Fig 1.
(a) schematic diagram of the pressurizing equipment. During pressurization, cells in Petri dishes sealed in polyethylene bags are placed in the pressure chamber, valve 3 is closed and water is pumped into the chamber through valve 2. (b) Ethidium bromide staining of RNA used for the microarray experiments separated in a 1% agarose gel. 500 ng of total RNA was run in each lane. (c) schematic diagram of the time course experiments: the yellow and red bars respectively represent the time the packed cells are placed in the water bath and in the pressure chamber under 25 MPa.
Table 1.
Primers used for real-time PCR.
Fig 2.
High HP modulates hundreds of genes.
(a) number of genes up- and down-regulated at least 2-fold after 1, 4 and 24 h of pressurization. (b) Venn diagrams showing the overlap between genes up-regulated (left) and down-regulated (right) at different time points.
Fig 3.
Volcano plot analysis of modulated genes at each time point.
Volcano plots showing genes modulated with a fold change above 2 of below 0.5 after a) 1 h, b) 4 h or c) 24 h of pressurization. The base 2 logarithm of the fold change for each gene is plotted against the base 10 logarithm of the p-value obtained from the rank product analysis. The names of the genes with a fold change greater than 16 or less than 0.0625 (represented by the vertical dotted lines) are written next to the corresponding points.
Table 2.
Most significantly up- and down-regulated genes.
Table 3.
Over-represented gene functional categories.
Fig 4.
Network of genes modulated by high HP.
Network representation of the protein-protein interactions between genes that were significantly (p<0.015) modulated after 1 h (a), 4 h (b) or 24 h (c) of pressurization. Each node represents a protein. Only networks with at least three nodes are shown. The fold change is indicated by the color scale. In order to highlight the more important nodes, the size of the node increases with the number of interacting partners.
Fig 5.
Real-time PCR validation of microarray results.
Expression of 17 genes in ATDC5 cells pressurized for 0, 1, 4 or 24 h measured by real-time PCR (solid line) and microarray (dashed line). The plots show the mean +/- SD of three (PCR results) or two (microarray results) independent experiments. *: p< 0.05 and **: p<0.01 compared to unpressurized cells.
Fig 6.
Normalized number of ATDC5 cells after 0, 6 or 24 h under 25 MPa. The graph shows the mean +/- SD of three independent experiments. *: p< 0.05 compared to unpressurized cells.
Table 4.
Comparison between the present work and previously published studies.