Fig 2: RKIP activates RhoA through GEF-H1. (a) Representative western blots of VAV2 GEF activity pull-down assays with lysates prepared from BT20 cells. Numbers are shown for quantified active VAV2 bands normalized with total VAV2 protein. (b) (Left panel) Representative western blots of lysates prepared from control knockdown (shCTR), or five different VAV2 knockdowns (shVAV22.1–2.5) BT20 cells. (Middle panel) Representative images of immunofluorescent staining with E-cad Ab and DRAQ5 of the same set of BT20 cells shown in the left top panel. (Right panel) E-cad intensity quantification on cell–cell junctions of immunofluorescent images shown in the left bottom panels using MetaMorph analysis software. (c) (Left panel) Representative western blots of lysates prepared from control knockdown (shCTR), or two different GEF-H1 knockdowns (shGEF-H12.2–2.3) BT20 cells. (Middle panel) Representative images of immunofluorescent staining with E-cad Ab and DRAQ5 of the same set of BT20 cells shown in the left top panel. (Right panel) E-cad intensity quantification on cell–cell junctions of immunofluorescent images shown in the left bottom panels using MetaMorph analysis software. (d) (Top left panel) Representative images of immunofluorescent staining with E-Cad Ab and DRAQ5 of BT20 cells with different combinations of control knockdown, GEF-H1 knockdown (shGEF-H1), empty vector control (EVC) or RKIP expression. (Bottom left panel) E-cad intensity quantification on cell–cell junctions of immunofluorescent images shown in the left bottom panels using MetaMorph analysis software. (Right panel) Representative western blots of lysates prepared from the same set of cells shown in the left panel. (e) (Left panel) Representative results of invasion assays with same set of cells used in (d) showing number of invaded cells through Matrigel in each indicated BT20 cell lines (mean ± SE). (Right panel) Representative images of the stained invaded cells in indicated cell lines shown at the left panel. Unpaired Student's t-test (two-tailed) was used for all analyses with p < 0.05 considered significant. ns not significant.

Fig 3: HV-107 inhibits Rac activity in breast cancer cell lines. To examine the effects of HV-107 on MDA-MB-231 (A–D) and MDA-MB-468 (E–H) triple negative breast cancer cells, a 24-h treatment with HV-107 ranging from 0 to 2000 nM was conducted. Pulldown assays utilizing the p21-binding domain of PAK were employed to isolate the active form of Rac (GTP bound), followed by western blot analysis. The western blot images presented in panels A and E illustrate the levels of both total Rac and GTP-bound Rac in MDA-MB-231 and MDA-MB-468 cells treated with various concentrations of HV-107. The quantification of total Rac levels is depicted in panels B and F, while the quantification of GTP-bound Rac levels is shown in panels C and G. Panels D and H represent the ratio of GTP-bound Rac to total Rac levels. I, J Pulldowns with RacG15A beads followed by western blots were performed to investigate Rac-Vav2 binding capacity in response to 250 nM HV-107. The western blot images and quantifications depict the levels of Vav2 bound to the RacG15A beads. Actin was used as a loading control. N = 3–4. Error bars represent ± SEM; * p ≤ 0.05

Fig 4: Role of DOCK180 in the effects of PTP1B inhibitor on EC motility.(A) Western blots showing the endogenous expression levels of Vav2, Tiam1 and DOCK180. IP, immunoprecipitation; WB, western blot. (B,C) Effects of gene silencing for DOCK180 with different siRNA constructs on the stimulatory effects of PTPI22 on EC spreading and migration. (D) Effects of gene silencing for Vav2 with different siRNA constructs on the stimulatory effect of PTPI22 on EC spreading. Data were mean ± S.E.M.; *P < 0.05 vs vehicle control, one-way ANOVA, n = 3–5.

Fig 5: The proposed mechanism of how CCL2 enhances cell motility of HNSCC.The proposed models described the effect of CCL2 on cell migration and tumor metastasis in HNSCC. From a macroscopic perspective, HNSCC can metastasize to distant sites through lymphatics and blood vessels, among which cervical lymph node metastasis was most frequent. From a microscopic perspective, HNSCC cells can secrete CCL2 which could bind to the membrane receptor CCR4 by autocrine or paracrine function. Then, the PI3K pathway transduces extracellular signals into the cell and activates the Vav2-Rac1-MLC signal axis. Finally, CCL2 promotes HNSCC cytoskeletal remodeling and pseudopodia formation, enhances the mobility of HNSCC cells, and promotes the metastasis of HNSCC.