Journal: Science Advances
Article Title: Acute contact with profibrotic macrophages mechanically activates fibroblasts via αvβ3 integrin–mediated engagement of Piezo1
doi: 10.1126/sciadv.adp4726
Figure Lengend Snippet: ( A ) 3D confocal-reflection image of a 60-μm-thick collagen gel (white) with red fluorescent beads on the surface. ( B ) Orthogonal view of collagen gel with fibroblast (center, gray) and Mϕ (small white) in plane with fluorescent beads on the surface. ( C ) Scanning electron microscope image of fibroblast with M IL-4/13 on collagen gel. ( D ) To visualize bead displacement, every frame was assigned a different color and overlaid to create a temporal color-code image of fibroblasts alone, fibroblasts with M LPS , and fibroblasts with M IL-4/13 over 80 min. ( E ) Fibroblast contractions were quantified by summing the vector norms of all bead displacements (in micrometers). Averages are shown ± SD for two to three fibroblasts from three independent experiments. ( F ) Speed of fibroblast contractions measured as the slope of the curves in (E), normalized to unstimulated contraction before t = 0. BD, before DMEM; AD, after DMEM; BC, before contact; AC, after contact. ( G ) Summary of contraction speeds. ( H ) The resulting contraction speeds are either classified as accelerated compared to fibroblasts alone or not changed and plotted as a percentage. ( I ) Maximum contraction for each fibroblast from curves in (E); each dot represents one fibroblast. ( J ) Traction force microscopy was performed to monitor local fibroblast contractions upon Mϕ contact, with local stress (pascals) shown color coded. M IL-4/13 (outlined in pink) contact the fibroblast (green outline) at various contact time points; outlines of the M IL-4/13 remain throughout all images when contact was made in the previous frame, with arrows indicating the point of contact. Scale bar, 20 μm. Fibroblast contraction analyzed as described for (E) to (G) from four to six fibroblasts from three biological repeats. Statistical significance was calculated using repeated measures ANOVA and Fisher’s LSD post hoc analysis with significance reached with P < 0.05 and “n.s.” indicating not significant.
Article Snippet: Samples were mounted and gold sputtered (Desk II sputter coater, Denton), and images were taken with a scanning electron microscope (FlexSEM1000, Hitachi, Tokyo, Japan) at a tilt angle of 45°.
Techniques: Microscopy, Plasmid Preparation
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