Each dish was then rinsed and stained for 20 min at room temperature with a 165 nM solution of Alexa Fluor 488 phalloidin (Invitrogen) in PBS containing 1% BSA. changes using comparable methodologies. We here investigate the hypothesis that this rate of depressurization, rather than Mutant IDH1-IN-1 elevated hydrostatic pressure itself, may be responsible for these reported changes. Hydrostatic pressure (100 mm Hg above atmospheric pressure) was applied to bovine aortic endothelial cells (BAECs) and PC12 neuronal cells using pressurized gas for periods ranging from 3 hours to 9 days, and then the system was either slowly (~30 minutes) or rapidly (~5 seconds) depressurized. Cell viability, apoptosis, proliferation, and F-actin distribution were then assayed. Our results did not show significant differences between rapidly and slowly depressurized cells that would explain differences previously reported in the literature. Moreover, we found no detectable effect of elevated hydrostatic pressure (with slow depressurization) on any measured variables. Our results do not confirm the findings of other groups that modest increases in hydrostatic pressure affect cell function, but we are not able to explain their findings. Introduction Cells are constantly exposed to a range of mechanical stresses due to the dynamic HESX1 nature of the biological environment in which they reside. It has been recognized that some of these physical stimuli can be sensed by cells and play a significant role in influencing cell signaling and behavior. Stretch-activated ion channels, membrane-bound enzymes, and internal cytoskeletal filaments have all been shown to respond to compressive, tensile, and shear stresses . Over the last two decades, a number of studies have also reported significant changes in cell behavior following the application of hydrostatic pressure in the range of 10C150 mm Hg to cells cultured in vitro on rigid substrates [2C31]. These changes include increases in cell proliferation and migration, increases in apoptosis, changes in cell morphology, and changes in gene expression. As biological cells and tissues are essentially incompressible , application of hydrostatic pressure will generate insignificant mechanical strain in these cells, and thus it is surprising that hydrostatic pressure would have any effect on them. It is possible that when hydrostatic pressure is usually applied to the cells, artifacts are introduced that affect cell function. Indeed, Lei et al.  showed that when hydrostatic pressure is usually applied by use of a hydrostatic fluid column, hypoxic conditions are created that Mutant IDH1-IN-1 alter cell function. Once the effects of hypoxia were controlled for, no effect of hydrostatic pressure on cell behavior was observed in these studies. Other methods of application of hydrostatic pressure, such as use of a pressurized chamber [2,5,11,13,26,34] and use of a pump-driven flow system [6,15,21] are not subject to this hypoxia artifact. Use of a pressurized chamber alters the gas composition in equilibrium with the culture medium , but the magnitude of these changes are small Mutant IDH1-IN-1 for modest changes in hydrostatic pressure. Agar et al.  proposed that application of hydrostatic pressure to a cell would necessarily involve transient changes in pressure during the initial pressurization step and the final depressurization step, and these transients might affect the cells. Resta et al.  provided data supporting this expectation. The objective of our study was to determine if the rate by which the system is usually depressurized, following application of hydrostatic pressure, might have an effect on cells in culture and potentially be responsible for the observed effects that had been previously attributed to hydrostatic pressure. We tested this hypothesis by replicating hydrostatic pressure experiments already reported in the literature [5,11,21,26] on bovine aortic endothelial cells (BAECs) and a PC12 neuronal cell line, while also examining the.