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Phys Chem Chem Phys. 2014 Oct 21;16(39):21595-21601. doi: 10.1039/c4cp03260h. Epub 2014 Sep 5.

Single-molecule quantification of lipotoxic expression of activating transcription factor 3.

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Division of Cardiovascular Medicine, Department of Internal Medicine, School of Medicine, University of California, Davis, CA 95616.
Biomolecular Photonics, Department of Physics, University of Bielefeld, 33615 Bielefeld, Germany.
Center for Biophotonics Science and Technology, UC Davis, 2700 Stockton Blvd, Sacramento, CA.
Department of Pathology, Microbiology, and Immunology, School of Veterinary Medicine, University of California, Davis, CA 95616.
Contributed equally


Activating transcription factor 3 (ATF3) is a member of the mammalian activation transcription factor/cAMP, physiologically important in the regulation of pro- and anti-inflammatory target genes. We compared the induction of ATF3 protein as measured by Western blot analysis with single-molecule localization microscopy dSTORM to quantify the dynamics of accumulation of intranuclear ATF3 of triglyceride-rich (TGRL) lipolysis product-treated HAEC (Human Aortic Endothelial Cells). The ATF3 expression rate within the first three hours after treatment with TGRL lipolysis products is about 3500 h(-1). After three hours we detected 33,090 ± 3491 single-molecule localizations of ATF3. This was accompanied by significant structural changes in the F-actin network of the cells at ∼3-fold increased localization precision compared to widefield microscopy after treatment. Additionally, we discovered a cluster size of approximately 384 nanometers of ATF3 molecules. We show for the first time the time course of ATF3 accumulation in the nucleus undergoing lipotoxic injury. Furthermore, we demonstrate ATF3 accumulation associated with increased concentrations of TGRL lipolysis products occurs in large aggregates.

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