No significant differences were observed, two-tailed Student's t-test. ( c) Quantification of reservoir size in control and ATP-depleted cells ( n=150/200 reservoirs from 3/4 cells). The effect of ATP depletion was significant ( P<0.001). ( b) Quantification of reservoir fluorescence after release of first stretch pulse and during application and release of the second pulse ( n=50/70 reservoirs from 5/5 cells). ( a) Examples of control and ATP-depleted pEYFP-mem-transfected cells before, during and after application of two 3-min constant stretch pulses. In all cases, zoomed insets (10 × 6 μm 2 in c, g and 10 × 10 μm 2 in d, h) show a magnification of the area marked in the main image. Scale bars are 5 μm in c, g and 20 μm in d, h. We note that reservoir heights are close to the axial resolution of our confocal microscope (0.9 μm) and thus represent upper estimates rather than accurate measurements. ( k) Quantification of mean height of structures formed after stretch release (reservoirs) and re-application of iso-osmotic medium (VLDs). ( j) Quantification of mean density of structures formed after stretch release (reservoirs) and re-application of iso-osmotic medium (VLDs). ( i) Quantification of mean diameter of structures formed after stretch release (reservoirs) and re-application of iso-osmotic medium (VLDs). Merged co-localization images are shown to the right. ( h) Staining images of cells fixed immediately after re-application of iso-osmotic medium showing the membrane (pEYFP-mem transfection), paxillin and actin. ( g) Confocal images of a pEYFP-mem-transfected cell before (top) and after (bottom) application of 50% hypo-osmotic medium for 3 min. ( f) Quantification of VLD fluorescence after re-application of iso-osmotic medium (1: initial fluorescence, 0: background). ( e) pEYFP-mem-transfected cells before, during and after application of 50% hypo-osmotic medium during 3 min. ( d) Staining images of cells fixed immediately after stretch release showing the membrane (pEYFP-mem transfection), paxillin and actin. ( c) Confocal vertical slice from a pEYFP-mem-transfected cell before (top) and after (bottom) application of 6% stretch for 3 min. ( b) Quantification of reservoir fluorescence after stretch release (1: initial fluorescence, 0: background). ( a) pEYFP-mem-transfected cells before, during and after constant stretch application during 3 min. ( f) % of cells showing membrane lysis after 3 min of application of medium with different osmolarity ( n=50 cells). ( e) % of cells showing membrane tearing after 3 min of constant stretch application ( n=70 cells). ( d) Corresponding quantification of the increase in cell volume and required membrane area ( n= 5 cells). ( c) Confocal slice showing a cell before (green) and after (red) application of medium with 50% osmolarity for 3 min. Yellow arrows indicate membrane ruffles, which either remain or flatten after applying 50 or 0% hypo-osmotic medium, respectively. Cells submitted to 0% osmolarity (de-ionized water) for 3 min sometimes rounded and flattened membrane ruffles (middle panel) and sometimes underwent membrane lysis (right panel). ( b) Cells transfected with pEYFP-mem before and after reducing medium osmolarity to either 50 or 0% of original medium. Yellow arrow indicates a membrane ruffle flattened by stretch. ( a) Cells transfected with pEYFP-mem before (top panel) and after (middle and bottom panels) applying different magnitudes of constant stretch.
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