Please use this identifier to cite or link to this item: http://dr.iiserpune.ac.in:8080/xmlui/handle/123456789/8967
Title: A high-throughput screen in mESCs to identify the cross-talk between signaling, endocytosis, and pluripotency
Authors: Mote, Ridim D.
Tiwari, Mahak
Yadavalli, Narayana
RAJAN, RAGHAV
Subramanyam, Deepa
Dept. of Biology
Keywords: Embryonic stem cells
High-throughput
Image analysis
Pluripotency
Screen
2024
2024-MAY-WEEK3
TOC-MAY-2024
Issue Date: May-2024
Publisher: Wiley
Citation: Cell Biology International.
Abstract: Embryonic stem cell fate is regulated by various cellular processes. Recently, the process of endocytosis has been implicated in playing a role in the maintenance of self-renewal and pluripotency of mouse embryonic stem cells. A previous siRNA-based screen interrogated the function of core components of the endocytic machinery in maintaining the pluripotency of embryonic stem cells, revealing a crucial role for clathrin mediated endocytosis. A number of other proteins involved in key signaling pathways have also been shown to both regulate and be regulated by endocytosis. We collated a list of 1141 genes in connection to the term “endocytosis” from Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Ontology (GO), excluding those previously interrogated, and examined the effect of their knockdown on the pluripotency of mouse embryonic stem cells (mESCs) using levels of green fluorescent protein driven by the Oct4 promoter. We used high-throughput screening followed by an automated MATrix LABoratory (MATLAB)-based analysis pipeline and assessed changes in GFP fluorescence as a readout for ESC pluripotency. Through this screen we identified a number of genes, many hitherto not associated with stem cell pluripotency, which upon knockdown either resulted in a significant increase or decrease of GFP fluorescence. We further present validation for some of these hits. We present a workflow aimed to identify genes involved in signaling pathways which can be regulated by endocytosis, and that affect the pluripotency of ESCs.
URI: https://doi.org/10.1002/cbin.12168
http://dr.iiserpune.ac.in:8080/xmlui/handle/123456789/8967
ISSN: 1065-6995
1095-8355
Appears in Collections:JOURNAL ARTICLES

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