Fig 1.
Uneven shear stress in adhesion assay and cell counting method.
(A) The nature of the orbital shaker causes a stronger shear stress at the periphery of the gel, causing a stronger cell detachment at these regions (denoted by the lighter color at the periphery in the diagram). (B) Cells are counted only at the periphery, from the edge up to 200 μm towards the center, before and after the shaking.
Fig 2.
Mechanical stimulation equipment and vibration characterization.
(A) Pictures of the vibration stimulation equipment with different components: XYZ stage, with micrometers attached, aluminum arm and aluminum rod with probe (top right panel); vibration mechanism and spacer ready to trigger the vibration (bottom right panel); setup with the aluminum rod already inserted in the aperture of the CO2 chamber and with probe positioned on the gel (left panel). (B) The left image shows the probe and gel substrate with 1 μm beads embedded. The right images show a typical displacement (in μm) and strain maps, with the cold and hot color representing the small and large displacement/strain, respectively. In both colormaps, the vectors are the displacements on the substrate. The black circle represents the typical position of a cell (~125 μm of diameter) during stimulation. The bars represent 25 μm. (C) Maximum displacement of the gel over time. (D) Maximum strain of the gel over time. For both graphs, the data points are plotted with 1 ms interval for the first 40 ms of vibration and, until 180 ms, for the frame with largest probe displacement in each vibration period.
Fig 3.
Typical calcium response upon mechanical vibration stimulation.
Typically, the cell responds with a rise in [Ca2+]i occurring mostly at a region close to the stimulation site. The DIC images show the location of the probe and the cell under stimulus. The time course represents the [Ca2+]i at the region of interest. The color images represent the fluorescence emission ratio of YPet/ECFP from the biosensor before stimulation, immediately after stimulation, and after [Ca2+]i re-stabilized. The bars represent 25 μm.
Table 1.
Displacement, strain and frequency of vibration for each gel thickness.
Fig 4.
Lower substrate adhesion inhibits response to stimulation.
Three different gel substrate treatments were used to assess the effect of adhesion on the calcium response to mechanical stimulation: regularly treated polyacrylamide gel substrates ("High Fn"), no Sulfo-SANPAH activation but coating with fibronectin at 5% of the normal amount per dish ("Low Fn"), no Sulfo-SANPAH and no fibronectin coating ("No Fn"). (A) Average intensities of rhodamine-labeled fibronectin of different gel substrates. All of them are statistically different among themselves (High Fn and Low Fn: p = 3x10-21, High Fn and No Fn: p = 2x10-23, Low Fn and No Fn: p = 0.007; Student's t-test). (B) Attachment ratios of HUVECs seeded on different gel substrates. All of them are statistically different among themselves (High Fn and Low Fn: p = 0.02, High Fn and No Fn: p = 7x10-10, Low Fn and No Fn: p = 0.0005; Student's t-test) (C) Relative difference of the biosensor fluorescence ratio of mechanically stimulated HUVECs seeded on different gel substrates. All of them are statistically different among themselves (High Fn and Low Fn: p = 0.04, High Fn and No Fn: p = 4x10-7, Low Fn and No Fn: p = 0.003; Student's t-test). The error bar on the bar graphs are the respective standard error of the samples.
Fig 5.
TRPM7 siRNA increases adhesion of HUVEC but inhibits response to stimulation.
HUVEC electroporated with non-targeting siRNA and TRPM7 siRNA had their adhesion and calcium response to mechanical stimulation compared. (A) Attachment ratios of HUVECs electroporated with non-targeting and TRPM7 siRNAs. These results are also compared to a control, HUVECs on "High Fn" dishes. Control and non-targeting siRNA are the only ones not statistically different (control and non-targeting siRNA: p = 0.2, control and TRPM7 siRNA: p = 0.03, non-targeting and TRPM7 siRNAs: p = 0.001; Student's t-test) (B) Relative difference of the biosensor fluorescence ratio of mechanically stimulated HUVECs non-targeting and TRPM7 siRNAs. They are statistically different (non-targeting and TRPM7 siRNAs: p = 0.01; Student's t-test). The error bar on the bar graphs are the respective standard error of the samples.