Membrane Blebbing Is Required for Mesenchymal Precursor Migration
Fig 5
MP wound-healing (migration) assay.
(A) Representative 10x images of wounding at 12h are shown. (B) Graphs of wound closure percentage from triplicate samples at each 2 hours over 12h; Statistical analysis were performed using 2 Way-ANOVA and Turkey´s multiple comparisons test to evaluated each condition each hour; * P < 0.05; ** P < 0.01; *** P < 0.001. (a) Comparison of mesenchymal precursors in control conditions (Asterisks above correspond to adipose precursors-cardiac precursors, and asterisk below correspond to muscle precursors-cardiac precursors). (b) Comparison between adipose precursors in control conditions and the same cells with blebbistatin and SDF1 (One row of asterisks refers to SDF1-blebbistatin and in two row of asterisks the above ones are related to control-blebbistatin and the below ones to SDF1-blebbisttatin). (c) Comparison of cardiac precursors in control blebbistatin and SDF1 conditions. (One row of asterisks refers to SDF1-blebbistatin and in two row of asterisks the above ones are related to control-blebbistatin and the below ones to SDF1-blebbisttatin). (d) Comparison of muscle precursors in control blebbistatin and SDF1 conditions. (One row of asterisks refers to SDF1-blebbistatin and in two row of asterisks the above ones are related to control-blebbistatin and the below ones to SDF1-blebbisttatin). (C) Higher magnification of F-actin staining of precursors (63x) in control and blebbistatin conditions. Blebs (marked with white arrows) can be visualized in the membrane of control cells at the leading edge of the cells localized at the front of the wound. (D) Representative phase contrast 20x images of the leading edge of cells that were migrating through the wound are shown. White discontinued lines defined lamellipodia and red lines delimited blebs in the cells. Higher magnification of these images is also shown at the below panel where blebs can be distinguished in black. Quantification of the percentage of cells with blebs at the front of the wound; non statistical differences were found using Mann-Whitney test.