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BioAssay: AID 743238

High Content Cell-Based Imaging Primary HTS to Identify Small Molecules Involved in X-Chromosome Reactivation and Reprogramming Measured in Cell-Based System Using Imaging - 7015-01_Activator_SinglePoint_HTS_Activity_Set2

In the mammalian female, nearly all genes on one of the two X chromosomes are transcriptionally silenced by X chromosome inactivation (XCI), the mechanism of dosage compensation that equalizes gene expression in XX females and XY males. In the inner cell mass of peri-implantation embryos, however, the inactivated X chromosome can be reactivated. In this assay, we aim to identify small molecule more ..
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 Tested Compounds
 Tested Compounds
All(359270)
 
 
Active(199)
 
 
Inactive(358649)
 
 
Inconclusive(432)
 
 
 Tested Substances
 Tested Substances
All(362415)
 
 
Active(199)
 
 
Inactive(361784)
 
 
Inconclusive(432)
 
 
 Related BioAssays
 Related BioAssays
AID: 743238
Data Source: Broad Institute (7015-01_Activator_SinglePoint_HTS_Activity_Set2)
BioAssay Type: Primary, Primary Screening, Single Concentration Activity Observed
Depositor Category: NIH Molecular Libraries Probe Production Network
Deposit Date: 2013-12-20

Data Table ( Complete ):           Active    All
BioActive Compounds: 199
Depositor Specified Assays
AIDNameTypeComment
743246Broad Institute A high throughput cell-based phenotypic screen for X-reactivation and reprogramming by small molecules Activator Probe Projectsummary
Description:
Keywords: X-Chromosome reactivation, Gene dosage, lncRNA

Assay Overview:
High content imaging assay (GFP)
In the mammalian female, nearly all genes on one of the two X chromosomes are transcriptionally silenced by X chromosome inactivation (XCI), the mechanism of dosage compensation that equalizes gene expression in XX females and XY males. In the inner cell mass of peri-implantation embryos, however, the inactivated X chromosome can be reactivated. In this assay, we aim to identify small molecule reactivators of a GFP reporter gene on the inactive X chromosome. This primary HTS is a high content screen using fibroblast cells in which GFP is under the control of the promoter on the inactive X (Xi) gene (XiGFP). The cells are immortalized with SV40 large T antigen. The fibroblast XiGFP cells will be exposed to compounds for 3 days before being imaged using a high content microscope (ImageXpress Micro automated microscope (Molecular Devices)). The re-induction of the inactive X gene by small molecules will be determined by calculating the percentage of GFP positive cells.

Expected Outcome:
Identify small molecule increasing the percentage of GFP positive cells. Compounds increasing the percentage of GFP positive by at least 10% in both replicates compared to the neutral and positive controls will be considered active hits. Compounds increasing the percentage by 10% in one of the two replicates will be considered inconclusive hits. Compounds increasing less than 10% in both replicates will be considered inactive hits.
Protocol
Protocol

Day 1:
- Plate 500 68-5-11 cells (provided by Dr. Derek Lessing from Jeannie Lee's lab at Massachusetts General Hospital, Boston MA) in 50 ml per well in DMEM media in presence of 5-aza-dC (Cayman Chemicals, 11166) (final conc. 0.5 mM)(5-aza-dc should theoretically sensitize cells to X-chromosome reactivator compounds).

Day 2
- Pin compounds (100 nl) and positive control Ellipticine (topisomerase inhibitor) (6uM final concentration) (Enzo Life Sciences, BML-GR315).

Day 5
- Aspirate media.
- Fix cells and stain nucleus by adding 50 ul PBS with 4% formaldehyde and Hoechst 33342 (Life technologies, H3570)(dilution 1:5000 (10 mg/ml))
- Incubate 15min at room temperature.
- Aspirate formaldehyde/Hoechst and wash fixed cells once with PBS (50ul) using plate washer
- Add 50ul PBS with Plate washer.

- Image cells on high content microscope (Molecular Devices, MetaXpress software)
analysis: number of Hoechst-positive total cells
and % of GFP-positive cells (nuclear).

DMEM media recipe:

500 mL DMEM (high glucose, glutamax, pyruvate) - Life Technologies 10569-044
60 mL Fetal Bovine Serum - Gibco, 16410
15 mL HEPES - Life Technologies 15630-130
6 mL MEM Non-essential amino acids - Life Technologies 11140-076
6 mL Pen/Strep/Glutamine: Life technologies: 16140-071
1 mL 2-Mercaptoethanol - Life Technologies 21985-023
Comment
PRESENCE OF CONTROLS: Neutral control wells (NC) and positive control wells (PC) were included on every plate.

EXPECTED OUTCOME: Active compounds result in increasing readout signal.

NORMALIZATION:
The raw signals of the plate wells were normalized using the 'Stimulators Minus Neutral Controls' 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: The plate pattern correction algorithm 'Runwise Pattern (Multiplicative)' in Genedata (v10.0.2) was applied to the normalized plate data.

PUBCHEM_ACTIVITY_SCORE:
This was set as equal to the mean of the normalized and corrected sample replicate activities, rounded to the nearest integer .
The minimum PUBCHEM_ACTIVITY_SCORE required for a compound to be called a hit (the activity threshold, or AT) was set at 10.

PERCENTAGE OF ACTIVE REPLICATES:
For each sample, the percentage of replicates (PCT_ACTIVE_REP) which had activity scores >= AT was determined.
The minimum percentage of replicates required for a compound to be called a hit (PAR_T) was set at 100.

PUBCHEM_ACTIVITY_OUTCOME:
Samples passing BOTH threshold criteria were assigned an outcome of 2 (active):
PUBCHEM_ACTIVITY_SCORE >= AT, and PCT_ACTIVE_REP >= PAR_T

Samples passing NEITHER threshold criteria were assigned an outcome of 1 (inactive):
PUBCHEM_ACTIVITY_SCORE < AT, and PCT_ACTIVE_REP < PAR_T

Samples passing AT only were assigned an outcome of 3 (inconclusive) :
PUBCHEM_ACTIVITY_SCORE >= AT, and PCT_ACTIVE_REP < PAR_T

Samples passing PAR_T only were assigned an outcome of 3 (inconclusive) :
PUBCHEM_ACTIVITY_SCORE < AT, and PCT_ACTIVE_REP >= PAR_T
Result Definitions
TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1REPRODUCIBILITY_COSINE_TRANSFORMA measure of how well the activity reproduced across the two samples. Computed as the absolute value of the cosine between the 'replicate vector' (ScoreA, ScoreB ---as well as ScoreC and/or ScoreD where applicable) and the vector (1, 1) representing perfect reproducibility. NULL will appear in this column if a sample was not run in duplicate or if the data produced by one of the replicates was Invalid# Float
2PCT_ACTIVE_REPLICATESThe percentage of replicates which pass the activity threshold.Float
3REPLICATE_A_ACTIVITY_SCORE_7.58uM_(%) (7.58μM**)The calculated activity for the indicated sampleFloat%
4REPLICATE_B_ACTIVITY_SCORE_7.58uM_(%) (7.58μM**)The calculated activity for the indicated sampleFloat%

** Test Concentration.
Additional Information
Grant Number: 1 R03 MH097478-01

Data Table (Concise)
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