Fig 2: Vimentin depletion results in increased activity and phosphorylation of GEF-H1. (A) Active GEF-H1 was co-sedimented with GST–RhoA-G17A, and detected by western blotting using an anti-GEF-H1 antibody. The lower panel shows the quantification of normalized relative levels of GEF-H1 co-sedimenting with GST–RhoA-G17A compared to total GEF-H1 levels in cell lysates from five western blots. ***P<0.001 (paired t-test). (B) Western blot analysis of GEF-H1 phosphorylated on Ser886 and total GEF-H1 levels in control and vimentin-knockout cells. The blots were also probed with vimentin antibody to confirm that the vimentin-knockout cell culture is not contaminated by wild-type cells, and with GADPH antibody to verify equal sample loading. The lower panel shows the quantification of normalized relative levels of P-GEF-H1 (Ser886) compared to total GEF-H1 levels from five western blots. ***P<0.001 (paired t-test). Molecular masses in kilodaltons (kDa) are indicated. (C) G-LISA analysis of the levels of active RhoA in wild-type, phospho-mimic (S886D) and phospho-deficient (S886A) GEF-H1-expressing cells. The data are from five independent experiments and were normalized to control cells. **P<0.01 (paired t-test). (D) Tpm4.2 levels are increased in cells expressing the phospho-mimic (S886D) GEF-H1 mutant, but not in cells expressing the phospho-deficient (S886A) mutant. Upper panels show representative images of control cells and cells expressing wild-type or mutant GEF-H1 (arrowheads). The lower panel shows the quantification of normalized relative Tpm4.2 fluorescence intensities (wild-type GEF-H1: 32 control cells from nine images and 31 transfected cells from nine images; GEF-H1-S886D: 32 control cells from ten images and 35 transfected cells from total ten images; GEF-H1-S886A: 32 control cells from nine images and 38 transfected cells from nine images). Mean intensity values of control and GEF-H1 overexpression cells from each image were used for statistical analysis. *P<0.05; ***P<0.001 (paired t-test). (E) P-MLC levels are increased in cells expressing the phospho-mimic (S886D) GEF-H1 mutant, but not in cells expressing the phospho-deficient (S886A) mutant. Upper panels show representative images of control cells and cells expressing wild-type or mutant GEF-H1 (arrowheads). The lower panel shows the quantification of normalized relative P-MLC fluorescence intensities (wild-type GEF-H1: 35 control cells from nine images and 29 transfected cells from nine images; GEF-H1-S886D: 35 control cells from ten images and 33 transfected cells from ten images; GEF-H1-S886A: 35 control cells from nine images and 28 transfected cells from nine images). Mean intensity values of control and GEF-H1 overexpression cells from each image were used for statistical analysis. *P<0.05, ***P<0.001 (paired t-test). The data are presented as mean±s.e.m. N.S., not significant. Scale bars: 10 µm.

Fig 3: GEF-H1 suppression by vimentin does not involve FAK and its downstream kinases. (A) Western blot analysis of GEF-H1 phosphorylated on Ser886 and total GEF-H1 levels in control and vimentin-depleted U2OS cells incubated in the presence or absence of FAK inhibitor FAK-14 (left panel) or MEK inhibitor U0126 (right panel). The blots were also probed with GADPH antibodies to verify equal sample loading. Molecular masses in kilodaltons (kDa) are indicated. (B) Quantification of normalized relative levels of P-GEF-H1 (Ser886) compared to total GEF-H1 levels from five western blots for each condition. *P<0.05, **P<0.01, ***P<0.001 (paired t-test). (C,D) Immunostainings demonstrating that neither FAK-14 nor U0126 compounds could suppress the increased Tpm4.2 and P-MLC levels that were induced by depletion of vimentin. Wild-type cells in each panel are indicated with arrowheads. Panels on the right show the quantifications of normalized relative Tpm4.2 (C, FAK14: 28 control cells from six images and 29 vimentin-knockout cells from six images; U0126: 31 control cells from seven images and 28 vimentin-knockout cells from seven images) and P-MLC (D, FAK14: 33 control cells from seven images and 34 vimentin-knockout cells from seven images; U0126: 28 control cells from six images and 27 vimentin-knockout cells from six images) fluorescence intensities. Mean intensity values of control and vimentin-knockout cells from each image were used for statistical analysis. ***P<0.001 (paired t-test). The data are presented as mean±s.e.m. N.S., not significant. Scale bars: 10 µm.

Fig 4: TTFields activate GEF-H1 and inhibit cancer cell motility in a Rho-dependent manner (A,B) A-172 and LN-229 glioblastoma cells were treated with TTFields for the indicated times and RhoA activation was assessed. Mean + SEM; paired t-test. (C) Western blot analysis of GEF-H1 phosphorylated on Ser886 and the total GEF-H1 levels in control and TTFields-treated cells (top panel). Quantification of normalized relative levels of P-GEF-H1 compared to total GEF-H1 levels from two western blots for each condition (bottom panel). (D) ROCK activity in A-172, LN-229, and U-87 MG cells treated with TTFields for 24 h. (E,F) Migration responses of (E) T-cells and (F) dendritic cells using the modified Boyden chamber either with or without CCL19. ns, not significant. Mean + SEM; paired t-test. * p < 0.05.

Fig 5: A model illustrating the mechanism by which TTFields modulates cancer cell motility. (1) Microtubules are required to specify the direction of cell movement. GEF-H1 catalytic activity is downregulated through microtubule binding. (2) TTFields exert directional forces on polar tubulins leading to their alignment in the direction of the field. This in turn, leads to the reorganization of the microtubule network resulting in changes in the number of microtubules, interference with the directionality of cellular migration, and initiation of the GEF-H1/RhoA/ROCK signaling pathway (3) to control actin dynamics (4) and formation of focal adhesions (5), which disrupt cell polarity and migration directionality. a, microtubule; b, TTFields; c, tubulin aligned with field; d, GEF-H1; e, active GEF-H1; f, Rho-A GDP; g, GTP; h, GDP; i, Rho-A GTP; j, actin fiber; k, integrin; l, focal adhesion; m, extracellular matrix.