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BioAssay: AID 588739

High Content Assay for Compounds that inhibit the Assembly of the Perinucleolar Compartment:Soft Agar Assay

The perinucleolar compartment (PNC) is a non-membrane-bound nuclear subdomain that is associated with but structurally distinct from the nucleolus. The PNC is a heritable trait, in which the number of PNCs per cell in daughter cells often mimics that of their mother cells. The PNC is heterogeneous in shape, is stable through interphase, disassembles during mitosis, and reassembles in early G1. more ..
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 Tested Compounds
 Tested Compounds
All(1)
 
 
Unspecified(1)
 
 
 Tested Substances
 Tested Substances
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Unspecified(1)
 
 
AID: 588739
Data Source: NCGC (PNC347)
Depositor Category: NIH Molecular Libraries Probe Production Network
Deposit Date: 2011-10-30
Hold-until Date: 2012-10-27
Modify Date: 2012-10-27

Data Table ( Complete ):           View All Data
Target
Tested Compound:
Related Experiments
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AIDNameTypeComment
2431High Content Assay for Compounds that inhibit the Assembly of the Perinucleolar Compartment: SummarySummarydepositor-specified cross reference
2417High Content Assay for Compounds that inhibit the Assembly of the Perinucleolar CompartmentScreeningsame project related to Summary assay
2729High Content Assay for Compounds that inhibit the Assembly of the Perinucleolar Compartment: WI-38 CytotoxicityConfirmatorysame project related to Summary assay
2730High Content Assay for Compounds that inhibit the Assembly of the Perinucleolar Compartment: Confirmation of PNC InhibitionConfirmatorysame project related to Summary assay
2731High Content Assay for Compounds that inhibit the Assembly of the Perinucleolar Compartment: PC3M Caspase 3/7 ActivationConfirmatorysame project related to Summary assay
2733High Content Assay for Compounds that inhibit the Assembly of the Perinucleolar Compartment: PC3M CytotoxicityConfirmatorysame project related to Summary assay
2734High Content Assay for Compounds that inhibit the Assembly of the Perinucleolar Compartment: DNA Intercalation/PicoGreen Displacement AssayConfirmatorysame project related to Summary assay
540362PC3M Cytotoxicity Assay for Compounds that Inhibit the Perinuclear CompartmentConfirmatorysame project related to Summary assay
540363PC3M Cytotoxicity Assay for Compounds that Inhibit the Perinuclear Compartment: Chemistry Optimization FollowupConfirmatorysame project related to Summary assay
588722High Content Assay for Compounds that inhibit the Assembly of the Perinucleolar Compartment:Cell MigrationOthersame project related to Summary assay
588738High Content Assay for Compounds that inhibit the Assembly of the Perinucleolar Compartment:Cell Migration SAROthersame project related to Summary assay
588740PC3M Cytotoxicity Assay for Compounds that Inhibit the Perinuclear Compartment: SARConfirmatorysame project related to Summary assay
602443High Content Assay for Compounds that inhibit the Assembly of the Perinucleolar Compartment: Aqueous Kinetic SolubilityOthersame project related to Summary assay
602444High Content Assay for Compounds that inhibit the Assembly of the Perinucleolar Compartment: Mouse Liver Microsome Stability without NADPHOthersame project related to Summary assay
602445High Content Assay for Compounds that inhibit the Assembly of the Perinucleolar Compartment: Mouse Liver Microsome Stability with NADPHOthersame project related to Summary assay
602446High Content Assay for Compounds that inhibit the Assembly of the Perinucleolar Compartment: Efflux RatioOthersame project related to Summary assay
Description:
The perinucleolar compartment (PNC) is a non-membrane-bound nuclear subdomain that is associated with but structurally distinct from the nucleolus. The PNC is a heritable trait, in which the number of PNCs per cell in daughter cells often mimics that of their mother cells. The PNC is heterogeneous in shape, is stable through interphase, disassembles during mitosis, and reassembles in early G1. The PNC is concentrated with newly synthesized RNA polymerase III (pol III) RNAs (MRP RNA, RNAse P H1 RNA, hY RNAs[hY1, 2, 5], Alu RNA, and SRP [7SL] RNA) and RNA binding proteins (nucleolin, PTB, CUG-BP, KSRP, Raver1, Raver2, and Rod). Continuous transcription by pol III is necessary for the structural integrity of the PNC and some protein components have been shown to bind RNAs within the PNC implicating involvement of PNCs in pol III RNA metabolism. Chemical biology studies have demonstrated that PNC maintenance is dependent on the integrity of DNA, specifically DNA base pairing, and cell biology investigations demonstrated that the PNC is nucleated upon a DNA locus, indicating that the components of the PNC are most likely directly interacting with the DNA locus.

In collaboration between Sui Huang from Northwestern University and the NCGC, a high content assay was developed and screened. The hits were then tested in follow-up assays. In vitro cellular transformation detection assays are semi-quantitative and measure the morphological transformation of cell colonies modulated by chemicals. This transformation is associated with certain phenotypic changes such as loss of contact inhibition (cells can grow over one another) and anchorage independence (cells form colonies in soft agar). Anchorage independence can be described in the light of primary fibroblasts and many other cell lines (e.g. BALB/c3T3, NIH-3T3, etc.) that must attach to a solid surface before they can divide. They fail to grow when suspended in a viscous fluid or gel (e.g. agar or agarose), however when these cell lines are transformed, they are able to grow in a viscous fluid or gel and become anchorage-independent. This process, by which these phenotypic changes occur, is assumed to be closely related, and hence a good predictor, of in vivo carcinogenesis.

NIH Chemical Genomics Center [NCGC]
NIH Molecular Libraries Probe Centers Network [MLPCN]

MLPCN Grant: MH082371
Assay Submitter (PI): Sui Huang, Northwestern University, Feinberg School of Medicine, Cell and Molecular Biology, Chicago, Illinois.
Protocol
PC3M cells were gently suspended in 0.35% agar in serum/antibiotic free RPMI, supplemented with 10% FBS, 1% penicillin-streptomycin, and compounds whose concentrations was based on the EC10 and EC20. Then, 5000 cells/mL per well were seeded onto 1:1 mixture of 1% agar RPMI containing supplements into 6-well plates. After the top layer had dried, the agarose was overlaid with 3 mL RPMI +10% FBS and drug concentration at EC10 or EC20. The overlaying media was changed twice per week. After 14 and 21 days, the number and size of colonies were visualized and measured, using 40x and 20x objectives. Triplicate wells were used for every treatment condition and the same field was used for analysis and then averaged.
Comment
The outcome of this assay is hard to quantify, hence the results are described.
Result Definitions
TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
1DescriptionString
Additional Information
Grant Number: MH082371

Data Table (Concise)
Data Table ( Complete ):     View All Data
Classification
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