Journal: Advanced Science

Article Title: Beyond Catalytic Therapy: Copper‐Paeonol Nanozymes Disrupt Fascin‐Mediated Actin Bundling to Suppress Tumor Growth and Metastasis

doi: 10.1002/advs.202512186

Figure Lengend Snippet: Schematic illustration. A) Scheme for the self‐assembly of Cu 2+ , Mg 2+ , Mn 2+ , Ca 2+ or Co 2+ with paeonol to form different nanostructures. B) CuPaeNs triggering oxidative damage and disrupting fascin‐mediated actin bundling for tumor therapy.

Article Snippet: Fascin polyclonal antibody (1:800) was purchased from Proteintech (Wuhan, China).

Techniques:

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: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Fascin1 Promotes Cell Migration of Mature Dendritic Cells §

doi: 10.4049/jimmunol.1001667

Figure Lengend Snippet: Characterization of wild type and fascin1 null DCs. A, FACS analyses of wild-type (red line) and fascin1-deficient (blue line), mature DCs. Black lines, controls without antibody labeling. a, CD11c; b, MHC-II; c, CD86; d, fascin1. e & f, FACS analyses of wild type (e) and KO (f) DCs double-labeled with CD86 and fascin1 antibodies. B, Western blot analyses. Immature (lM, lanes 1 and 3) and mature (M, lanes 2 & 4) DCs prepared from wild type (lanes 1 & 2) and fascin1 KO (lanes 3 & 4) mice were analyzed with the indicated antibodies. C, Immunofluorescent localization of fascin1 and CD86 in mature wild-type (a–c) and fascin1 KO (d–f) DCs. Mature DCs were double labeled with anti-CD86 (a and d, green) and mouse anti-fascin1 (b and e, red) antibodies. c & f, merged images. Bar, 10µm. D, Localization of fascin1 and F-actin in wild type (a–f) and fascin1 KO (g–l) DCs. Cells were labeled with chick anti-fascin1 (a, d, g & j, green) and rhodamine phalloidin (b, e, h & k, red). Images were taken at the ventral surface (a–c & g–i), as well as at the middle (d–f & j–l) of the same cells. c, f, i, & l, merged images. Arrowheads indicate co-localization of fascin1 and F-actin at the cortex of veil-like protrusions. Bar, 10µm.

Article Snippet: Mature or immature DCs were transfected with a human GFP-fascin1 fusion construct ( 26 ) using an Amaxa Nucleofector II according to the manufacture’s instructions.

Techniques: Antibody Labeling, Labeling, Western Blot

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Fascin1 Promotes Cell Migration of Mature Dendritic Cells §

doi: 10.4049/jimmunol.1001667

Figure Lengend Snippet: Fascin1 expression and actin-bundling activity is critical for podosome disassembly of mature DCs. A, Immunofluorescence of immature (a & b) and mature (c & d) DCs from wild-type (a & c) and fascin1 null (b & d) mice labeled with anti-vinculin (red) and anti-CD11c (green) antibodies. Arrows, podosomes with the characteristic ring structure; arrowheads, focal adhesions. Bar, 15µm. B, Mature fascin1high DCs (arrow) show no podosome assembly. Bar, 10µm. C, Statistical analyses of podosome assembly. CD11c-positive immature (blue bar) and mature (red) DCs with at least five vinculin-positive podosomes, were judged as DCs with podosomes. Wild-type, mature DCs with very high fascin1 expression (more than 10 times higher than background, pink bar) were also examined for podosome assembly. D, Effects of forced expression of GFP control (a–c), GFP-Wild-type-fascin1 (d–f), GFP-A-fascin1 (g–i) and GFP-D-fascin1 (j–l) on podosome assembly. Fascin1 null DCs were transfected with GFP control (a–c), as well as with wild type and fascin1 mutants, and stained with anti-vinculin antibody (red, b, e, h, k). GFP signal (green, a, d, g, j); merged images (c, f, i, l). Arrowheads in j–l show podosomes. Bar, 10µm. E, Statistical analyses of podosome loss. Podosomes were counted in DCs exogenously expressing control GFP, GFP-W-fascin1, GFP-A-fascin or GFP-D-fascin, and cells were categorized as having less than 4 podosomes or more than 5 podosomes.

Article Snippet: Mature or immature DCs were transfected with a human GFP-fascin1 fusion construct ( 26 ) using an Amaxa Nucleofector II according to the manufacture’s instructions.

Techniques: Expressing, Activity Assay, Immunofluorescence, Labeling, Control, Transfection, Staining

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Fascin1 Promotes Cell Migration of Mature Dendritic Cells §

doi: 10.4049/jimmunol.1001667

Figure Lengend Snippet: Fascin1 is critical for membrane dynamics. A–C, Time-lapse, phase-contrast microscopy was performed with wild type (A) and fascin1-null (B,C) DCs that had been plated on glass coverslips. B shows a fascin1-null DC with a spread shape, the dominant phenotype; C shows a cell with a rounded shape, a less common phenotype. Numbers are in sec. Bar, 5 µm. D–F, kymographs generated from the white one-pixel lines at time 0 in A–C, respectively. Dashed lines in D–F indicate the boundary of the membrane protrusions. G & H, box plot analyses of protrusion (G) and retraction (H) rates calculated from kymographs. The bottom and top of each box indicate the first and third quartiles, respectively, and dots indicate outliers. Four asterisks, p<0.0001. I & J, Phase-contrast, time-lapse images of fascin1-null mature DCs exogenously expressing GFP (I) or GFP-fascin1 (J). The last images show fluorescent images at the end (20min) of imaging to confirm GFP-expression. Numbers are in sec. Bar, 5 µm. K & L, kymographs of GFP (K)-, and GFP-fascin1 (L)-expressing DCs generated from the time-lapse images in I and J, respectively. M & N, box plot analyses of protrusion (M) and retraction (N) rates of fascin1-null DCs exogenously expressing GFP or GFP-fascin1.

Article Snippet: Mature or immature DCs were transfected with a human GFP-fascin1 fusion construct ( 26 ) using an Amaxa Nucleofector II according to the manufacture’s instructions.

Techniques: Membrane, Microscopy, Generated, Expressing, Imaging

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Fascin1 Promotes Cell Migration of Mature Dendritic Cells §

doi: 10.4049/jimmunol.1001667

Figure Lengend Snippet: Fascin1 is critical for chemotaxis and Langerhans cell migration into draining lymph nodes. A, In vitro chemotaxis toward the chemokine CCL19, measured with a modified Boyden chamber. (p=0.0095). B, Immunofluorescence imaging of Langerhans cells in ear epidermal sheets from wild type (a & c) and fascin1 null (b & d) mice without (control, a & b) and with FITC painting (+allergen, c & d). Langerhans cells (indicated by arrowheads) were labeled with the MHC-II antibody. Representative images from four independent experiments. C, Box plot analyses of Langerhans cell distribution without or with allergen treatment. NS, no statistical significance; one asterisk, p<0.05; two asterisks, p<0.01, three asterisks, p<0.001; four asterisks, p<0.0001. D, Box plot analysis of FITC-bearing DCs migrated into draining lymph nodes. P=0.0059.

Article Snippet: Mature or immature DCs were transfected with a human GFP-fascin1 fusion construct ( 26 ) using an Amaxa Nucleofector II according to the manufacture’s instructions.

Techniques: Chemotaxis Assay, Migration, In Vitro, Modification, Immunofluorescence, Imaging, Control, Labeling

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Fascin1 Promotes Cell Migration of Mature Dendritic Cells §

doi: 10.4049/jimmunol.1001667

Figure Lengend Snippet: Morphological characterization of wild type and fascin1 null DCs. A, Scanning electron micrographs of wild-type (a) and fascin1-deficient (b) mature DCs. Bar, 10 µm. B, Orthogonal views of wild-type (a) and fascin1-deficient (b), CD86high DCs. Mature DCs were double stained with phalloidin and anti-CD86 antibody. Only phalloidin staining is shown here. The xy images are on the ventral surface. Both xz and yz images are shown with the top and bottom of cells indicated by dashed lines. Bar, 5 µm. C, D and E, Box blot analyses of thickness (C), area (D) and circularity (E) of wild-type and fascin1-deficient DCs. Thickness was determined without (w/o cfg) or with (w. cfg) cytospin.

Article Snippet: Mature or immature DCs were transfected with a human GFP-fascin1 fusion construct ( 26 ) using an Amaxa Nucleofector II according to the manufacture’s instructions.

Techniques: Staining

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Fascin1 Promotes Cell Migration of Mature Dendritic Cells §

doi: 10.4049/jimmunol.1001667

Figure Lengend Snippet: Fascin1 is localized to podosomes in THP-1 cells and microinjection of fascin1 induces podosome disassembly. A, Co-localization of fascin1 and α-actinin at podosomes. THP-1 cells differentiated with TPA were labeled with anti-fascin1 (b & e, green) and anti-α-actinin (red, a & d) antibodies. c & f, merged images. Images in d–f show enlargements of the boxed areas in a–c. Arrowheads, podosomes. B, Disassembly of podosomes by microinjection of fascin1. THP-1 cells were microinjected with FITC-labeled BSA (a) or GFP-fascin1 (c) and counter-stained with phalloidin (b & d). Arrows indicate injected cells while arrowheads indicate podosomes. C, Percentage of cells with podosome arrays of un-injected cells (green); or after microinjection of fascin1 (red) or FITC-BSA (blue). Representative data from three independent experiments is shown. Approximately 20 injected cells for each condition were counted for each set of experiments.

Article Snippet: Mature or immature DCs were transfected with a human GFP-fascin1 fusion construct ( 26 ) using an Amaxa Nucleofector II according to the manufacture’s instructions.

Techniques: Microinjection, Labeling, Staining, Injection

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