Journal: Cellular and molecular life sciences : CMLS

Article Title: PI(3,4,5)P3-mediated Cdc42 activation regulates macrophage podosome assembly.

doi: 10.1007/s00018-025-05664-2

Figure Lengend Snippet: Fig. 3 Identification of VAV1 as a PI(3,4,5)P3-dependent Cdc42 GEF. A RT-qPCR analysis of GEF expression in pre-differentiated monocyte and TGF-β1 induced macrophage. Among 19 Cdc42 GEFs, VAV1 exhibits the highest expression. B, C EBFP-VAV1 is actively recruited to the plasma membrane upon the stimulation of EGF (100 ng/mL) in MEF. D Endogenous VAV1 is enriched at the podosome in THP-1 macrophage. Podosome core and ring are identi- fied by the staining of CF680R-phalloidin and anti-Paxillin, respec- tively. The insets represent the boxed regions. Anti-VAV1 staining can be found at the podosome core (inset), as well as other paxillin- positive adhesion sites (arrowheads). E Intensity line scan along the

Article Snippet: EGFP-Cdc42 (plasmid #20142), pC.HA VAV1 (plasmid #14553), pC.HA VAV3 (plasmid #14555), GFP-wGBD (plasmid #26734) and pDONR223PIK3CB (plasmid #23543) were obtained from Addgene.

Techniques: Quantitative RT-PCR, Expressing, Clinical Proteomics, Membrane, Staining

Journal: Cellular and molecular life sciences : CMLS

Article Title: PI(3,4,5)P3-mediated Cdc42 activation regulates macrophage podosome assembly.

doi: 10.1007/s00018-025-05664-2

Figure Lengend Snippet: Fig. 4 Membrane association of VAV1 is essential for macrophage migration and gelatin degradation. A, B Reintroductions of wildtype GFP-VAV1, rather than GFP-VAV1-W495L or GFP-VAV1-E201A restore the podosome assembly in VAV1-knockdown THP-1 mac- rophages. Podosome core and ring are identified by the staining of CF680R-phalloidin and anti-Paxillin, respectively. The insets repre- sent the boxed regions. C, D Degradation of Cy3-labeled gelatin in the indicated conditions. Representative images are stitched from 5 × 5 tile scan. Gelatin degradation activity of VAV1-knockdown THP-1 macrophage is suppressed, compared to the control. Express- ing wildtype GFP-VAV1, as opposed to VAV1-W495L or GFP- VAV1-E201A restores the gelatin degradation (arrowheads). F-actin

Article Snippet: EGFP-Cdc42 (plasmid #20142), pC.HA VAV1 (plasmid #14553), pC.HA VAV3 (plasmid #14555), GFP-wGBD (plasmid #26734) and pDONR223PIK3CB (plasmid #23543) were obtained from Addgene.

Techniques: Membrane, Migration, Knockdown, Staining, Labeling, Activity Assay, Control

Journal: Cellular and molecular life sciences : CMLS

Article Title: PI(3,4,5)P3-mediated Cdc42 activation regulates macrophage podosome assembly.

doi: 10.1007/s00018-025-05664-2

Figure Lengend Snippet: Fig. 5 Summary of PI(3,4,5)P3-VAV1-Cdc42 signaling cascade. The production of PI(3,4,5)P3 by PIK3CB is an upstream signal to recruit VAV1 to the plasma membrane. Activated VAV1 then promotes the level of GTP-bound Cdc42. Together with anionic lipids, locally con- centrated Cdc42-GTP activates WASP-mediated podosome assembly in the macrophage

Article Snippet: EGFP-Cdc42 (plasmid #20142), pC.HA VAV1 (plasmid #14553), pC.HA VAV3 (plasmid #14555), GFP-wGBD (plasmid #26734) and pDONR223PIK3CB (plasmid #23543) were obtained from Addgene.

Techniques: Clinical Proteomics, Membrane

Journal: Molecular Biology of the Cell

Article Title: The scaffold RhoGAP protein ARHGAP8/BPGAP1 synchronizes Rac and Rho signaling to facilitate cell migration

doi: 10.1091/mbc.e21-03-0099

Figure Lengend Snippet: FIGURE 5: BPGAP1 induces cancer cell motility, invadopodium formation, and extravasation in zebrafish larvae via the RacGEF activity of Vav1. (A, B) Vav1 is required for BPGAP1-induced Rac1 activation. MCF7 cells expressing vector control or BPGAP1 were (A) treated with Vav inhibitor, azathioprine (5 μM) or (B) transfected with siRNA specific for Vav1. Cells were seeded on collagen-coated plates for 30 min, followed by active Rac1 pull-down assay. All data are represented as mean ± SEM (n = 4 [A] and 3 [B] independent experiments). ** represents P < 0.01, and * represents P < 0.05. A one-way ANOVA test was used to compare the different conditions to the control group. (C) Vav1 is required for BPGAP1-induced cell migration. MDA-MB-231 cells stably expressing wild-type and mutants of BPGAP1 and Vav1 were seeded on collagen-coated dishes and imaged by time-lapse microscopy for 2 h. Three independent experiments were performed and are represented as mean ± SEM. Data sharing different letters are statistically significant at P < 0.05, two-way ANOVA test. (D) BPGAP1 promotes longer invadopodia via Vav. MDA-MB-231-mCherry or mCherry-BPGAP1 cells were seeded on the micropit topographic features for 6 h. Cells were treated with azathioprine 2 h after seeding. All data are represented as mean ± SEM (n = 3 independent experiments >1000 data points). ** represents P < 0.01. Unpaired two-tailed T test. (E) BPGAP1 promotes cancer cell extravasation in zebrafish larvae via Vav1. (i) MDA-MB-231-mCherry or mCherry-BPGAP1 cells were injected into the yolk of transgenic zebrafish (fli-1-EGFP) larvae 48 hpf and fixed at 70 hpf. Cancer cell extravasation is indicated by white arrows in the representative merged images. Scale bar: 100 μm. (ii) Cancer cell extravasation was quantified. Data sharing different letters are statistically significant at P < 0.05, two-way ANOVA test.

Article Snippet: 16 | D. C. P. Wong, C. Q. Pan, et al. Molecular Biology of the Cell Immunohistochemical staining of breast tissue microarrays Immunohistochemical staining of TMAs was performed with BPGAP1 (in-house production) and VAV1 antibodies (Acris-OriGene; Catalogue: AP06359PU-N) using the Bond Max automated immunohistochemistry vision Biosystem (Leica Microsystems GmbH, Germany) based on the manufacturer’s protocol.

Techniques: Activity Assay, Activation Assay, Expressing, Plasmid Preparation, Control, Transfection, Pull Down Assay, Migration, Stable Transfection, Time-lapse Microscopy, Two Tailed Test, Injection, Transgenic Assay

Journal: Molecular Biology of the Cell

Article Title: The scaffold RhoGAP protein ARHGAP8/BPGAP1 synchronizes Rac and Rho signaling to facilitate cell migration

doi: 10.1091/mbc.e21-03-0099

Figure Lengend Snippet: FIGURE 7: Schematic model depicting BPGAP1 as a switch that synchronizes Rac and Rho signaling during cancer cell motility and invasion. (A) Model for how BPGAP1 synchronizes Rho/ Rac signaling during cell motility. The autoinhibited BPGAP1 is released upon growth factor stimulation and cell spreading (step 1). BPGAP1 recruits Rac1 and Vav1 (step 2; asterisk represents the “open conformation” of BPGAP1) to activate Rac1 (step 3) while inactivating RhoA, leading to reduced actomyosin contractility (step 4) and enhanced protrusive lamellipodia that collectively drive cancer cell motility and invasion (step 5). (B) BPGAP1 synchronizes Rac1/ RhoA signaling. Upon growth factor stimulation and cell spreading, active BPGAP1 promotes Rac1 activation and RhoA inactivation (left panel), whereas the inactive BPGAP1 maintains a high level of RhoA activity that also impairs Rac1 binding to BPGAP1, thus preventing Rac1 from being activated (right panel). Please refer to the text for more details. “T” symbol refers to inhibition and the symbol refers to inactive RhoGAP.

Article Snippet: 16 | D. C. P. Wong, C. Q. Pan, et al. Molecular Biology of the Cell Immunohistochemical staining of breast tissue microarrays Immunohistochemical staining of TMAs was performed with BPGAP1 (in-house production) and VAV1 antibodies (Acris-OriGene; Catalogue: AP06359PU-N) using the Bond Max automated immunohistochemistry vision Biosystem (Leica Microsystems GmbH, Germany) based on the manufacturer’s protocol.

Techniques: Activation Assay, Activity Assay, Binding Assay, Inhibition