Journal: Laboratory investigation; a journal of technical methods and pathology

Article Title: Fascin is involved in tumor necrosis factor-alpha-dependent production of MMP9 in cholangiocarcinoma.

doi: 10.1038/labinvest.2009.89

Figure Lengend Snippet: Figure 1 Immunohistochemical expression of fascin and MMP9 in extrahepatic, hilar, and intrahepatic cholangiocarcinoma (CC). Fascin and MMP9 were expressed in CC cells in a granular cytoplasmic pattern. Images of fascin and MMP9 are in almost the same field of CC. (All images, original magnification 200.)

Article Snippet: Fascin siRNA (h) (sc-35359) was purchased from Santa Cruz Biotech.

Techniques: Immunohistochemical staining, Expressing

Journal: Laboratory investigation; a journal of technical methods and pathology

Article Title: Fascin is involved in tumor necrosis factor-alpha-dependent production of MMP9 in cholangiocarcinoma.

doi: 10.1038/labinvest.2009.89

Figure Lengend Snippet: Figure 2 Frequencies and intensities of expressions of fascin, MMP9, and MMP2 in cholangiocarcinoma (CC). *, Po0.05 vs non-neoplastic epithelium; **, Po0.05 vs extrahepatic CC.

Article Snippet: Fascin siRNA (h) (sc-35359) was purchased from Santa Cruz Biotech.

Techniques:

Journal: Laboratory investigation; a journal of technical methods and pathology

Article Title: Fascin is involved in tumor necrosis factor-alpha-dependent production of MMP9 in cholangiocarcinoma.

doi: 10.1038/labinvest.2009.89

Figure Lengend Snippet: Figure 4 Postoperative survival rates of patients with extrahepatic, hilar, and intrahepatic cholangiocarcinoma (CC). When all cases were examined, the patients with fascin expression showed more unfavorable prognosis compared with those without fascin expression. However, fascin expression did not influence postoperative survival of patients with extrahepatic or hilar CC, when examined separately. In contrast, fascin expression was clearly an unfavorable prognostic factor in the patients with intrahepatic CC.

Article Snippet: Fascin siRNA (h) (sc-35359) was purchased from Santa Cruz Biotech.

Techniques: Expressing

Journal: Laboratory investigation; a journal of technical methods and pathology

Article Title: Fascin is involved in tumor necrosis factor-alpha-dependent production of MMP9 in cholangiocarcinoma.

doi: 10.1038/labinvest.2009.89

Figure Lengend Snippet: Figure 5 Expression of fascin and MMP9 in two cholangiocarcinoma (CC) cell lines (CCKS-1 and HuCCT1). (a) Fascin and MMP9 were expressed in both cell lines at an mRNA level (RT–PCR). CCKS-1 had more intense expression levels of fascin and MMP9 mRNAs compared with HuCCT1. (b) Western blotting showed fascin protein expression in both cell lines. (c) Zymography also showed MMP9 expression in two cell lines, although active form of MMP (arrow) was expressed in only CCKS-1.

Article Snippet: Fascin siRNA (h) (sc-35359) was purchased from Santa Cruz Biotech.

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Western Blot, Zymography

Journal: Laboratory investigation; a journal of technical methods and pathology

Article Title: Fascin is involved in tumor necrosis factor-alpha-dependent production of MMP9 in cholangiocarcinoma.

doi: 10.1038/labinvest.2009.89

Figure Lengend Snippet: Figure 6 Alterations of fascin and MMP9-expression levels in cholangiocarcinoma (CC) cell lines after TNF-a treatment. (a) TNF-a treatment (12 h) dose-dependently upregulated fascin and MMP9 expression at the mRNA level. (b) Real-time PCR clearly showed TNF-a-induced overexpressions of fascin and MMP were dose dependent. (c) Expression of fascin protein was increased by TNF-a treatment (48 h) in HuCCT1. In contrast, its expression was not significantly changed in CCKS-1, which had intense expression of fascin mRNA even before TNF-a treatment. (d) TNF-a (50 ng/ml) time-dependently upregulated fascin and MMP9 expression at the RNA level. (e) Real-time PCR also showed that TNF-a time-dependently upregulated expressions of fascin and MMP9, and they peaked around at 12 h after the treatment of TNF-a. (f) Zymography showed that TNF-a treatment dose-dependently and time-dependently upregulated expression of MMP9 protein including active forms. *, Po0.05 vs without TNF-a treatment; w, Po0.01 vs without TNF-a treatment.

Article Snippet: Fascin siRNA (h) (sc-35359) was purchased from Santa Cruz Biotech.

Techniques: Expressing, Real-time Polymerase Chain Reaction, Zymography

Journal: Laboratory investigation; a journal of technical methods and pathology

Article Title: Fascin is involved in tumor necrosis factor-alpha-dependent production of MMP9 in cholangiocarcinoma.

doi: 10.1038/labinvest.2009.89

Figure Lengend Snippet: Figure 7 Expression of fascin in CCKS-1 after transfection of siRNA against fascin. (a, b) Transfection of fascin siRNA successfully induced a knockdown of fascin expression at mRNA and protein levels. (c) Knockdown of the fascin expression was observed from 24 to 72 h post transfection at mRNA level, and from 48 to 120 h post transfection at protein level.

Article Snippet: Fascin siRNA (h) (sc-35359) was purchased from Santa Cruz Biotech.

Techniques: Expressing, Transfection, Knockdown

Journal: Laboratory investigation; a journal of technical methods and pathology

Article Title: Fascin is involved in tumor necrosis factor-alpha-dependent production of MMP9 in cholangiocarcinoma.

doi: 10.1038/labinvest.2009.89

Figure Lengend Snippet: Figure 8 Alteration of fascin or MMP9 expression in cultured cholangiocarcinoma cell line after treatment with TNF-a alone, with fascin siRNA alone, or with TNF-a and fascin siRNA. At 12 h after transfection of siRNA, some groups of cells were treated with TNF-a (50 ng/ml). At 48 h after transfection, the cells were used for RNA or protein extraction. (a) In both CCKS-1 and HuCCT1, TNF-a treatment (50 ng/ml) induced the overexpression of both fascin and MMP9. (b) Real-time PCR showed that knockdown of the fascin expression by siRNA significantly inhibited TNF-a-induced overexpression of fascin and MMP9. (c) Zymography also showed that fascin siRNA inhibited overexpression of MMP9 protein including active forms (arrow). *, Po0.05 vs control and control siRNA; w, Po0.05 vs with TNF-a treatment and control siRNA with TNF-a treatment.

Article Snippet: Fascin siRNA (h) (sc-35359) was purchased from Santa Cruz Biotech.

Techniques: Expressing, Cell Culture, Transfection, Protein Extraction, Over Expression, Real-time Polymerase Chain Reaction, Knockdown, Zymography, Control

Journal: Laboratory investigation; a journal of technical methods and pathology

Article Title: Fascin is involved in tumor necrosis factor-alpha-dependent production of MMP9 in cholangiocarcinoma.

doi: 10.1038/labinvest.2009.89

Figure Lengend Snippet: Figure 9 Inhibitory studies using TNFR antibodies and intracellular signal inhibitors. (a) TNFR1 antibody inhibited TNF-a-induced overexpressions of fascin and MMP9 more clearly compared with TNFR2 antibody. (b) Real-time PCR showed that TNFR1 antibody significantly inhibited overexpression of fascin and MMP9 in both CCKS-1 and HuCCT1. In contrast, TNFR2 antibody inhibited overexpression of MMP9 only in CCKS-1. (c) MG132 (an NF-kB inhibitor), U0126 (an Erk1/2 inhibitor), and SB203580 (a p38 MAPK inhibitor) inhibited TNF-a-induced overexpressions of fascin and MMP9. (d) Those inhibitions were significant based on findings of real-time PCR. In contrast, LY294002 (a PI3K inhibitor) did not influence TNF-a-induced overexpressions of fascin and MMP9 (MG, MG132; LY, LY294002; U, U0126; SB, SB203580). *, Po0.05 vs control; w, Po0.05 vs with TNF-a treatment.

Article Snippet: Fascin siRNA (h) (sc-35359) was purchased from Santa Cruz Biotech.

Techniques: Real-time Polymerase Chain Reaction, Over Expression, Control

Journal: Laboratory investigation; a journal of technical methods and pathology

Article Title: Fascin is involved in tumor necrosis factor-alpha-dependent production of MMP9 in cholangiocarcinoma.

doi: 10.1038/labinvest.2009.89

Figure Lengend Snippet: Figure 10 Schematic diagrams of the possible involvement of fascin in the activation process of cholangiocarcinoma invasion.

Article Snippet: Fascin siRNA (h) (sc-35359) was purchased from Santa Cruz Biotech.

Techniques: Activation Assay

Journal: Science Advances

Article Title: Mesothelial cells promote peritoneal invasion and metastasis of ascites-derived ovarian cancer cells through spheroid formation

doi: 10.1126/sciadv.adu5944

Figure Lengend Snippet: ( A ) Schematic showing the comparison between RNA expression in OV90 and mesothelial cells. ( B and C ) PCA plot of (B) OV90 and (C) HPMCs. ( D ) Volcano plot and clustering of RNA expression changes in OV90. The red line indicates an adjusted P value <0.05. ( E ) Volcano plot and clustering of RNA expression changes in HPMCs. The red line represents an adjusted P value <0.05. The right side of the volcano plot represents a fold change. ( F ) Significant up-regulated pathway changes in mesothelial cells after interaction with OV90 in KEGG. ( G ) Significant up-regulated pathway changes in mesothelial cells after interaction with OV90 in the GO term. ( H ) Significant down-regulated pathway changes in mesothelial cells after interaction with OV90 in KEGG. ( I ) Significant down-regulated pathway changes in mesothelial cells after interaction with OV90 in the GO term. ( J and K ) PROGENy pathway activity analysis of the ascites samples of the Zheng et al. EOC scRNA-seq dataset revealed high TGF-β pathway activity in both EOC and mesothelial cells. ( L ) Bar plot showing the concentration of TGF-β1 in the supernatant from HPMCs, TGF-β1–stimulated HPMCs, and OV90 cells. ( M ) Scheme of an invadopodium in a mesothelial cell. ( N ) Immunofluorescence images of a single cell invading the collagen layer using invadopodium formation. Green, cortactin; red, phalloidin. Scale bars, 10 μm. ( O ) The number of invadopodia was significantly higher in TGF-β1–stimulated mesothelial cells. ( P ) Strategy to detect candidates with a high invasion ability in mesothelial cells. ( Q ) Western blot analysis of fascin-1 and several proteins related to invadopodium formation. ( R ) Immunofluorescence images of fascin-1 or myosin X (green) in TGF-β1–stimulated mesothelial cells. Scale bars, 5 μm. FACS, fluorescence-activated cell sorting; FC, fold change. *** P < 0.001.

Article Snippet: In some experiments, a FI (NP-G2-044, T9107, TargetMol, MA) was added to evaluate the role of fascin-1 in mesothelial cell–mediated collagen degradation.

Techniques: Comparison, RNA Expression, Activity Assay, Concentration Assay, Immunofluorescence, Single Cell, Western Blot, Fluorescence, FACS

Journal: Science Advances

Article Title: Mesothelial cells promote peritoneal invasion and metastasis of ascites-derived ovarian cancer cells through spheroid formation

doi: 10.1126/sciadv.adu5944

Figure Lengend Snippet: ( A and B ) Violin plots showing the expression of invadopodium-related genes across cell components in ascites on the basis of two different scRNA-seq datasets from Izar et al. and Zheng et al. . In the dataset of (A), mesothelial cells are classified as fibroblasts. ( C ) Collagen degradation assay. The thickness represents the cell invasion ability. Scale bars, 400 μm. ( D ) Bar graph showing the thickness of remnant collagen 48 hours after incubation. ( E ) Bar graph showing the number of invadopodia. sh-Fascin-1 or sh-myosin X inhibited invadopodium maturation. ( F and G ) 3D images and bar graph showing that spheroids invade collagen with shRNA-induced mesothelial cells (green) and OV90 (red). The invasion ability of mesothelial cells was significantly inhibited by sh- FSCN1 or sh- MYO10 . Scale bars, 200 μm. ( H ) Scheme of the malignant ascites in vivo model using shRNA-treated HPMCs. ( I and J ) Images and bar graph showing the differences in the metastasis area on the omentum from mice 1 week after the injection of OV90 with or without sh-induced mesothelial cells. Scale bars, 1 mm. ( K ) Representative IHC image of mouse tissue with fascin-1. Invasive stromal cells strongly expressed fascin-1. Scale bar, 100 μm. ( L ) IHC of metastasis samples in clinical samples. Fascin-1–positive stromal cells were present in the tumor-invasive regions. Scale bar, 100 μm. ( M ) Kaplan-Meier plot showing the patient’s progression-free survival depending on fascin-1 expression in stromal cells or cancer cells. Fascin-1 expression in stromal cells in metastasis samples was significantly related to a worse prognosis ( P = 0.030). * P < 0.05, ** P < 0.01, and *** P < 0.001.

Article Snippet: In some experiments, a FI (NP-G2-044, T9107, TargetMol, MA) was added to evaluate the role of fascin-1 in mesothelial cell–mediated collagen degradation.

Techniques: Expressing, Degradation Assay, Incubation, shRNA, In Vivo, Injection

Journal: Science Advances

Article Title: Mesothelial cells promote peritoneal invasion and metastasis of ascites-derived ovarian cancer cells through spheroid formation

doi: 10.1126/sciadv.adu5944

Figure Lengend Snippet: Almost all the EOC cells identified in the ascites were in a spheroids formation and 65% were accompanied by mesothelial cells, referred to as ACMSs. The formation of ACMSs enabled EOC cells to alter the RNA expression profiles of mesothelial cells via TGF-β related pathway. These alternations increased the expression of fascin-1 in this pathway, which caused invadopodia formations in mesothelial cells to mature, and this degraded collagen with MMP14. Mesothelial cells interacted with EOC cells, which aggressively invaded the collagen and mesothelial layer. These results show that EOC cells can induce peritoneal metastasis without direct dynamic RNA expression changes. EOC cells then followed the route created by the mesothelial cells. This model explains that EOC cells control the unique tumor microenvironment in ascites to rapidly induce abdominal dissemination.

Article Snippet: In some experiments, a FI (NP-G2-044, T9107, TargetMol, MA) was added to evaluate the role of fascin-1 in mesothelial cell–mediated collagen degradation.

Techniques: RNA Expression, Expressing, Control

Journal: Toxicology letters

Article Title: Fascin2 regulates cisplatin-induced apoptosis in NRK-52E cells

doi: 10.1016/j.toxlet.2016.11.021

Figure Lengend Snippet: Fascin2 Knockdown Increases Susceptibility to Cisplatin-Induced Nephrotoxicity. A. shRNA knockdown of fascin2 expression by four different targeting constructs; significant knockdown was seen with A1, B1, and D1 constructs. The impact on fascin1 and fascin2 expression is shown in B. C. Cell viability was determined by the MTT assay in cells treated 150 μM cisplatin for the indicated time periods. The results are presented as the percent viability of untreated NRK/V1 cells in SF media. D. The activities of caspase 3/7 in cells treated with 150 μM cisplatin for the indicated time periods were determined by luminescence. Data points represent the mean ± SE of four samples; *indicates a significant difference from control; similar results were seen in replicate experiments.

Article Snippet: The following antibodies were used: fascin1 (GeneTex, GTX63842; 1:1000), fascin2 (NOVUS; Ab78599, 1:1000), BH3 interacting-domain death agonist (BID; NOVUS, NB100–56106, 1:1000), B-cell lymphoma 2 (bcl-2; Cell Signaling, 2876, 1:1000), cleaved poly (ADP-ribose) polymerase (PARP; Sigma, SAB4500487, 1:1000), α(E)-catenin (GeneTex, GTX 61621, 1:1000) and anti-β-actin (Sigma, A2228, 1:2500).

Techniques: shRNA, Expressing, Construct, MTT Assay

Journal: Toxicology letters

Article Title: Fascin2 regulates cisplatin-induced apoptosis in NRK-52E cells

doi: 10.1016/j.toxlet.2016.11.021

Figure Lengend Snippet: Fascin2 Colocalizes with α-Catenin and the Actin Cytoskeleton; Overexpression of Fascin2 Rescues Stress Fibers in C2Cells. A. Immunofluorescence images (60x) of NRK-52E cells treated with rabbit anti-fascin2 (red), FITC-phalloidin (green) or anti-α-catenin (green) and DAPI (blue) demonstrates that fascin2 colocalizes with both actin and α-catenin. B. Overexpression of fascin1 (C2/Fscn1) or fascin2 (C2/Fscn2) in C2 cells. C. Phalloidin staining demonstrates increased actin stress fibers in C2/Fscn2 cells, while the bottom panel shows stress fibers using cell surface scanning with AFM. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: The following antibodies were used: fascin1 (GeneTex, GTX63842; 1:1000), fascin2 (NOVUS; Ab78599, 1:1000), BH3 interacting-domain death agonist (BID; NOVUS, NB100–56106, 1:1000), B-cell lymphoma 2 (bcl-2; Cell Signaling, 2876, 1:1000), cleaved poly (ADP-ribose) polymerase (PARP; Sigma, SAB4500487, 1:1000), α(E)-catenin (GeneTex, GTX 61621, 1:1000) and anti-β-actin (Sigma, A2228, 1:2500).

Techniques: Over Expression, Immunofluorescence, Staining

Journal: Scientific Reports

Article Title: Hydrogen Sulfide Recruits Macrophage Migration by Integrin β1-Src-FAK/Pyk2-Rac Pathway in Myocardial Infarction

doi: 10.1038/srep22363

Figure Lengend Snippet: ( a ) Surface proteins of RAW264.7 cells were biotinylated and separated by streptavidin beads. Integrin β1 and integrin β3 levels were detected by western blot (I: internalized proteins from cell surface; II: total cell lysates; III: cell surface proteins). ( b ) RAW264.7 cells were incubated with NaHS (100 μM) for the indicated time and surface levels of integrin β1 were labeled with the FITC-conjugated secondary antibody, followed by Flow cytometric analysis. ( c ) RAW264.7 cells were incubated with NaHS (100 μM) for the indicated time. Surface Integrin β1 was stained with FITC-conjugated antibody (green); F-actin bundles were detected with Rhodamine (Red); Nuclei were counterstained with DAPI (blue). Scale bar, 5 μm. All the data are representative of three independent experiments. ( d ) After siRNA-integrin β1 or siRNA-NC transfection for 24 h, RAW264.7 cells were incubated without or with NaHS (100 μM) for 6 h, and then the migratory capacities of the cells were evaluated by transwell assay. Values are the mean ± SD from three independent experiments. ( e ) RAW264.7 cells transfected with siRNA-integrin β1 were treated with NaHS (100 μM) for 6 h and the phosphor-Src, -Pyk2, -FAK 397 , -FAK 925 , total Src and FAK were analyzed by western blot (NC, negative control; p, phosphorylated; t, total).

Article Snippet: And the siRNA to mouse integrin β1 were chemically synthesised by Shanghai GenePharma Co., Ltd.

Techniques: Western Blot, Incubation, Labeling, Staining, Transfection, Transwell Assay, Negative Control

Journal: Scientific Reports

Article Title: Hydrogen Sulfide Recruits Macrophage Migration by Integrin β1-Src-FAK/Pyk2-Rac Pathway in Myocardial Infarction

doi: 10.1038/srep22363

Figure Lengend Snippet: ( a ) After specific siRNA transfection for 24 h, RAW264.7 cells were treated with NaHS (100 μM) for 6 h, the interference efficiency of CSE was confirmed by quantitative real-time PCR. ( b , c ) After siRNA-CSE or siRNA-NC transfection for 24 h, RAW264.7 cells were incubated without or with NaHS (100 μM) for 6 h, and then the migratory capacities of the cells was evaluated by transwell assay. Values are the mean ± SD from three independent experiments. Scale bar, 50 μm. ( d ) RAW264.7 cells transfected with siRNA-CSE were treated with NaHS (100 μM) for 6 h and the expression of phospho-Pyk2, -FAK, total Src and FAK and integrin β1 in total cell lysates (I) or cell surface (II) were analyzed by western blot (NC, negative control; p, phosphorylated; t, total).

Article Snippet: And the siRNA to mouse integrin β1 were chemically synthesised by Shanghai GenePharma Co., Ltd.

Techniques: Transfection, Real-time Polymerase Chain Reaction, Incubation, Transwell Assay, Expressing, Western Blot, Negative Control