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. 2016 Jun 15;27(12):1911–1920. doi: 10.1091/mbc.E15-09-0645

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© 2016 Kushner et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

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FIGURE 4: — ECs with excess centrosomes have ectopic Rac1 activation and centrosome orientation defects. (A) Images of EC with one or two (1-2C) or more than two (>2C) centrosomes expressing the Rac1 biosensor and pseudocolored to indicate Rac1 activity. Scale bar, 10 μm. (B) Scatter plot of average FRET/CFP ratios between EC with 1-2 or >2 centrosomes and with or without nocodazole treatment (NOC). –NOC: 1-2C, 60 ECs; >2C, 58 ECs. +NOC: 1-2C, 19 ECs; >2C, 19 ECs. (C) Percentage of forward-oriented ECs with indicated treatments between ECs with 1-2 or >2 centrosomes, normalized to controls. CA, constitutively active; DN, dominant negative. Comparisons are to 1-2C group by c² analysis. (D) Percentage of forward-oriented ECs with indicated treatments between ECs with one or two or more than two centrosomes, normalized to controls. Rac inhibitor (NSC23766); Arp 2/3 inhibitor (Ck-666). Comparisons are to 1-2C group compared by c² analysis. (E) Images of front cortex of HUVECs on micropatterns stained for α-tubulin; treatments as indicated. Scale bar, 10 μm. Right, higher magnification of leading edge. (F) Quantification of whole-cell and cortex MTs (α-tubulin intensity) with indicated treatments, normalized to 1-2C EC controls. >2C, 20 ECs; >2C + Rac Inh, 20 ECs; >2C + Arp 2/3 Inh, 18 ECs. Error bars, 95% confidence intervals. (G) Time-lapse images of a representative EC on micropattern expressing mCherry-PA-Rac1 without 488-nm light exposure (dark; left) and subsequent to light exposure (light; right). Red boxes, higher-magnification areas to left. Time is indicated in seconds. (H) Time-lapse images of a representative EC on a micropattern expressing Venus-PA-Rac1 (C450A) light-insensitive mutant in conditions similar to those in G. Red boxes, higher-magnification areas to left. Time is indicated in seconds. (I) Time-lapse images of EC in sprouts expressing centrin::tdTomato to mark centrosomes, GFP::Rac1 (DN), and N-C axis superimposed. Scale bar, 10 μm. (J) Percentage of sprouting ECs that repolarize with expression of DN Rac::GFP. Control: 1-2C, 22 ECs; >2C, 17 ECs. DNRac: 1-2C, 10 ECs; >2C, 12 ECs. All p values are from two-tailed Student’s t test from at least three experiments, unless indicated otherwise. *p < 0.05; **p ≤ 0.01; ***p ≤ 0.001; NS, not significant; N, number of ECs analyzed.