anti fascin  (Cell Signaling Technology Inc)

Journal: Oncotarget

Article Title: PCAIs stimulate MAPK, PI3K/AKT pathways and ROS-Mediated apoptosis in aromatase inhibitor-resistant breast cancer cells while disrupting actin filaments and focal adhesion

doi: 10.18632/oncotarget.28759

Figure Lengend Snippet: Cells were grown in 8-well ibidi μ-slides overnight and treated with the respective concentrations of PCAIs for 48 h, then fixed and permeabilized. Images of the cells treated with 0 and 5 μM NSL-YHJ-2-27 are shown at higher magnification whereby yellow arrows indicating retracting lamellipodia indicative of collapsing actin filaments. ( A ) Triplicates of the fixed cells were stained with Alexa Fluor ™ 568 Phalloidin for actin filaments. Yellow arrows indicate retracting lamellipodia indicative of the collapsing actin filaments (magnified images). ( B ) Vinculin punctates were probed using vinculin antibody and visualized using rabbit IgG Alexa Fluor 555 conjugate. White arrows indicate vinculin punctates. ( C ) Fascin was probed with fascin antibody and visualized using mouse IgG Alexa Fluor 488. White arrows indicate defined fascin spots. Images were captured using Keyence BX-X800 microscope at 40X magnification. ( D ) Cells treated for 48 h with the indicated concentrations of NSL-YHJ-2-27 or 5 μM of the non-farnesylated PCAIs analog, NSL-YHJ-2-62. Lysis of the cells and analysis by western blotting for vinculin and fascin were conducted as described in the methods. Western blot images and plots of chemiluminescence intensities of the bands following quantification using Image Lab Software normalized against GAPDH against concentration. Data are representative of three independent experiments. Statistical significance ( ** p < 0.01 in relation to the negative control, 0 μM) was determined by one-way ANOVA with post hoc Dunnett’s test.

Article Snippet: Primary antibodies specific to proteins: total MEK 1/2 (Cat #8727S), phosphorylated MEK 1/2 (p-MEK 1/2, Cat #9154), total ERK 1/2 (Cat#4695S), phosphorylated ERK 1/2 (p-ERK1/2, Cat #4370), total P90RSK (Cat #9355S), total AKT (Cat #4691S), phosphorylated AKT(pAKT(Ser473)(Cat#4060S), phosphorylated P90RSK (p-P90RSK, Cat #11989), RAC1/2/3 (Cat #2465), CDC42 (Cat #2462), vinculin (Cat #18799), fascin (Cat #54545), caspase 7 (Cat #9492S), HER2/ErbB2 (Cat #60388S), BAX (Cat #5023S), P-p53 (9284S), PARP (9542T), p-ERα (2511T), t-ERα (13258S); secondary antibodies: anti-mouse IgG, HRP-linked Antibody (Cat #7076), anti-rabbit IgG, HRP-linked Antibody (Cat #7074) and housekeeping protein, GAPDH (Cat #5174) were purchased from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Staining, Microscopy, Lysis, Western Blot, Software, Concentration Assay, Negative 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