Sequestration in Speckles

The HIF (hypoxia-inducible factor) family of oxygen-sensing proteins are a crucial element of cells’ responses to alterations in their immediate environment, kicking off a signaling cascade involving more than 1000 genes. HypOxystation users Taylor and See at the University of Liverpool describe novel insights into the subcellular localization of some of the HIF proteins and why the “where” determines the “how”.

HIF-2α and HIF-1α both form heterodimers with HIF-1β, and while similarities abound between the isoforms, the two subunits are differentially expressed and regulated and have distinctly separate target genes. Taylor and See triggered HIF activation using microscope stage incubators and the Hypoxystation by Don Whitley Scientific to incubate HeLa cells in hypoxia (1%). They found that while HIF-1α distributes homogenously in the nucleus, HIF-2α diffuses freely through the nucleus but is concentrated in speckles that are tethered to nuclear structures close to active RNA polymerases. This distribution is not significantly altered by low oxygen levels.

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Avoiding Spikes of Normoxia

“Using the HypOxystation is critical to our work because… unlike an incubator, it maintains the O2 levels when cells are taken in and out, avoiding spikes of normoxia.” Timothy Audas, University of Miami.

Responses to physiological cues are governed on a cellular level by changes in protein levels, and HypOxystation users Ho et al. describe their research on mechanisms mediating these responses in their newest paper "Systemic Reprogramming of Translation Efficiencies on Oxygen Stimulus”  (Cell Reports 14, 1293-1300, 2016).  Contrary to the widely held belief that transcription and mRNA levels are the main regulators of protein expression, Dr. Ho and Dr. Timothy Audas assert that translation efficiency Te of mRNA’s that are already present in the cell exerts a much larger influence than previously thought, especially in response to a stimulus such as hypoxia.

Glioblastoma and renal clear cell carcinoma cells were maintained at hypoxia (1%) in the H35 HypOxystation and at normoxia, and mRNA levels and protein expression were compared. The correlation between mRNA and protein output was quite weak, leading the authors to postulate that cellular response to oxygen stimulus occurs through a switch in Te rather than in transcription levels. Gene silencing of hypoxic and normoxic cell cultures via transient transfection with siRNA confirmed the existence of an alternative translation initiation complex binding to the 5’ cap of the mRNA, termed hypoxic eIF4F (eIF4FH), that is not in use at normoxia. The HypOxystation reliably creates physiological conditions for cells habituated to much lower oxygen than the ambient 21%, while also controlling CO2, temperature and humidity. Working inside the HypOxystation via gloveless sleeves allows users to culture and manipulate cells for extended periods of time, without ever exposing them to the “oxygen shock” of the lab atmosphere. In an interview with the University of Guelph, co-author and HypOxystation user Jim Uniacke describes using the HypOxystation to design a suicide mRNA strategy targeting hypoxic tumors.

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Examining the Role of Autophagy in Hypoxic Tumors

HypOxystation users Tan et al. at the University of Toronto published a paper in June examining the significance of autophagy in cancer development (“Role of Autophagy as a Survival Mechanism for Hypoxic Cells in Tumors”, Neoplasia (2016) 18, 347–355). Autophagy as a means of recycling cell components is induced under stress conditions such as hypoxia, and Tan et al. investigated the correlation of hypoxia and autophagy in solid tumors in the context of resistance to cancer therapeutics. 

Cells were cultured in the H35 HypOxystation  for up to 48 hours at hypoxia (0.2 %) and compared to cells grown at ambient oxygen level. Gene silencing of autophagy proteins ATG7 and BECLIN1 with shRNA resulted in decreased cell survival under hypoxia, and inhibition of autophagy with pantoprazole exacerbated the loss of viability in the knock-down cells under hypoxia, demonstrating the cyto-protective effects of these autophagy proteins. Using the Seahorse XFe Analyzer to assess oxygen consumption in wild-type and silenced cells, Dr. Tan’s lab found reduced respiration when autophagy is disrupted, possibly due to accumulation of dysfunctional mitochondria in these mutant cells. The H35 HypOxystation  Dr. Tan’s lab used for these studies creates a closed environment with controlled temperature, humidity, CO2 and oxygen, in which cells are cultured and manipulated over the course of days and weeks without the need to transfer into ambient conditions, ever. The combination of HypOxystation and i2 Instrument Workstation is custom-designed to accommodate the specific requirements of the Seahorse XFe Analyzer for the duration of the metabolism assays investigating oxygen consumption and extracellular acidification.

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Physiological Oxygen is Healthier for Cell Cultures

“Culturing cells in ambient air could be far from physiological with respect to oxygen. Oxygen is a surprisingly neglected factor [in cell culture].” Timpano and Uniacke

Drs. Timpano and Uniacke, HypOxystation users at University of Guelph in Ontario, have published a very thorough study examining the molecular basis of cells’ reactions to differing levels of hypoxia. In their paper “Human Cells Cultured Under Physiological Oxygen Utilize Two Cap-binding Proteins to Recruit Distinct mRNAs for Translation” (Journal of Biological Chemistry 291:20; 2016), they examine 2 different translation initiation proteins, eiF4E and eiF4E2, that are activated under either high (>8% O2) or low (<1% O2) oxygen levels, with the aid of mTORC1 or HIF-2α, respectively, and activated simultaneously in an area of low- to mid-level physioxia (1-8% O2). Timpano and Uniacke were able to stably and accurately create low oxygen in their HypOxystation by HypOxygen, which provides a closed workstation environment that enables researchers to culture and manipulate cells inside the chamber through gloveless sleeves, eliminating the negative consequences of spikes of higher oxygen and lower temperatures encountered in an incubator as cell cultures are growing. Their research into translational modulation of the proteome using the HypOxystation gives seminal insights into physioxia as the natural condition for cells, both in vitro and in vivo.

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H35 HypOxystation used in heart regeneration project

Dr Vaibhao Janbandhu is a Postdoctoral Research Scientist at the Victor Chang Cardiac Research Institute (VCCRI) in Sydney. He has been in contact with Don Whitley Scientific to explain how his lab’s work has benefited from the use of a Whitley H35 HypOxystation. Vaibhao uses the HypOxystation to isolate, culture and characterise adult cardiac stem cells (CSCs).

Dr Janbandhu had already been using a H35 that was set up at the institute for almost three years before he got his own unit installed last year. Specifically, his project is to find new ways to stimulate heart regeneration during ageing and after heart attack. For this he needs a way to isolate, culture and characterise adult CSCs. In Vaibhao’s words the H35 Hypoxystation seems well suited for this application: “the DWS HypOxystation provides a highly stabilised environment in which levels of oxygen, carbon dioxide, temperature and humidity are precisely controlled and it will be an integral part of the project to advance the project aims”.

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