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. 2014 Oct 1;127(19):4186–4200. doi: 10.1242/jcs.150227

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© 2014. Published by The Company of Biologists Ltd

This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution and reproduction in any medium provided that the original work is properly attributed.

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Fig. 6. — GEF-H1-mediated FA recruitment of NMIIB controls stress fiber polarization and FA formation. (A) Serum-starved non-silencing (a–c), GEF-H1-silencing MSCs expressing pLKO vector (mock) (d–f), pLKO-GEFH1 (g–i) or pLKO-GEF-H1_DH(m) (j–l), and NMIIB-silencing MSCs (m–o) were treated with Dex (0.1 µM, 6 h), and immunostained with FITC–phalloidin, to localize F-actin (a, d, g, j and m). Scale bar: 20 µm. After image segmentation, the image was rotated to set the median stress-fiber-orientation as horizontal (b, e, h, k and n). The orientation of each fiber was depicted with a specific color as indicated by the color at the right-hand side of each panel (c, f, i, l and o). (B,C) The polar histograms (B) and spread distribution (C) of stress fiber orientations were statistically calculated from non-silencing (n = 19 cells), or GEF-H1-silencing MSCs expressing pLKO vector (mock) (n = 18 cells), pLKO-GEFH1 (n = 15 cells) or pLKO-GEF-H1_DH(m) (n = 15 cells), and NMIIB-silencing MSCs (n = 14 cells). Data are mean±s.e.m. ***P<0.0001 (C, top: compared with non-silencing; bottom: compared with mock). (D) Serum-starved non-silencing, NMIIB-silencing, and GEF-H1-silencing MSCs expressing pLKO-vector (mock), pLKO-GEFH1 or pLKO-GEF-H1_DH(m) were treated with Dex (0.1 µM, 6 h), immunostained with paxillin, to visualize FAs, and imaged by TIRF microscopy. Scale bar: 20 µm. (E) The number of segmented paxillin-marked FAs of MSCs, as described in D. Data are mean±s.e.m. (non-silencing, n = 8 cells; NMIIB-silencing, n = 6 cells; GEF-H1-silencing cells expressing mock, n = 9 cells; expressing pLKO-GEF-H1, n = 7 cells; expressing pLKO-GEF-H1_DH(m), n = 6 cells). **P<0.005, ***P<0.0001 (top, compared with non-silencing; bottom, compared with mock). (F) Size distribution of segmented paxillin-marked FAs of MSCs, as described in D. Data are mean±s.e.m. (non-silencing, n = 8 cells and 1506 FAs; NMIIB-silencing, n = 6 cells and 581 FAs; GEF-H1-silencing expressing mock, n = 9 cells and 835 FAs; expressing pLKO-GEF-H1, n = 7 cells and 1358 FAs; expressing pLKO-GEF-H1_DH(m), n = 6 cells and 537 FAs). *P<0.05; **P<0.005 (top, compared with non-silencing; bottom, compared with mock).