Fig 2: Schematic illustration of the Rho signaling pathway at the TGN.GPCR signaling activates GEF-H1 by releasing it from microtubules. GEF-H1 in turn promotes an increase in cellular RhoA activity. At the TGN, active RhoA binds to its effector PLCe, which hydrolyzes PtdIns(4)P to generate DAG and IP2. DAG recruits and activates nPKCs and PKD, which gets further activated by nPKC-mediated phosphorylation. The activation of PKD by RhoA is counterbalanced by the RhoGAP DLC3. Active PKD recruits Rab8 to TGN membranes and induces the fission of Rab6-positive vesicles containing cargo such as TNFa destined for FA-targeted transport along microtubules.

Fig 3: 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 4: Lfc regulates microtubule-mediated adhesion resolution. (a–d) Cell shape outlines of non-treated control cells migrating under agarose under adhesive (a) or non-adhesive (PEG-coated; b) conditions. Cell shape outlines of nocodazole-treated cells migrating under agarose under adhesive (c) or non-adhesive (PEG-coated; d) conditions. Upper panels show littermate control wild-type cells. Lower panels show Lfc−/− cells. Scale bar, 10 µm. See also Video 10. (e) Cell lengths of non-treated control cells migrating under adhesive and non-adhesive conditions (n = minimum of 80 cells per condition from N = 5 experiments). Boxes extend from 25th to 75th percentiles. Whiskers span minimum to maximum values. Annotation above columns indicates results of Kruskal-Wallis with Dunn’s test; ****, P ≤ 0.0001. (f) Cell lengths of nocodazole-treated cells migrating under adhesive and non-adhesive conditions (n = minimum of 80 cells per condition from N = 5 experiments). Boxes extend from 25th to 75th percentiles. Whiskers span minimum to maximum values. Annotation above columns indicates results of Kruskal-Wallis with Dunn’s test; ****, P ≤ 0.0001. (g) Migration distance of Lfc+/+ and Lfc−/− DCs migrating under agarose under non-adhesive (PEG-coated) conditions of n = minimum of 80 cells per condition from N = 5 experiments. Cells were either non-treated or treated with nocodazole. Boxes extend from 25th to 75th percentiles. Whiskers span minimum to maximum values. Annotation above columns indicates results of one-way ANOVA; *, P ≤ 0.05; ****, P ≤ 0.0001. (h) Persistence of Lfc+/+ and Lfc−/− DCs migrating under agarose under non-adhesive conditions (PEG-coated). Cells were either non-treated or nocodazole-treated (n = minimum of 80 cells per condition from N = 5 experiments). Boxes extend from 25th to 75th percentiles. Whiskers span minimum to maximum values. Annotation above columns indicates results of Kruskal-Wallis with Dunn’s test; ****, P ≤ 0.0001. non-adhes., non-adhesive; n.s., not significant.

Fig 5: Inhibition of the CX3CL1/ICAM-1-mediated feedback cycle between circulating NSCLC cells and VBMECs by silencing CX3CR1 in A549 cells reduced NSCLC spinal metastasis and prolonged survival in mice. (A) Western blotting of CX3CR1 and LFA-1 protein levels in the indicated A549 cells. (B) Kaplan-Meier spinal metastasis-free curve of mice inoculated intracardially with control, CX3CR1-KD, CX3CR1-KD and Vector (CX3CR1-KD+Vector) or CX3CR1-KD and LFA-1-overexpression (CX3CR1-KD+LFA-1) A549 cells (n = 10). (C) Normalized BLI signals of spinal metastases. Data represent the mean ± standard deviation (SD) (n = 10). *P < 0.05 and NSP > 0.05. (D) Kaplan-Meier survival curve of mice. (E) Representative BLI, microCT, and histological (HE) images of bone lesions (B) invaded by tumors (T) in each group. Arrows indicate osteolytic bone lesions and dashed rectangles are used to mark the regions of interest for vertebral bone scan and analysis. (F) IHC images of p-PI3K, p-AKT, CX3CL1, and MMP-2 in tumor areas from serial sections of the indicated samples. (G, H) Quantification of osteolytic areas of spine and bone volume relative to total volume from microCT scans. Data represent the mean ± SEM (n = 5). **P < 0.01, ***P < 0.001, and NSP > 0.05. (I) mRNA levels of CX3CR1, LFA-1, Icam-1, and Gef-h1 in tumors of mice by RT-qPCR. Data represent the mean ± SEM (n = 10). *P < 0.05, **P < 0.01, ***P < 0.001, and NSP > 0.05.