HTS for PAX8 inhibitors using PAX8 luciferase reporter gene assay in RMG-I cells Measured in Cell-Based System Using Plate Reader - 7054-01_Inhibitor_Dose_CherryPick_Activity
A cellular assay that measures Pax8 activity using a Luciferase-based reporter of a well characterized PAX8 transcriptional target, thyroperoxidase (TPO), was used in the primary assay. A 420 bp fragment of the rat TPO promoter that was sensitive to Pax8 levels in RMG-I ovarian cancer cells was stably introduced to create a reporter cell line (RMG-1:TPO-Luc)with a biologically-relevant context. Cells were treated with compound and controls for 24 hours. Following compound treatment, TPO promoter activity (via luciferase light production) was measured using Promega SteadyGlo. ..more
BioActive Compounds: 1635
Keywords: ovarian cancer, Pax8, luciferase, SteadyGlo, RMG-1 cells, thyroperoxidase
A cellular assay that measures Pax8 activity using a Luciferase-based reporter of a well characterized PAX8 transcriptional target, thyroperoxidase (TPO), was used in the primary assay. A 420 bp fragment of the rat TPO promoter that was sensitive to Pax8 levels in RMG-I ovarian cancer cells was stably introduced to create a reporter cell line (RMG-1:TPO-Luc)with a biologically-relevant context. Cells were treated with compound and controls for 24 hours. Following compound treatment, TPO promoter activity (via luciferase light production) was measured using Promega SteadyGlo.
Expected Outcome: If a compound inhibits Pax8 function, there will be less TPO expression in RMG-1 cells and this will lead to a decrease in luciferase signal.
PAX8 Primary Assay Protocol
Cell maintenance and passaging:
RMG-I-Pax8Luc cells: Stably express a Pax8-Luciferase reporter that contains a portion of the thyroperoxidase (TPO) gene promoter. Cells are grown in phenol red free RPMI-1640 media supplemented with 10% FBS (Sigma, F4135), Hygromycin (75 ug/mL) and 1X Pen/Strep (Gibco 15140-122). Cells are grown in a humidified incubator, 37 degrees C, 5% CO2.
Upon thawing, cells are allowed to recover for at least 1 week before any experimental procedure. When flasks reach ~80% confluent, cells are passaged, at a 1:4 ratio:
2.Briefly wash with PBS.
3.Add Trypsin (0.25%,Gibco 25200-056)
4.Place flask in incubator for 2-3 minutes.
5.Stop trypsin by adding serum containg medium. Transfer cells to a 50 or 250 mL conical tube. Centrifuge 5 minutes at 1200 rpm.
6.Re-plate 25% of the cells into a new plate containing fresh medium.
Note: The cells are very sensitive to long incubation with trypsin or to insufficient dilution of trypsin in FBS-containing medium.
Important: Medium needs to be replaced every 2-3 days between passages (Avoid yellow color of medium, highly acidic).
Day 1: Preparing cells for experiment:
Cells are trypsinized as stated above, centrifuged, aspirate trypsin/media, resuspended in fresh media and counted. Cells are diluted in fresh medium to a final concentration of 167,000 cells/ml. Cells are dispensed into barcoded 384 well plates (Corning 8867 white, opaque), 30 ul/well (5000 cells/well) using a Thermo-Fisher Multidrop Combi and a standard Combi cassette.
Day 2: Treatment with compounds:
100 nL of compounds or controls are added to the plates by pinning method. The positive control used is 2 uM mitoxantrone (BRD-K21680192-001-01-5), a known cytotoxin that will reduce signal of luciferase in the reporter assay due to loss of cells.
Day3: SteadyGlo Luciferase assay:
Measure luciferase activity 24h after compound pinning. Steady-Glo solution (Promega E2550 for assay development and med. chem experiments; X1006 pre-made solution for the HTS) is added using a Thermo-Fisher Multidrop Combi, 10 ul/well (standard cassette at medium speed). Plates are shaken briefly and allowed to incubate for 10 minutes at room temperature. Luminescence levels are read on a Perkin-Elmer EnVision plate reader, using the USLum (ultra-sensitive luminescence) protocol. (Time= 0.5s/well)
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.
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 (v10.0.2):
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.10.0.2) 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
Assay Cell Type: RMG-I
Assay Format: Cell-based
Assay Type: Functional
* Activity Concentration. ** Test Concentration.
Data Table (Concise)