Fig 1.
Morphological appearance of mesenchymal precursors in culture.
(A) Representative phase contrast images (20x) of MPs at 6 and 24h in culture, where blebbing can be observed. (B) Time-lapse imaging of bleb formation (arrow) in a cardiac precursor, 6h after seeding. Images (20x) show bleb expansion (15” and 25”) and bleb retraction (60”). (C) Quantification of the percentage of cells with blebs in mesenchymal precursors; Statistical analysis were performed using Mann-Whitney test; ** P < 0.01; *** P < 0.001. (D) Blebbistatin-treated muscle precursors (magnification of 20x images), added at the same time of seeding and 24h later respect to control muscle precursors. Images were taken 1h and 24h hours after blebbistatin was added.
Fig 2.
Characterization of bleb structure by immunofluorescence.
Confocal images of MPs at 63x using zoom in. Higher magnifications of selected blebs are shown in the upper left corner. All precursors are stained for F-actin (in red) and DAPI (in blue). Cell fixed after 6 hours of seeding the cells were stained for RhoA in green (d-f). Cells were stained for pERM in green at 6 hours (g-i) and 24 (j-l) hours after seeding the cells. Merged of pERM in green, F-actin in red and nuclei in blue at 6h (g-i) and 24h (j-l). Confocal images of MPs at 63x and zoom in. Bar 5 μm.
Fig 3.
Mesenchymal precursors cultured on different matrices.
(A) Representative phase contrast images (10x) of MPs without matrix, on gelatin, collagen and Matrigel. Higher magnifications of selected cells are shown in the upper left corner. (B) Quantification of the percentage of cells with blebs and a comparison of muscle precursor images. Blebbing can be observed in control (no matrix) conditions.; Statistical analysis were performed using One-way ANOVA, Bonferroni; ** P < 0.01; *** P < 0.001.
Fig 4.
(A) Representative images from five randomly selected 10x fields of MPs with/without blebbistatin are shown. (B) Data are from representative experiment out of three performed and denote mean±SEM of migrated cells/field; Statistical analysis were performed using Mann-Whitney test; *** P < 0.001.
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.
Fig 6.
(A) Representative images from five randomly selected 10x fields of MPs with/without blebbistatin are shown. (B) Data are from representative experiment out of three performed and denote mean±SEM of migrated cells/field; Statistical analysis were performed using One-way ANOVA, Bonferroni post-test; ** P < 0.01; *** P < 0.001. (C) Zoom of cells located at the lower surface of the Transwell pore, showing fan-shaped lamellipodia containing blebs (marked with asterisks) at the leading edge. (D) Higher magnifications of muscle precursors are shown. (E) Quantification of the percentage of cells with blebs in migrating cells; Statistical analysis were performed using Mann-Whitney test; * P < 0.05.