Fig. 7 | Nature Communications

Fig. 7

From: In vivo topology converts competition for cell-matrix adhesion into directional migration

Fig. 7

Activation of adhesion and actin dynamics are required for rescue of exposure to Sema3A. a Effects of Manganese (Mn2+) and CucurbitacinE (CuE) on adhesion and actin. b Rac1 activity (FRET) in control/treated cells with Mn2+ (2 mM) for 2 h, n = 24 cells (ncontrols = 11, nMn2+ = 13) from two independent experiments, unpaired Student t-test, p value on the graph. c Cells cultured on Fibronectin with/without Mn2+ (2 mM) or CuE (1 nM), Phalloidin. d Normalised cell area, n = 104 cells (ncontrols = 15, nMn2+ = 17, nCuE = 14, nSema = 20, nSema+Mn2+ = 20, nSema+CuE = 18), ANOVA, multiple comparisons; **p(ctl vs sema) = 0.0036; **p(sema vs sema+Mn) = 0.0053; and *p(ctl vs sema+CuE) = 0.0210. e PPax immunostaining, cells cultured on Fibronectin with/without Sema3A at 60 ng/mL with/without Mn2+ (2 mM) and/or CuE (1 nM). f Focal adhesion per cell per condition depicted in e, n = 178 cells (ncontrols = 29, nMn2+ = 29, nSema = 30, nSema+Mn2+ = 30, nSema+CuE = 30, and nSema+Mn2++CuE = 30) from two independent experiments; ANOVA, multiple comparisons; *p(ctl vs sema) = 0.0348; **p(ctl vs Mn) = 0.0053; **p(ctl vs sema+Mn) = 0.0026; ****p(sema vs sema+Mn) < 0.0001. g, h FA length and frequency distribution from e, n of individual FA analysed: ncontrols = 2197, nMn2+ = 1688, nSema = 3101, nSema+Mn2+ = 2708, nSema+CuE = 2469, nSema+Mn2++CuE = 2328, from two independent experiments. Colour code same as in g. i Explants cultured on Fibronectin or Fibronectin plus Sema3A at 15 ng/mL with/without Mn2+ (2 mM), stained with DAPI. j Mean distance between nearest neighbours from i, n = 106 explants (ncontrols = 47, nMn2+ = 26, nSema = 17, and nSema+Mn2+ = 16), from two independent experiments; ANOVA, multiple comparisons, ***p = 0.0005. k Percentage of adherent and dispersing explants after a 3-hour culture on Fibronectin or Fibronectin plus Sema3A at 15, 30 or 60 ng/mL with/without Mn2+ (2 mM) and/or CuE (1 nM), n = 268 explants (ncontrols = 52, nMn2+ = 26, nCuE = 26, nSema15 = 17, nSema15+Mn2+ = 17, nSema30 = 22, nSema30+Mn2+ = 22, nSema60 = 43, nSema60+Mn2+ = 17, and nSema60+Mn2++CuE = 26) from five independent experiments. Comparisons of proportions were made using contingency tables60. Null hypothesis rejected if T > 3.841 (*α = 5%); T > 6.635 (**α = 1%); T > 10.83 (***α = 0.1%). l Normalised area overtime for explants cultured on Fibronectin or Fibronectin plus Sema3A at 60 ng/mL with/without Mn2+ (2 mM), n = 145 explants (ncontrols = 38, nSema = 33, nSema+Mn = 42, and nSema+Mn+CuE = 32) from four independent experiments. m Normalised explant areas after 8 h for conditions shown in l; ANOVA, multiple comparisons, ***p = 0.0005; ****p < 0.0001. Scale bars (c, e), 10 μ. Scale bar in i, 100 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

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