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Fig 4. Sdf1 and Sema3A have opposite effects on spreading, adhesions and protrusions. a DAPI (blue) and Phalloidin staining (green) on NC cells cultured in control conditions or exposed to Sdf1 and/or Sema3A. b Colour-coded time projection of time-lapse movies of cells transfected with life-Act-GFP (see Supplementary Movie 8). c–e Area occupied by the cells, normalised to control conditions (c), circularity (d) and aspect ratio (e), n = 105 cells (ncontrols = 25; nSdf1 = 21; nSema3A15ng.mL = 25; nSema3A15ng.mL+Sdf1 = 34) from two independent experiments, from fixed cells counterstained with Phalloidin. c–e ANOVA followed by multiple comparisons; ****p < 0.0001. f Average area occupied per protrusion (from Life-Act movies), normalised to controls, n = 122 protrusions (ncontrols = 24; nSdf1 = 23; nSema3A15ng.mL = 35; and nSema3A15ng.mL+Sdf1 = 40) analysed from two independent experiments. ANOVA followed by multiple comparisons ** (ctl vs sdf), p = 0.0014, ** (ctl vs Sema), p = 0.0048, ***p = 0.0009. g Phospho-paxillin (PPax) immunostaining, cells adhering on Fibronectin, Sema3A/Sdf1 were added in solution 30 min before fixation. h Ratio of area occupied by PPax divided by area of the cell, n = 51 cells from one experiment. Student’s t-test; *p = 0.0427. **p = 0.0144. i PPax intensity plot from cell tip to cell centroid (mean+s.e.m). Colour code corresponds to conditions shown in graphs e and h. j Frequency distribution of FA length (main axis). Note that exposure to Sema3A reduces the number of large FAs. Colour code corresponds to conditions shown in graphs e and h. k Average FA length, ANOVA followed by multiple comparisons; ****p value <0.0001. Data shown in h–k were gathered from two independent experiments, n = 30 cells per conditions. Scale bars 10 μ. Box and whiskers plot: the box extends from the 25th to the 75th percentile; the whiskers show the extent of the whole dataset. The median is plotted as a line inside the box. Source data are provided as a Source Data file
Image published in: Bajanca F et al. (2019) © The Author(s) 2019. This image is reproduced with permission of the journal and the copyright holder. This is an open-access article distributed under the terms of the Creative Commons Attribution license Larger Image Printer Friendly View |