Dataset Information

Alternative to the soft-agar assay that permits high-throughput drug and genetic screens for cellular transformation.

ABSTRACT: Colony formation in soft agar is the gold-standard assay for cellular transformation in vitro, but it is unsuited for high-throughput screening. Here, we describe an assay for cellular transformation that involves growth in low attachment (GILA) conditions and is strongly correlated with the soft-agar assay. Using GILA, we describe high-throughput screens for drugs and genes that selectively inhibit or increase transformation, but not proliferation. Such molecules are unlikely to be found through conventional drug screening, and they include kinase inhibitors and drugs for noncancer diseases. In addition to known oncogenes, the genetic screen identifies genes that contribute to cellular transformation. Lastly, we demonstrate the ability of Food and Drug Administration-approved noncancer drugs to selectively kill ovarian cancer cells derived from patients with chemotherapy-resistant disease, suggesting this approach may provide useful information for personalized cancer treatment.

SUBMITTER: Rotem A

PROVIDER: S-EPMC4426412 | biostudies-literature | 2015 May

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Alternative to the soft-agar assay that permits high-throughput drug and genetic screens for cellular transformation.

Rotem Asaf A Janzer Andreas A Izar Benjamin B Ji Zhe Z Doench John G JG Garraway Levi A LA Struhl Kevin K

Proceedings of the National Academy of Sciences of the United States of America 20150420 18

Colony formation in soft agar is the gold-standard assay for cellular transformation in vitro, but it is unsuited for high-throughput screening. Here, we describe an assay for cellular transformation that involves growth in low attachment (GILA) conditions and is strongly correlated with the soft-agar assay. Using GILA, we describe high-throughput screens for drugs and genes that selectively inhibit or increase transformation, but not proliferation. Such molecules are unlikely to be found throug ...[more]

PMID: 25902495

Similar Datasets

Project description:The alphaproteobacterium Magnetospirillum gryphiswaldense has the intriguing ability to navigate within magnetic fields, a behavior named magnetotaxis, governed by the formation of magnetosomes, intracellular membrane-enveloped crystals of magnetite. Magnetosomes are aligned in chains along the cell's motility axis by a dedicated multipart cytoskeleton ("magnetoskeleton"); however, precise estimates of its significance for magnetotaxis have not been reported. Here, we estimated the alignment of strains deficient in various magnetoskeletal constituents by live-cell motility tracking within defined magnetic fields ranging from 50 μT (reflecting the geomagnetic field) up to 400 μT. Motility tracking revealed that ΔmamY and ΔmamK strains (which assemble mispositioned and fragmented chains, respectively) are partially impaired in magnetotaxis, with approximately equal contributions of both proteins. This impairment was reflected by a required magnetic field strength of 200 μT to achieve a similar degree of alignment as for the wild-type strain in a 50-μT magnetic field. In contrast, the ΔmamJ strain, which predominantly forms clusters of magnetosomes, was only weakly aligned under any of the tested field conditions and could barely be distinguished from a nonmagnetic mutant. Most findings were corroborated by a soft agar swimming assay to analyze magnetotaxis based on the degree of distortion of swim halos formed in magnetic fields. Motility tracking further revealed that swimming speeds of M. gryphiswaldense are highest within the field strength equaling the geomagnetic field. In conclusion, magnetic properties and intracellular positioning of magnetosomes by a dedicated magnetoskeleton are required and optimized for bacterial magnetotaxis and most efficient locomotion within the geomagnetic field.IMPORTANCE In Magnetospirillum gryphiswaldense, magnetosomes are aligned in quasi-linear chains in a helical cell by a complex cytoskeletal network, including the actin-like MamK and adapter MamJ for magnetosome chain concatenation and segregation and MamY to position magnetosome chains along the shortest cellular axis of motility. Magnetosome chain positioning is assumed to be required for efficient magnetic navigation; however, the significance and contribution of all key constituents have not been quantified within defined and weak magnetic fields reflecting the geomagnetic field. Employing two different motility-based methods to consider the flagellum-mediated propulsion of cells, we depict individual benefits of all magnetoskeletal constituents for magnetotaxis. Whereas lack of mamJ resulted almost in an inability to align cells in weak magnetic fields, an approximately 4-fold-increased magnetic field strength was required to compensate for the loss of mamK or mamY In summary, the magnetoskeleton and optimal positioning of magnetosome chains are required for efficient magnetotaxis.

Dataset Information

Alternative to the soft-agar assay that permits high-throughput drug and genetic screens for cellular transformation.

Publications

Alternative to the soft-agar assay that permits high-throughput drug and genetic screens for cellular transformation.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets