High-content, full genome siRNA screen for regulators of oncogenic HRAS-driven macropinocytosis Journal Article
| Authors: | Fennell, M.; Commisso, C.; Ramirez, C.; Garippa, R.; Bar-Sagi, D. |
| Article Title: | High-content, full genome siRNA screen for regulators of oncogenic HRAS-driven macropinocytosis |
| Abstract: | Uptake of nutrients, such as glucose and amino acids, is critical to support cell growth and is typically mediated by cell surface transporters. An alternative mechanism for the bulk uptake of nutrients from the extracellular space is macropinocytosis, a nonclathrin, and nonreceptor-mediated endocytic process, in which extracellular fluid is taken up into large intracellular vesicles called macropinosomes. Oncogenic transformation leads to the increased metabolic activity of tumor cells, and in the Ras-driven tumor part of this enhanced activity is the stimulation of macropinocytosis. To measure oncogene-dependent macropinocytosis, we used HeLa cells expressing oncogenic HRASG12D driven from a Tet-regulated promoter. Upon oncogenic HRAS expression, the cells undergo metabolic changes that include the elevation of macropinocytosis. We detected macropinocytosis through the uptake of lysine-fixable tetramethyl rhodamine (TMR)-Dextran (70kDa) from the cell media into nascent intracellular macropinosomes. These macropinosomes were quantified by image-based high-content analysis, with the size, intensity, and position of macropinosomes measured. Using this model system, we ran a full genome-wide siRNA screen (siGenome™; GE) to identify genes involved in controlling oncogenic HRAS-dependent macropinocytosis. Hits from the primary screen were confirmed with siRNA reagents from a different library (GE, OTP), which allowed us to mitigate potential off-target effects. Candidate genes from this screen include known regulators of macropinocytosis as well as novel targets. © Copyright 2015, Mary Ann Liebert, Inc. |
| Keywords: | controlled study; human cell; comparative study; cell death; gene expression; small interfering rna; oncogene h ras; genetic transfection; cytoplasm; doxycycline; genetic screening; extracellular fluid; molecular weight; gene ontology; rac1 protein; pinocytosis; transport vesicle; human; female; article; hela cell line |
| Journal Title: | Assay and Drug Development Technologies |
| Volume: | 13 |
| Issue: | 7 |
| ISSN: | 1540-658X |
| Publisher: | Mary Ann Liebert, Inc |
| Date Published: | 2015-09-01 |
| Start Page: | 347 |
| End Page: | 355 |
| Language: | English |
| DOI: | 10.1089/adt.2015.660 |
| PROVIDER: | scopus |
| PMCID: | PMC4554932 |
| PUBMED: | 26267765 |
| DOI/URL: | |
| Notes: | Export Date: 2 October 2015 -- Source: Scopus |
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