Dataset Information

Automated analysis of NF-?B nuclear translocation kinetics in high-throughput screening.

ABSTRACT: Nuclear entry and exit of the NF-?B family of dimeric transcription factors plays an essential role in regulating cellular responses to inflammatory stress. The dynamics of this nuclear translocation can vary significantly within a cell population and may dramatically change e.g. upon drug exposure. Furthermore, there is significant heterogeneity in individual cell response upon stress signaling. In order to systematically determine factors that define NF-?B translocation dynamics, high-throughput screens that enable the analysis of dynamic NF-?B responses in individual cells in real time are essential. Thus far, only NF-?B downstream signaling responses of whole cell populations at the transcriptional level are in high-throughput mode. In this study, we developed a fully automated image analysis method to determine the time-course of NF-?B translocation in individual cells, suitable for high-throughput screenings in the context of compound screening and functional genomics. Two novel segmentation methods were used for defining the individual nuclear and cytoplasmic regions: watershed masked clustering (WMC) and best-fit ellipse of Voronoi cell (BEVC). The dynamic NF?B oscillatory response at the single cell and population level was coupled to automated extraction of 26 analogue translocation parameters including number of peaks, time to reach each peak, and amplitude of each peak. Our automated image analysis method was validated through a series of statistical tests demonstrating computational efficient and accurate NF-?B translocation dynamics quantification of our algorithm. Both pharmacological inhibition of NF-?B and short interfering RNAs targeting the inhibitor of NF?B, I?B?, demonstrated the ability of our method to identify compounds and genetic players that interfere with the nuclear transition of NF-?B.

SUBMITTER: Di Z

PROVIDER: S-EPMC3531459 | biostudies-literature | 2012

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Automated analysis of NF-κB nuclear translocation kinetics in high-throughput screening.

Di Zi Z Herpers Bram B Fredriksson Lisa L Yan Kuan K van de Water Bob B Verbeek Fons J FJ Meerman John H N JH

PloS one 20121227 12

Nuclear entry and exit of the NF-κB family of dimeric transcription factors plays an essential role in regulating cellular responses to inflammatory stress. The dynamics of this nuclear translocation can vary significantly within a cell population and may dramatically change e.g. upon drug exposure. Furthermore, there is significant heterogeneity in individual cell response upon stress signaling. In order to systematically determine factors that define NF-κB translocation dynamics, high-throughp ...[more]

PMID: 23300644

Similar Datasets

Project description:BackgroundMultiple myeloma (MM) is an incurable hematological malignancy. Although proteasome inhibitors and immunomodulators have significantly improved patient outcomes, some patients respond poorly to treatment and almost all patients will relapse. Mechanisms of proteasome inhibitor resistance in multiple myeloma have not been fully elucidated. ZHX2 is a transcription regulator degraded via proteasome and presents both oncogenic or tumor suppressive effect in different cancers, however, it is still unknown that the role of ZHX2 in myeloma. In this study, we aim to demonstrate the effect and mechanism of ZHX2 on proteasome inhibitor resistance in MM.MethodsGSE24080 gene expression profile datasets from Gene Expression Omnibus (GEO) were analyzed to evaluate the relationship between ZHX2 expression level and survival in MM. Expression of ZHX2 in human MM cell lines at baseline and after bortezomib (BTZ) treatment was determined by Western blotting (WB). The proliferation and apoptosis rate of MM cells treated with BTZ after the knockdown of ZHX2 were analyzed by flow cytometry. Nuclear translocation of NF-κB after the knockdown of ZHX2 was evaluated by WB and immunofluorescence, and the expression of NF-κB target genes was measured by real-time quantitative PCR. Co-immunoprecipitation (Co-IP) and WB were used to detect the interaction of ZHX2 with NF-κB.ResultsWe found that higher ZHX2 expression was correlated with poorer clinical outcomes of patients. In addition, ZHX2 expression was relatively higher in RPMI-8226 and MM.1S cell lines and the level of ZHX2 protein was upregulated after BTZ treatment. Knockdown of ZHX2 significantly enhanced the sensitivity of MM cells to BTZ, inhibited nuclear translocation of NF-κB, and reduced mRNA expression of NF-κB target genes. It was also revealed that ZHX2 directly binds to NF-κB.ConclusionOur study showed that ZHX2 can promote proteasome inhibitor resistance in MM cells by regulating the nuclear translocation of NF-κB.

Dataset Information

Automated analysis of NF-?B nuclear translocation kinetics in high-throughput screening.

Publications

Automated analysis of NF-κB nuclear translocation kinetics in high-throughput screening.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets