Journal: Frontiers in cell and developmental biology

Article Title: Ena/VASP proteins at the crossroads of actin nucleation pathways in dendritic cell migration.

doi: 10.3389/fcell.2022.1008898

Figure Lengend Snippet: FIGURE 6 The VASP-EVH1 domain interacts with Wave2 and mDia1 in DCs. (A) Domain structure of Ena/VASP proteins and derived GST-fusion protein. EVH1, Ena/VASP homology 1 domain; EVH2, Ena/VASP homology 2 domain; PRR, Proline-rich region; GAB, G-actin binding site; FAB, F-actin binding site; GST: Glutathione S-transferase; FPPPP, Pro-rich motif recognized by EVH1 domain. (B–D) Recombinant GST-VASP-EVH1 and GST as control were purified from bacteria with GST-beads and incubated with detergent-extracted lysate of immature or mature DCs in the presence or absence of an EVH1-specific inhibitor. After thorough washing, proteins were eluted and analyzed by mass spectrometry (B,C) or by immunoblotting with the indicated antibodies (D). Volcano plots illustrate the inhibitor-sensitive hits obtained by mass spectrometry for iDCs (B) and mDCs (C). (D) Immunoblotting confirms that Abi1 and Wave2 (as representatives of the WRC) and mDia1 from iDCs and mDCs show specific binding to the EVH1 domain of VASP which is inhibited by the EVH1 domain-specific inhibitor.

Article Snippet: Antigen Species Clone name or catalog # Supplier IF WB FACS Comments α-Tubulin Mouse DM1A Cell signalling -- 1:500 -- unlabeled β-Actin Mouse Ac-15; A5441 Sigma-Aldrich -- 1:1,000 -- unlabeled CD11c Hamster N418; MCD11c05 Thermo Fisher Scientific -- -- 1:400 APC-labeled CD40 Mouse 3/23; #562846 BD Biosciences -- -- 1:400 Pacific Blue-labeled CD86 Rat GL1 eBioscience -- -- 1:100 APC-labeled EVL Rabbit TA343847 Origene -- 1:500 -- unlabeled GAPDH Mouse G8795 Sigma -- 1:1,000 -- unlabeled mDia1 Rabbit Ab129167; Ab96784 Abcam -- 1:1,000 -- unlabeled mDia1 Mouse Clone 51; 610,848 BD Biosciences 1:50 -- -- unlabeled Lamellipodin Rabbit -- Matthias Krause, Kings College London -- 1:20,000 -- unlabeled Mena Rabbit LS-C170270 LSBio -- 1:500 -- unlabeled p34/ARPC2 Rabbit 07-227-I Sigma-Aldrich 1:100 1:1,000 -- unlabeled Paxillin Rabbit Y113; ab32084 Abcam 1:400 -- -- unlabeled VASP Rabbit 9A2 Cell signalling -- 1:500 -- unlabeled Vinculin Mouse V9264 Sigma -- 1:1,000 -- unlabeled Vinculin Rabbit Ab73412 Abcam 1:400 -- -- unlabeled Wave2 Rabbit D2C8 Cell signalling -- 1:500–1:1,000 -- unlabeled Wave2 Mouse C-6; sc-373889 Santa Cruz Biotechnology 1:100 -- -- unlabeled Zyxin Rabbit Z4751 Sigma 1:400 1:500 -- unlabeled Frontiers in Cell and Developmental Biology frontiersin.org04 (Visweshwaran and Maritzen, 2019) with the following modifications: The migration chamber was coated with 10 μg/ml fibronectin for 1 h at 37°C using 150 μl of solution.

Techniques: Derivative Assay, Binding Assay, Recombinant, Control, Bacteria, Incubation, Mass Spectrometry, Western Blot

Journal: International Journal of Molecular Sciences

Article Title: Gα i 2 Induces Cell Migration in PC3 Prostate Cancer Cells in the Absence of Rac1 Activation

doi: 10.3390/ijms26062663

Figure Lengend Snippet: ( A ). Rac1-dependent activation of WAVE2 is impaired in the absence of Ga i 2. Western blot analysis of Gα i 2, p-Wave2 in PC3 cells constitutively overexpressing Rac1, with (+) or without (−) Gα i 2 siRNA. Total WAVE2 and α-Tubulin were used as loading controls. ( B ). Ratios of optical densities (mean ± SEM) of Phosho-Wave2/total Wave2 after different treatments (data from three replicate experiments).

Article Snippet: WAVE2 (Cat# WP1791) were obtained from ECM Biosciences (Versailles, KY, USA).

Techniques: Activation Assay, Western Blot

Journal: Oncotarget

Article Title: Stimulus-dependent dissociation between XB130 and Tks5 scaffold proteins promotes airway epithelial cell migration.

doi: 10.18632/oncotarget.13261

Figure Lengend Snippet: Figure 3: Stimulus-dependent translocation of endogenous XB130 and Tks5 to the cell membrane indicates distinct signaling roles. A. Immunoblots of cytoplasm (C) and membrane (M) fractionated BEAS-2B cell lysates. Cells were treated with or without 50 ng/mL EGF, 500 nM PDBu or 0.1 μM NNK. XB130 and WAVE2 expression and translocation from the cytoplasm to the cell membrane are more dependent on EGF stimulation, whereas, Tks5 and N-WASP expression and translocation are more dependent on PDBu and NNK stimulation. B. Ratio of normalized membrane expression to normalized cytoplasm expression. Expression of Na+/K+ ATPase was used to normalize membrane fractions and expression of GAPDH was used to normalize cytoplasmic fractions. Data is summarized from three independent experiments and presented as mean ± SD. * represents p < 0.01 compared to the corresponding no treatment group.

Article Snippet: Small interfering RNA (siRNA) targeting human AFAP1L2, human Tks5 and control siRNA and rabbit anti-N-WASP (H100) (1:750), WAVE2 (H110) (1:500), PAK1 (C-19) (1:250) and mouse anti-Tks5 (M300) (1:500) antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Translocation Assay, Membrane, Western Blot, Expressing

Journal: Oncotarget

Article Title: Stimulus-dependent dissociation between XB130 and Tks5 scaffold proteins promotes airway epithelial cell migration.

doi: 10.18632/oncotarget.13261

Figure Lengend Snippet: Figure 4: XB130 colocalizes robustly with WAVE2 at lamellipodia but not at podosomes with N-WASP, after stimulation. A.-B. Co-immunofluorescence staining of XB130 (green), actin (blue) and either WAVE2 (A) or N-WASP (B) (red). BEAS2B cells were treated with or without 50 ng/mL EGF, 500 nM PDBu or 0.1 μM NNK. No treatment control shows normal stress fibers. Stimulation with EGF, PDBu and NNK produces actin-rich ruffled areas at the cell membrane, which are indicative of lamellipodia via WAVE2 staining (A). These areas are also enriched with XB130 (A and B). PDBu and NNK induce formation of podosomes (white arrows) which are enriched by N-WASP but not XB130 (B ). D. Mander’s overlap co-efficient (MOC) of the cell periphery displays the relative colocalization of XB130 with WAVE2, Tks5 or N-WASP, where 0 represents no colocalization and 1 represents perfect colocalization. XB130 colocalizes robustly with WAVE2 at the lamellipodia and to a lesser extent with Tks5 and N-WASP, indicating it translocates to and is involved in lamellipodia formation. Data is summarized from 10 different cells per group from 3 different experiments and presented as mean ± SD. * represents p < 0.01 for XB130/N-WASP and XB130/Tks5 MOCs compared to XB130/WAVE2 MOCs.

Article Snippet: Small interfering RNA (siRNA) targeting human AFAP1L2, human Tks5 and control siRNA and rabbit anti-N-WASP (H100) (1:750), WAVE2 (H110) (1:500), PAK1 (C-19) (1:250) and mouse anti-Tks5 (M300) (1:500) antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Immunofluorescence, Staining, Control, Membrane

Journal: Oncotarget

Article Title: Stimulus-dependent dissociation between XB130 and Tks5 scaffold proteins promotes airway epithelial cell migration.

doi: 10.18632/oncotarget.13261

Figure Lengend Snippet: Figure 8: Schematic diagram of the role of XB130 and Tks5 in Rac1 and Cdc42-associated cytoskeletal remodeling for lung epithelial cell migration. Cell migration requires cytoskeleton remodeling mediated by the Arp2/3 complex, which results in the formation of branched, F-actin rich structures (red ball and sticks), such as lamellipodia and podosomes. This diagram shows the A. Top- down view and B. Side-view of a cell displaying lamellipodia and podosome. We demonstrated a novel mechanism for lung epithelial cell migration, in which extracellular factors stimulate a sub-population of XB130 to dissociate from Tks5 and translocate to the cell periphery to promote Rac1-activated signaling of WAVE2-associated lamellipodia formation for cell extension and Tks5 mediation of Cdc42 activity via PAK1 interaction for the promotion of N-WASP-associated podosome assembly and function for ECM-dependent cell migration. Dashed black lines represent translocation of XB130 and Tks5 to the cell membrane.

Article Snippet: Small interfering RNA (siRNA) targeting human AFAP1L2, human Tks5 and control siRNA and rabbit anti-N-WASP (H100) (1:750), WAVE2 (H110) (1:500), PAK1 (C-19) (1:250) and mouse anti-Tks5 (M300) (1:500) antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Migration, Activity Assay, Translocation Assay, Membrane

Journal: Cancer Management and Research

Article Title:

WAVE2 Enhanced Hepatic Stellate Cells Activity in Colorectal Liver Metastases

doi: 10.2147/cmar.s259125

Figure Lengend Snippet: Figure 1 Association between WAVE2 protein expression and hepatic micro-metastasis in human CLM. (A) Profiles of sinusoid-associated micro-metastasis (SAM) and portal-associated micro-metastasis (PAM) following H&E staining. (B) Detection of CLM in hepatic tissue sections via immunohistochemical staining with CK20. (C) Profiles of CD31-labeled MVD in CLM. (D) Immunohistochemical staining of WAVE2 protein expression in SAM and PAM. Scale bar=50 μm. (E) PAM lesions exhibit higher WAVE2 protein levels than SAM lesions. Quantification of WAVE2 expression by H-score is also shown. *** P<0.001. (F) The relationship between WAVE2 and MVD in CLM. *** P<0.001.

Article Snippet: Packaging lentiviruses encoding non-targeting short hairpin RNAs (sh-NT) and WAVE2 shRNA (shWAVE2) were purchased from Santa Cruz Biotechnology (NM_001201404-Q9Y6W5 and NM_006990-605875; Cat# sc-36833-V), whereas si-YAP was acquired from Guangzhou RiboBio (Guangzhou, China).

Techniques: Expressing, Staining, Immunohistochemical staining, Labeling

Journal: Cancer Management and Research

Article Title:

WAVE2 Enhanced Hepatic Stellate Cells Activity in Colorectal Liver Metastases

doi: 10.2147/cmar.s259125

Figure Lengend Snippet: Figure 3 WAVE2/YAP1 signaling is a critical driver of activation of HSC processes. (A) Western blots of HSC activation showing lower levels of WAVE2, α-SMA and p-SMAD2 in Si-YAP1-transfected HSC cells relative to controls. (B) IF analysis for WAVE2 (green) and p-SMAD2 (red).

Article Snippet: Packaging lentiviruses encoding non-targeting short hairpin RNAs (sh-NT) and WAVE2 shRNA (shWAVE2) were purchased from Santa Cruz Biotechnology (NM_001201404-Q9Y6W5 and NM_006990-605875; Cat# sc-36833-V), whereas si-YAP was acquired from Guangzhou RiboBio (Guangzhou, China).

Techniques: Activation Assay, Western Blot, Transfection

Journal: Cancer Management and Research

Article Title:

WAVE2 Enhanced Hepatic Stellate Cells Activity in Colorectal Liver Metastases

doi: 10.2147/cmar.s259125

Figure Lengend Snippet: Figure 2 WAVE2 knockdown inhibits activation of HSCs into tumor-associated myofibroblasts. (A) Downregulation of WAVE2 expression via shWAVE2 lentiviral knockdown in human HSCs cells. Western blots showing efficiently-knocked down WAVE2. (B) HSCs transduced with shNC or shWAVE2 lentiviruses, treated with TGF-β1 (2.5 ng/mL) and subjected to IF for α-SMA (green). WAVE2 knockdown consistently suppressed TGF-β1 activation of HSCs into myofibroblasts. Bar=50 mm. **P<0.01, ***P<0.001; n=5 randomly picked microscopic fields. (C) Control and WAVE2 knockdown HSCs, serum-starved and treated with TGF-β1 after 24 hours. Cell lysates were subjected to Western blot analysis for detection of HSC activation markers, α-SMA and p-SMAD2.

Article Snippet: Packaging lentiviruses encoding non-targeting short hairpin RNAs (sh-NT) and WAVE2 shRNA (shWAVE2) were purchased from Santa Cruz Biotechnology (NM_001201404-Q9Y6W5 and NM_006990-605875; Cat# sc-36833-V), whereas si-YAP was acquired from Guangzhou RiboBio (Guangzhou, China).

Techniques: Knockdown, Activation Assay, Expressing, Western Blot, Transduction, Control

Journal: Cancer Management and Research

Article Title:

WAVE2 Enhanced Hepatic Stellate Cells Activity in Colorectal Liver Metastases

doi: 10.2147/cmar.s259125

Figure Lengend Snippet: Figure 4 WAVE2 knockdown reduces stimulatory effects of HSCs on tumor prognosis in mice. (A) HT29 cells (0.5×106) mixed with 0.5×106 a-HSCs expressing either sh- NC or sh-WAVE2 were implanted into nude mice by orthotropic transplantation. Images of dissected tumors from mice (left) and the tumor size between the HT29+HSC- shNC and HT29+HSC-shWAVE2 groups (right) were compared. Each bar represents mean ± SD of six mice per group. (B) IF staining for α-SMA detection showing that WAVE2 knockdown HSCs impairs HT29 tumor growth in mice. *** P<0.001 (C) IHC staining of CD31 in xenograft. *** P<0.001.

Article Snippet: Packaging lentiviruses encoding non-targeting short hairpin RNAs (sh-NT) and WAVE2 shRNA (shWAVE2) were purchased from Santa Cruz Biotechnology (NM_001201404-Q9Y6W5 and NM_006990-605875; Cat# sc-36833-V), whereas si-YAP was acquired from Guangzhou RiboBio (Guangzhou, China).

Techniques: Knockdown, Expressing, Transplantation Assay, Staining, Immunohistochemistry

Journal: Cancer Management and Research

Article Title:

WAVE2 Enhanced Hepatic Stellate Cells Activity in Colorectal Liver Metastases

doi: 10.2147/cmar.s259125

Figure Lengend Snippet: Figure 5 Schematic diagram showing the effect of WAVE2 on HSC processes and paracrine signaling in the tumor microenvironment. WAVE2 was associated with regulatory functions of the TGF-β1/YAP1 signaling in CLM pathogenesis.

Article Snippet: Packaging lentiviruses encoding non-targeting short hairpin RNAs (sh-NT) and WAVE2 shRNA (shWAVE2) were purchased from Santa Cruz Biotechnology (NM_001201404-Q9Y6W5 and NM_006990-605875; Cat# sc-36833-V), whereas si-YAP was acquired from Guangzhou RiboBio (Guangzhou, China).

Techniques:

Journal: Frontiers in Oncology

Article Title: BTG1 Overexpression Might Promote Invasion and Metastasis of Colorectal Cancer via Decreasing Adhesion and Inducing Epithelial–Mesenchymal Transition

doi: 10.3389/fonc.2020.598192

Figure Lengend Snippet: Antibodies used for Western blot.

Article Snippet: WAVE2 (C-6) , Mouse , Santa Cruz Biotechnology.

Techniques: Western Blot

Journal: Heliyon

Article Title: Mangiferin inhibits chronic stress-induced tumor growth in colorectal liver metastases via WAVE2 signaling pathway

doi: 10.1016/j.heliyon.2023.e13753

Figure Lengend Snippet: MGF reduces promoting effects of HSCs on tumor growth in mice. (A) HT-29 cells (0.5 × 10 6 ) mixed with 0.5 × 10 6 a-HSCs expressing tumors were dissected from nude mice, and tumor size was compared between the HT29+HSC-Control and HT29+HSC-MGF groups (left). (B) MVD in HT29+HSC-CUMS and HT29+HSC- MGF group. (C) Masson's trichrome staining of xenograft. (D) IF staining for α-SMA detection shows that MGF inhibits HT-29 tumor cell growth in mice. (E)Western blot showed that expression of β2-AR, WAVE2, VEGF and α-SMA decreased significantly in HT29+HSC-MGF group. Data are presented as the mean ± S.E.M. *P < 0.05, **P < 0.01, ***P < 0.001.

Article Snippet: The stock solution was used to prepare different concentrations in the test media. including β2-AR polyclonal antibody (Cat. #8513, CST), WAVE2 polyclonal antibody (Cat. # YT4898, ImmunoWay), VEGF polyclonal antibody (Cat. # sc-7269, Santa Cruz Biotechnology), α-smooth muscle actin (α-SMA) (Abcam, Cat. # ab5694), a polyclonal antibody against p -SMAD2 (Cat. #3104, CST) at a 1:1000 and GAPDH mouse monoclonal antibody (Cat. # ab8245, Abcam) at 1:5000 dilution.

Techniques: Expressing, Control, Staining, Western Blot

Journal: Heliyon

Article Title: Mangiferin inhibits chronic stress-induced tumor growth in colorectal liver metastases via WAVE2 signaling pathway

doi: 10.1016/j.heliyon.2023.e13753

Figure Lengend Snippet: MGF inhibits activation of HSCs via the WAVE2 signaling pathway. (A) Transduced HSCs treated with TGF-1 (2.5 ng/mL) and IF for SMA show that MGF consistently suppresses TGF-1-induced myofibroblast differentiation. (B) Following 24 h of serum starvation and treatment with TGF-1, control and MGF-treated HSCs are shown. Western blot analysis for detection of HSC activation markers, α-SMA and p -SMAD2, and activator β2-AR and TGF-β1. Data are presented as the mean ± S.E.M. *P < 0.05, **P < 0.01, ***P < 0.001.

Article Snippet: The stock solution was used to prepare different concentrations in the test media. including β2-AR polyclonal antibody (Cat. #8513, CST), WAVE2 polyclonal antibody (Cat. # YT4898, ImmunoWay), VEGF polyclonal antibody (Cat. # sc-7269, Santa Cruz Biotechnology), α-smooth muscle actin (α-SMA) (Abcam, Cat. # ab5694), a polyclonal antibody against p -SMAD2 (Cat. #3104, CST) at a 1:1000 and GAPDH mouse monoclonal antibody (Cat. # ab8245, Abcam) at 1:5000 dilution.

Techniques: Activation Assay, Control, Western Blot