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: International Journal of Molecular Medicine

Article Title: Identification of novel molecules and pathways associated with fascin actin-bundling protein 1 in laryngeal squamous cell carcinoma through comprehensive transcriptome analysis

doi: 10.3892/ijmm.2024.5363

Figure Lengend Snippet: Screening of differentially expressed genes using microarray profiling. (A) Validation of FSCN1 knockdown. TU-177 cells were transfected with FSCN1 (si-FSCN1) or negative (si-NC) siRNAs for 48 h, and the expression level of FSCN1 was determined using RT-qPCR and western blot analysis. In the RT-qPCR experiment, data are presented as the mean ± SD of three independent experiments. *** P<0.001. (B) Volcano plots of differentially expressed mRNAs. The location of DHCR24 is marked. (C) Hierarchical clustering heatmap of differentially expressed mRNAs. (D) Number of differentially expressed genes, including mRNAs, lncRNAs and circRNAs. (E and F) Expression of the top 10 up- and 10 downregulated genes in TU-177 cells in which FSCN1 was knocked down compared with control TU-177 cells based on microarray data (n=3). * P<0.05, ** P<0.01 and *** P<0.001. siRNA, small interfering RNA; lncRNA, long non-coding RNA; mRNA, messenger RNA; FSCN1, fascin actin-bundling protein 1; RT-qPCR, reverse transcription-quantitative PCR; MUC20, mucin 20, cell surface associated; SLC26A1, solute carrier family 26 member 1; ZNF85, zinc finger protein 85; HLA-DRA, major histocompatibility complex, class II, DR alpha; ZNF618, zinc finger protein 618; SLC22A7, solute carrier family 22 member 7; DOCK2, dedicator of cytokinesis 2; ACOX3, acyl-CoA oxidase 3, pristanoyl; PLD5, phospholipase D family member 5; RNF165, ring finger protein 165; GABRD, gamma-aminobutyric acid type A receptor delta subunit; PTPN, protein tyrosine phosphatase, non-receptor type 3; ADPRM, ADP-ribose/CDP-alcohol diphosphatase, manganese dependent; OSCAR, osteoclast associated, immunoglobulin-like receptor; USP9Y, ubiquitin specific peptidase 9, Y-linked; WISP1, WNT1 inducible signaling pathway protein 1; CAGE1, cancer antigen 1; ZNF518A, zinc finger protein 518A; DCT, dopachrome tautomerase; ZBBX, zinc finger B-box domain containing; DHCR24, 24-dehydrocholesterol reductase.

Article Snippet: The protein concentration was determined using a BCA Protein Assay kit (Thermo Fisher Scientific, Inc.), and total protein was separated using 10% SDS-PAGE gels, transferred to a PVDF membrane and blocked with 10% w/v non-fat milk powder in TBST at room temperature for 2 h. The membranes were incubated with primary antibodies against Flag (1:1,000; cat. no. F1804; Merck KGaA), FSCN1 (1:1,000; cat. no. 54545S; Cell Signaling Technology, Inc.), and GAPDH (1:1,000; cat.no.

Techniques: Microarray, Biomarker Discovery, Knockdown, Transfection, Expressing, Quantitative RT-PCR, Western Blot, Control, Small Interfering RNA, Reverse Transcription, Real-time Polymerase Chain Reaction, Immunopeptidomics, Ubiquitin Proteomics

Journal: International Journal of Molecular Medicine

Article Title: Identification of novel molecules and pathways associated with fascin actin-bundling protein 1 in laryngeal squamous cell carcinoma through comprehensive transcriptome analysis

doi: 10.3892/ijmm.2024.5363

Figure Lengend Snippet: Analysis of genes co-expressed with FSCN1 in LSCC. (A) Flow chart of constructing a network of genes co-expressed with FSCN1 in LSCC. (B) Constructed network based on the 48 differentially expressed mRNAs (10 up- and 38 downregulated) that were co-expressed with FSCN1 in LSCC. (C and D) Gene Ontology biological process terms and Kyoto Encyclopedia of Genes and Genomes pathways enriched by the 48 differentially expressed mRNAs co-expressed with FSCN1 in LSCC. DEGs, differentially expressed genes; FSCN1, fascin actin-bundling protein 1; LSCC, laryngeal squamous cell carcinoma.

Article Snippet: The protein concentration was determined using a BCA Protein Assay kit (Thermo Fisher Scientific, Inc.), and total protein was separated using 10% SDS-PAGE gels, transferred to a PVDF membrane and blocked with 10% w/v non-fat milk powder in TBST at room temperature for 2 h. The membranes were incubated with primary antibodies against Flag (1:1,000; cat. no. F1804; Merck KGaA), FSCN1 (1:1,000; cat. no. 54545S; Cell Signaling Technology, Inc.), and GAPDH (1:1,000; cat.no.

Techniques: Construct

Journal: International Journal of Molecular Medicine

Article Title: Identification of novel molecules and pathways associated with fascin actin-bundling protein 1 in laryngeal squamous cell carcinoma through comprehensive transcriptome analysis

doi: 10.3892/ijmm.2024.5363

Figure Lengend Snippet: PPI network of DEGs in cells in which FSCN1 was knockdown. (A) The PPI network obtained from the STRING database with a confidence score of >0.7. The network contained 455 nodes and 570 edges. (B-D) The three tightly connected network clusters obtained with MCODE were rendered as separate modules. (E and F) Validation of FSCN1 affected functional or signaling pathway molecules. (E) FD-LSC-1 and (F) TU-177 LSCC cells were transfected with FSCN1 siRNAs or NC siRNAs for 48 h, and then the expression level of genes involved in defense response to virus and steroid biosynthesis the pathway was determined using RT-qPCR. In the RT-qPCR experiment, data are presented as the mean ± SD of three independent experiments. * P<0.05, ** P<0.01 and *** P<0.001. PPI, protein-protein interaction; DEGs, differentially expressed genes; FSCN1, fascin actin-bundling protein 1; MCODE, Molecular Complex Detection; LSCC, laryngeal squamous cell carcinoma; siRNA, small interfering RNA; RT-qPCR, reverse transcription-quantitative PCR; IFIT2, tetratricopeptide repeats 2; IFIT3, tetratricopeptide repeats 3; OASL, 2′-5′-oligoadenylate synthetase like; SQLE, squalene epoxidase; FDFT1, farnesyl-diphosphate farnesyltransferase 1; DHCR24, 24-dehydrocholesterol reductase.

Article Snippet: The protein concentration was determined using a BCA Protein Assay kit (Thermo Fisher Scientific, Inc.), and total protein was separated using 10% SDS-PAGE gels, transferred to a PVDF membrane and blocked with 10% w/v non-fat milk powder in TBST at room temperature for 2 h. The membranes were incubated with primary antibodies against Flag (1:1,000; cat. no. F1804; Merck KGaA), FSCN1 (1:1,000; cat. no. 54545S; Cell Signaling Technology, Inc.), and GAPDH (1:1,000; cat.no.

Techniques: Knockdown, Biomarker Discovery, Functional Assay, Transfection, Expressing, Virus, Quantitative RT-PCR, Small Interfering RNA, Reverse Transcription, Real-time Polymerase Chain Reaction

Journal: International Journal of Molecular Medicine

Article Title: Identification of novel molecules and pathways associated with fascin actin-bundling protein 1 in laryngeal squamous cell carcinoma through comprehensive transcriptome analysis

doi: 10.3892/ijmm.2024.5363

Figure Lengend Snippet: Crosstalk analysis and validation. (A) Venn diagram confirmed 7 target genes that overlapped between the DEGs in FSCN1-knockdown TU-177 cells and FSCN1 interacting proteins in TU-177 cells. (B) Validation of the expression of FSCN1 regulated genes. FD-LSC-1 and TU-177 LSCC cells were transfected with FSCN1 siRNAs or NC siRNAs for 48 h, and the expression level of PTGR1 and SLC38A2 was then determined using RT-qPCR. In the RT-qPCR experiment, data are presented as the mean ± SD of three independent experiments ( * P<0.05). (C) Validation of the interactions between PTGR1, DHCR24 and SLC38A2 with FSCN1. HA-tagged FSCN1 and Flag-tagged PTGR1, DHCR24 and SLC38A2 were transiently co-expressed in 293T cells, respectively. For western blot analysis, antibodies against Flag, HA and GAPDH were used to detect protein expression. Total cell lysates were used to validate the interaction by co-IP. Antibodies against Flag tag were used to capture proteins-FSCN1 complexes, and normal mouse IgG served as a negative control. On western blot analysis, rabbit anti-HA antibody was used to detect FSCN1. DEGs, differentially expressed genes; FSCN1, fascin actin-bundling protein 1; siRNAs, small interfering RNA; LSCC, laryngeal squamous cell carcinoma; RT-qPCR, reverse transcription-quantitative PCR; PTGR1, prostaglandin reductase 1; DHCR24, 24-dehydrocholesterol reductase; SLC38A2, solute carrier family 38 member 2; PRDX4, peroxiredoxin 4; TPM4, tropomyosin 4; ABHD16A, abhydrolase domain containing 16A; CARMIL1, capping protein regulator and myosin 1 linker 1.

Article Snippet: The protein concentration was determined using a BCA Protein Assay kit (Thermo Fisher Scientific, Inc.), and total protein was separated using 10% SDS-PAGE gels, transferred to a PVDF membrane and blocked with 10% w/v non-fat milk powder in TBST at room temperature for 2 h. The membranes were incubated with primary antibodies against Flag (1:1,000; cat. no. F1804; Merck KGaA), FSCN1 (1:1,000; cat. no. 54545S; Cell Signaling Technology, Inc.), and GAPDH (1:1,000; cat.no.

Techniques: Biomarker Discovery, Knockdown, Expressing, Transfection, Quantitative RT-PCR, Western Blot, Co-Immunoprecipitation Assay, FLAG-tag, Negative Control, Small Interfering RNA, Reverse Transcription, Real-time Polymerase Chain Reaction

Journal: The Journal of Cell Biology

Article Title: MicroRNA control of podosome formation in vascular smooth muscle cells in vivo and in vitro

doi: 10.1083/jcb.200912096

Figure Lengend Snippet: miR-143 and -145 targets. (A) Representative immunoblot of primary VSMCs isolated from miR-143(145) KO mice transduced with adenovirus (Ad-miR) with an empty expression cassette (−) or expressing miR-143, -145, or -208. (B) Luciferase reporter assay on 3T3 cells performed by cotransfection of 20 nM miR-143, miR-145, or scrambled oligonucleotide (+) with a renilla reporter gene linked to 10 ng WT (wt) or mutated (mt) 3′ UTR of PKC-ε, PDGF-Rα, or fascin. All measurements were calculated as the percentage of control (WT 3′ UTR), and error bars were calculated as propagated standard errors of the mean of triplicate measurements from each experiment. *, P < 0.03 versus control scrambled miR. (C) Morphology of KO VSMCs with a knockdown of PKC-ε and fascin by lentiviral shRNA interference. (D) Migration of KO VSMCs with a knockdown of PKC-ε and fascin by lentiviral shRNA interference. All measurements were calculated as the percentage of scrambled control, and error bars were calculated as propagated standard errors of the mean of triplicate measurements from each experiment. *, P < 0.05. Bar, 10 µm.

Article Snippet: Lentiviruses with scrambled, PKC-ε, and fascin short hairpin RNA (shRNA) were purchased from Santa Cruz Biotechnology, Inc., and KO SMCs were infected at MOI 5 using 5 μg/ml polybrene.

Techniques: Western Blot, Isolation, Transduction, Expressing, Luciferase, Reporter Assay, Cotransfection, Control, Knockdown, shRNA, Migration

Journal: The Journal of Cell Biology

Article Title: MicroRNA control of podosome formation in vascular smooth muscle cells in vivo and in vitro

doi: 10.1083/jcb.200912096

Figure Lengend Snippet: Model for the role of miR-143 and -145 in VSMC migration and podosome formation. Vascular stress triggers the PDGF response, which activates Src, which in turn inhibits p53 and thus represses miR-143 and -145 expression. This relieves miR-143 repression of the expression of PKC-ε and PDGF-Rα and miR-145 repression of fascin, allowing the formation of podosomes and an increased migratory capacity. Thus, loss of both miRs increases the activity of pathways involved in cell migration.

Article Snippet: Lentiviruses with scrambled, PKC-ε, and fascin short hairpin RNA (shRNA) were purchased from Santa Cruz Biotechnology, Inc., and KO SMCs were infected at MOI 5 using 5 μg/ml polybrene.

Techniques: Migration, Expressing, Activity Assay