Flow cytometry data for differentiated PC12 cells stained with Annexin V and DAPI.

<p>Results are for 24 hrs after exposure to hydrostatic pressure for 3 hrs and then rapid (A) or slow (B) depressurization as compared to a negative control (C: atmospheric pressure) and a positive control (D: 1 μM staurosporine for 24 hrs). Plots are representative of two independent experimental runs.</p

Schematic of the pressure chamber set-up.

<p>Chambers depicted with the lids off. Lids were tightly screwed into place and sealed with a silicone O-ring prior to pressurization. Tubing colored in blue represents the inlet gas streams while tubing colored in red represents the outlet gas streams. Dotted vs. solid colored lines are used to distinguish tubing attached to the left vs. right pressure chambers respectively. The two black dots near the top and bottom of each pressure chamber represent the positions of the outlet and inlet gas streams, respectively. The two large rectangles within each chamber represent the shelves used to hold cell culture plates, while the smaller rectangles at the bottoms of each chamber represent the water reservoirs. The dotted black line represents the 37°C incubator.</p

Percentages of BAEC populations in each of the four Annexin V/DAPI quadrants indicated in the flow cytometry plots in Fig 6.

<p>Percentages of BAEC populations in each of the four Annexin V/DAPI quadrants indicated in the flow cytometry plots in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0189890#pone.0189890.g006" target="_blank">Fig 6</a>.</p

Percentage of viable PC12 cells (Q3 of flow cytometry data show in Fig 12) of cells exposed to elevated hydrostatic pressure for 3 hours and then rapid (A) or slow (B) depressurization as compared to a negative control (C: atmospheric pressure) and a positive control (D: 1 μM staurosporine for 24 hrs).

<p>Values represent the means of two independent experimental runs. Error bars represent standard errors.</p

Confocal images of F-actin distribution in BAECs after exposure to elevated hydrostatic pressure for 24 hours followed by either rapid (A) or slow (B) depressurization as compared with a negative control (C: atmospheric pressure).

<p>F-actin is stained in red; nuclear DNA is stained in blue. The first two columns, which both contain images of confluent monolayers within each sample, were taken at z heights approximately 0.5–1 μm apart, moving from the surface (first column) down into the interior of the cell (second column). The third column contains images of cell multilayers which were found in all samples. Scale bar = 40 μm.</p

F-actin distribution in BAECs after 3 hrs of exposure to elevated hydrostatic pressure followed by rapid depressurization or slow depressurization as compared with a negative control (atmospheric pressure).

<p>F-actin is stained in red; nuclear DNA is stained in blue. Subconfluent regions of each sample are depicted in the top row while confluent regions of each sample are depicted in the bottom row. Scale bar = 40 μm.</p

Cell proliferation assays for BAECs exposed to elevated hydrostatic pressure for indicated periods and then subjected to rapid depressurization or slow depressurization along with a negative control (atmospheric pressure).

<p>(A) CyQUANT assay results after 24 hr application of elevated hydrostatic pressure as a function of initial cell seeding density within single wells of a 96 multiwell plate. Data represents mean +/-SE for 3–5 replicates within each experimental group. (B) Cell counts within 6 well plates taken using a hemocytometer as a function of days exposed to elevated hydrostatic pressure. Data displayed is an average of two individual experimental runs (exception is the 9 day cell count point for the atmospheric pressure negative control data set; this represents a single run). Two to four cell counts were taken at each time for each run. Error bars represent standard error.</p

Schematic of the pressure chamber set-up.

<p>Chambers depicted with the lids off. Lids were tightly screwed into place and sealed with a silicone O-ring prior to pressurization. Tubing colored in blue represents the inlet gas streams while tubing colored in red represents the outlet gas streams. Dotted vs. solid colored lines are used to distinguish tubing attached to the left vs. right pressure chambers respectively. The two black dots near the top and bottom of each pressure chamber represent the positions of the outlet and inlet gas streams, respectively. The two large rectangles within each chamber represent the shelves used to hold cell culture plates, while the smaller rectangles at the bottoms of each chamber represent the water reservoirs. The dotted black line represents the 37°C incubator.</p

Fluorescent LIVE/DEAD images of BAECs after exposure to hydrostatic pressure for either 2 days or 4 days.

<p>Cells were depressurized by either rapid depressurization (A) or slow depressurization (B) or exposed only to ambient atmospheric pressure (negative control) (C) and then stained as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0189890#pone.0189890.g002" target="_blank">Fig 2</a>. The vast majority of observed cells were live as indicated by the green stain. Scale Bar = 100 μm.</p