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- Kundu, Snehangshu, et al.
(författare)
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Linking FOXO3, NCOA3, and TCF7L2 to Ras pathway phenotypes through a genome-wide forward genetic screen in human colorectal cancer cells
- 2018
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Ingår i: Genome Medicine. - : Springer Science and Business Media LLC. - 1756-994X .- 1756-994X. ; 10
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Tidskriftsartikel (refereegranskat)abstract
- Background:The Ras pathway genes KRAS, BRAF, or ERBBs have somatic mutations in similar to 60% of human colorectal carcinomas. At present, it is unknown whether the remaining cases lack mutations activating the Ras pathway or whether they have acquired mutations in genes hitherto unknown to belong to the pathway.Methods:To address the second possibility and extend the compendium of Ras pathway genes, we used genome-wide transposon mutagenesis of two human colorectal cancer cell systems deprived of their activating KRAS or BRAF allele to identify genes enabling growth in low glucose, a Ras pathway phenotype, when targeted.Results:Of the 163 recurrently targeted genes in the two different genetic backgrounds, one-third were known cancer genes and one-fifth had links to the EGFR/Ras/MAPK pathway. When compared to cancer genome sequencing datasets, nine genes also mutated in human colorectal cancers were identified. Among these, stable knockdown of FOXO3, NCOA3, and TCF7L2 restored growth in low glucose but reduced MEK/MAPK phosphorylation, reduced anchorage-independent growth, and modulated expressions of GLUT1 and Ras pathway related proteins. Knockdown of NCOA3 and FOXO3 significantly decreased the sensitivity to cetuximab of KRAS mutant but not wild-type cells.Conclusions:This work establishes a proof-of-concept that human cell-based genome-wide forward genetic screens can assign genes to pathways with clinical importance in human colorectal cancer.
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| 2. |
- Padhan, Narendra
(författare)
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Highly Sensitive and Quantitative Detection of Proteins and Their Isoforms by Capillary Isoelectric Focusing Method
- 2018
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Ingår i: Journal of Visualized Experiments. - : JOURNAL OF VISUALIZED EXPERIMENTS. - 1940-087X. ; :139
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Tidskriftsartikel (refereegranskat)abstract
- Immunoblotting has become a routine technique in many laboratories for protein characterization from biological samples. The following protocol provides an alternative strategy, capillary isoelectric focusing (cIEF), with many advantages compared to conventional immunoblotting. This is an antibody-based, automated, rapid, and quantitative method in which a complete western blotting procedure takes place inside an ultrathin capillary. This technique does not require a gel to transfer to a membrane, stripping of blots, or x-ray films, which are typically required for conventional immunoblotting. Here, proteins are separated according to their charge (isoelectric point; pl), using less than a microliter (400 nL) of total protein lysate. After electrophoresis, proteins are immobilized onto the capillary walls by ultraviolet light treatment, followed by primary and secondary (horseradish peroxidase (HRP) conjugated) antibody incubation, whose binding is detected through enhanced chemiluminescence (ECL), generating a light signal that can be captured and recorded by a charge-coupled device (CCD) camera. The digital image can be analyzed and quantified (peak area) using software. This high throughput procedure can handle 96 samples at a time; is highly sensitive, with protein detection in the picogram range; and produces highly reproducible results because of automation. All of these aspects are extremely valuable when the quantity of samples (e.g., tissue samples and biopsies) is a limiting factor. The technique has wider applications as well, including screening of drugs or antibodies, biomarker discovery, and diagnostic purposes.
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