Journal: Carcinogenesis

Article Title: Abl interactor 1 regulates Src-Id1-matrix metalloproteinase 9 axis and is required for invadopodia formation, extracellular matrix degradation and tumor growth of human breast cancer cells

doi: 10.1093/carcin/bgp251

Figure Lengend Snippet: Abi1 is found in invadopodia in MDA-MB-231 cells. (A) Expression and complex formation of Abi1 with Sra, Nap1, WAVE2 and N-WASP. The MDA-MB-231 cells were lysed and immunoprecipitated (IP) with pre-immune serum (Pre-IP) or anti-Abi1-specific antibodies (Abi1-IP), as indicated. The immunoprecipitates, along with total cell lysate (TCL), were separated on 8% SDS–polyacrylamide gel electrophoresis (PAGE), transferred to nitrocellulose membranes and subjected to western blot (WB) analysis using the indicated antibodies. (B) Abi1 colocalizes with invadopodia. The MDA-MB-231 cells were incubated with anti-Abi1 specific antibody and subsequently stained with Alexa-conjugated secondary antibodies (green). The cells were then counterstained with Alexa-conjugated phalloidin to visualize F-actin (red). Abi1 location in invadopodia is shown by an arrow in the merged image (merge); bar: 10 μm. (C) Abi1 is found in ECM degradation sites. MDA-MB-231 cells were grown on coverslips coated with a thin layer of FITC-conjugated gelatin (green). Cells were incubated with Abi1 antibody and subsequently stained with Alexa-conjugated secondary antibody (red). Degradation is indicated as dark patches within the fluorescent monolayer (upper panel). Abi1 is found in degraded area, as shown by arrows. (D) Expression of GFP and GFP–Abi1 in MDA-MB-231 cells. The MDA-MB-231 cells were transfected with plasmids expressing GFP alone or GFP-tagged Abi1, as indicated. Total cell lysate containing 50 μg protein was separated on SDS–PAGE and analyzed by western blot using indicated antibodies. (E) GFP–Abi1 is found in invadopodia-like structures. The MDA-MB-231 cells expressing GFP–Abi1 were counterstained with Alexa-conjugated phalloidin (red). The subcellular localization of GFP–Abi1 and F-actin was examined by fluorescence microscopy. GFP–Abi1 was found in F-actin-enriched puncta, as indicated by arrow. (F) F-actin-enriched puncta colocalize with ECM degradation sites. MDA-MB-231 cells were grown on coverslips coated with a thin layer of FITC-conjugated gelatin (green). Cells were stained with Alexa-conjugated phalloidin (red) and examined by fluorescence microscopy. Degradation is indicated as dark patches within the fluorescent monolayer. The F-actin-enriched puncta locate in the degraded area, as indicated by arrows; bar: 10 μm. (G) GFP–Abi1 colocalizes with cortactin. The MDA-MB-231 cells expressing GFP–Abi1 were incubated with anti-cortactin-specific antibody and subsequently stained with Alexa-conjugated secondary antibodies (red). The subcellular localization of GFP–Abi1 and cortactin was examined by fluorescence microscopy. The colocalization of Abi1 with cortactin is shown by the merged image (merge). The arrows indicate Abi1, invadopodia and their colocalization; bar: 10 μm.

Article Snippet: The polyclonal antibodies against N-WASP, WAVE2 and c-Src were purchased from Santa Cruz Biotechnology (Santa Cruz, CA).

Techniques: Expressing, Immunoprecipitation, Polyacrylamide Gel Electrophoresis, Western Blot, Incubation, Staining, Transfection, SDS Page, Fluorescence, Microscopy

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