF-1 mediated luciferase expression is measured using a luminescence detection reagent.
Expected Outcome: 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. 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, or act solely through interaction with MG132 to potentiate stress response.
HGL HSF-1 luciferase 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 Luke Whitesell.
The HGL cell line is propagated in Opti-MEM (Invitrogen, cat 31985-088) supplemented with 5% heat inactivated fetal bovine serum (FBS) (Invitrogen, cat 16140089), 1% penicillin/streptomycin/glutamine (Invitrogen, cat 10378-016) at 37C 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. 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 200,000 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 4,000 cells per well in final volume of 20uL. 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 CO2 incubator 9 (General automation system (GS)) (Liconic Instruments) calibrated at 5% CO2, 21% O2, and 95% humidity.
Day 2 (Compound pinning into assay plate):
3.The MLPCN test compounds plates are transferred from the Compound Management incubators STX1000 1 and 2 system (Liconic Instruments) on the GS to the Liconic CO2 incubator 8 before the initiation of the pinning run. The In-plate positive control compound plate (sentinel) (25uM Rocaglamide A (RocA), Alexis Biochemicals ALX-350-121-C100), or vehicle (DMSO only) are already present in the Liconic incubator 8 at the beginning of the pinning. Both STX and Liconic 8 CO2 incubator temperature are kept at 20C. MLPCN test compounds plates are pinned as well as the in-plate positive control (32 wells, 50nM final conc. RocA) are pinned consecutively one after the other and transferred into one assay plate. Each compound plate is pinned into duplicate assay plates. The final concentration for the MLPCN test compounds is 7.5uM with final concentration no more than 1% DMSO.
4. After 30min incubation with the compounds, 50M MG132 (Enzo, cat PI-102) in 1X PBS (Invitrogen, cat 10010) is added in 1L with the CombinL (Thermo) (2.5uM final conc. MG132) to induce HSF-1. The cells are incubated in the presence of MG132 for a further 8hrs.
(Reading luminescence from assay plates with Envision):
5. After 8 hr incubation, 20L of Steady-Glo luciferase (Promega, cat E2550) is added to each assay plate using a MultiDrop Combi (Thermo). Luminescence is measured in each well (0.1 second/well) using the Envision plate reader (Perkin Elmer)(Corning plate setting).
Dose Data Analysis
Neutral control (NC) wells were included on every plate.
Active inducer compounds result in increased signal.
The raw signals of the plate wells were normalized using the 'Neutral Controls' method in Genedata Assay Analyzer (v7.0.3):
The median raw signal of the intraplate NC wells was set to a normalized activity value of 0.
A normalized activity of -50% was set to equal (0.5)(median raw NC).
A normalized activity of 100% was set to equal (2)(median raw NC).
Experimental wells were scaled to this range, giving an activity score as percent change in signal relative to the intraplate controls.
The plate pattern correction algorithm 'Runwise Pattern (Multiplicative)' from Genedata Condoseo (v7.0.3) was applied.
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.
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)