Journal: Breast Cancer Research and Treatment

Article Title: Study of breast cancer incidence in patients of lymphangioleiomyomatosis

doi: 10.1007/s10549-016-3737-8

Figure Lengend Snippet: Positivity for mTORC1 signaling and metastatic markers in breast tumors of LAM patients. a Results of phospho-Ser235/236-ribosomal protein S6 (pS6) staining in two available breast tumors. Heterogeneity (i.e., positive and negative tumor cells in case #1) and positive cells with a spindle phenotype (case #2, right panels , depicted in insets ) can be observed ( arrows mark magnified regions). b Results of the analysis of the metastatic markers (FSCN1, ID1, and SOX9) in both cases. Heterogeneity (particularly in case #1) and spindle-like phenotypes (particularly in case #2) can be observed ( arrows mark magnified regions)

Article Snippet: The antibodies used in this study were anti-ERα (#IR151, Dako), anti-FSCN1 (#SC-56531, Santa Cruz Biotechnology), anti-HMB-45 (#SC-59305, Santa Cruz Biotechnology), anti ID1 (#SC-488, Santa Cruz Biotechnology), anti-PR (#IR168, Dako), anti-phospho-Ser235-236 S6 ribosomal protein (anti-pS6; clone 91B2, Cell Signaling Technology), anti-SMA (#A2547, Sigma-Aldrich), and anti-SOX9 (#AB5535, Millipore).

Techniques: Staining

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: The following primary antibodies were used: fascin-1 (Merck Millipore, MAB3582), myosin X (Novus Biologicals, 22430002), integrin β1 (BD Biosciences, 610467), cortactin (BD Biosciences, 610049), Tks5 (Santa Cruz Biotechnology, sc-30122), and HIF1A (R&D Systems, 241809).

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: The following primary antibodies were used: fascin-1 (Merck Millipore, MAB3582), myosin X (Novus Biologicals, 22430002), integrin β1 (BD Biosciences, 610467), cortactin (BD Biosciences, 610049), Tks5 (Santa Cruz Biotechnology, sc-30122), and HIF1A (R&D Systems, 241809).

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: The following primary antibodies were used: fascin-1 (Merck Millipore, MAB3582), myosin X (Novus Biologicals, 22430002), integrin β1 (BD Biosciences, 610467), cortactin (BD Biosciences, 610049), Tks5 (Santa Cruz Biotechnology, sc-30122), and HIF1A (R&D Systems, 241809).

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 Expression is Decreased During Acute Injury. A. A loss of fascin2 protein expression was seen in C2, but not NT3, cells following challenge with 150 μM cisplatin for 12–24 h. B. Densitometric analysis of three replicate experiments; protein expression is shown as the fold decrease from control (C2, 0 h). Each data point represents the mean + SE of three samples, * indicates a significant difference from control. C. Fascin2 expression is decreased in aged (24 mon), but not young (4 mon) kidney 72 h (peak of injury) after cisplatin challenge (2.75 mg/kg). Each lane is a sample from an individual rat. D. Densitometric analysis of four replicate experiments; protein expression is shown as the fold decrease from control (4 mon control). Each data point represents the mean + SE of four samples, * indicates a significant difference from control.

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: 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: Knockdown, shRNA, Expressing, Construct, MTT Assay, 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 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: 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 Rescues the Increased Susceptibility to Cisplatin-Induced Nephrotoxicity. A. Overexpression of fascin2 attenuates loss of viability in C2 cells relative to NT3. B. Overexpression of fascin2 attenuates markers of apoptosis in C2 cells, including BID, PARP cleavage and Bcl-2 expression. C. Activation of caspase3/7 is decreased in C2 cells by fascin2 overexpression. 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: Over Expression, Expressing, Activation Assay, 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 Rescues Mitochondrial Dysfunction in Cisplatin-Induced Nephrotoxicity. A. Fascin2 attenuates loss of ATP following cisplatin challenge. B. Oxygen consumption of complex I and II in C2 cells is corrected by fascin2 overexpression. C. Cisplatin induces increased oxidative stress in C2 cells; an effect attenuated by fascin2. Data points represent the mean ± SE of 12 replicates; *indicates a significant difference from control (NT3/V).

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, Control

Journal: Technology in cancer research & treatment

Article Title: Fascin Overexpression Promotes Cholangiocarcinoma RBE Cell Proliferation, Migration, and Invasion.

doi: 10.1177/1533034615580696

Figure Lengend Snippet: Figure 4. Fascin overexpression inhibited E-cadherin expression in cholangiocarcinoma RBE cells. A, E-cadherin mRNA expression was detected in each group of cells by real-time PCR. B, E-cadherin was detected by immunofluorescence. Fluorescence microscopy showed that E-cadherin was highly expressed in the cells of parental group and the pcDNA3.1 group (red), and the nuclei were stained blue by DAPI. Scale bars 20 mm. C, E-cadherin protein expression levels were detected by Western blot, and b-actin served as an internal control for the grayscale analysis. The data are presented as the mean + standard deviation. Compared with the pcDNA3.1 transfection group, ** indicates P < .01. Compared with the parental group, ## indicates P < .01. mRNA indicates messenger RNA; PCR, polymerase chain reaction; cDNA, comple- mentary DNA; DAPI, 40,6-diamidino-2-phenylindole.

Article Snippet: The fascin (1:100 dilution, Boster) and E-cadherin antibodies (1:100 dilution, Wanleibio) were added, followed by incubation at 4 C overnight.

Techniques: Over Expression, Expressing, Real-time Polymerase Chain Reaction, Immunofluorescence, Fluorescence, Microscopy, Staining, Western Blot, Control, Standard Deviation, Transfection, Polymerase Chain Reaction

Journal: Technology in cancer research & treatment

Article Title: Fascin Overexpression Promotes Cholangiocarcinoma RBE Cell Proliferation, Migration, and Invasion.

doi: 10.1177/1533034615580696

Figure Lengend Snippet: Figure 5. The regulation of E-cadherin expression by fascin was independent of NF-kB signaling pathway. A, Cytoplasmic p-IkB and nuclear NF-kB p65 protein expression levels were detected by Western blot, and the grayscale analysis was performed with b-actin and Lamin A as the internal controls, respectively. The data are presented as the mean + standard deviation. B and C, E-cadherin protein expression was detected by Western blot. For the NF-kB inhibitor treatment group, BAY11-7082 was added to a final concentration of 3 mmol/L and incubated for 24 hours before analyzing E-cadherin expression. For the NF-kB siRNA intervention group, cell transfection was performed according to the manufacturer’s instructions. Representative results are shown in this figure. The data are presented as the mean + standard deviation. Compared with the pcDNA3.1 transfection group, ** indicates P < .01. Compared with the parental group, ## indicates P < .01. Compared with the pcDNA3.1þBAY11-7082 group, $$ indicates P < .01. NF-kB indicates nuclear factor-kB; siRNA, small interfering RNA; cDNA, comple- mentary DNA.

Article Snippet: The fascin (1:100 dilution, Boster) and E-cadherin antibodies (1:100 dilution, Wanleibio) were added, followed by incubation at 4 C overnight.

Techniques: Expressing, Western Blot, Standard Deviation, Concentration Assay, Incubation, Transfection, Small Interfering RNA

Journal: Oncology Letters

Article Title: Analyses on K-ras mutations and fascin expression in patients with cardia cancer

doi: 10.3892/ol.2018.9750

Figure Lengend Snippet: Primer sequences of fluorescence quantitative PCR.

Article Snippet: Then, the sections were stained using immunohistochemistry MaxVision two-step method blocked with 5% milk at 20°C for 2 h. After that, mouse anti-human fascin monoclonal antibodies (dilution, 1:200; MAB-0228, clone no. FCN01) and MaxVision immunohistochemistry kits (both from Fuzhou Maxim Biotech, Inc., Fuzhou, China) were used.

Techniques: Fluorescence

Journal: Oncology Letters

Article Title: Analyses on K-ras mutations and fascin expression in patients with cardia cancer

doi: 10.3892/ol.2018.9750

Figure Lengend Snippet: Results of immunohistochemistry for fascin in cardia cancer (p<0.05). Results of immunohistochemistry in (A) the non-mutant and (B) mutant groups. The number of positive cells in the mutant group is greater than that in the non-mutant group (p<0.05).

Article Snippet: Then, the sections were stained using immunohistochemistry MaxVision two-step method blocked with 5% milk at 20°C for 2 h. After that, mouse anti-human fascin monoclonal antibodies (dilution, 1:200; MAB-0228, clone no. FCN01) and MaxVision immunohistochemistry kits (both from Fuzhou Maxim Biotech, Inc., Fuzhou, China) were used.

Techniques: Immunohistochemistry, Mutagenesis

Journal: Oncology Letters

Article Title: Analyses on K-ras mutations and fascin expression in patients with cardia cancer

doi: 10.3892/ol.2018.9750

Figure Lengend Snippet: Results of qPCR. The expression level of fascin gene in the mutant group is higher than that in the non-mutant group. qPCR, quantitative polymerase chain reaction. *p<0.05, compared with the non-mutant group.

Article Snippet: Then, the sections were stained using immunohistochemistry MaxVision two-step method blocked with 5% milk at 20°C for 2 h. After that, mouse anti-human fascin monoclonal antibodies (dilution, 1:200; MAB-0228, clone no. FCN01) and MaxVision immunohistochemistry kits (both from Fuzhou Maxim Biotech, Inc., Fuzhou, China) were used.

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