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: 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: bioRxiv

Article Title: Nuclear fascin regulates cancer cell survival

doi: 10.1101/2022.06.17.496538

Figure Lengend Snippet: (A) Example images of WT and fascin KD HeLa (left panels) and MDA-MB-231 (right panels) cells fixed and stained for endogenous fascin. Inset panels show nuclear fascin localisation. Scale bars are 10 µm. (B) Schematic diagram of fascin domain structure indicating identified NLS and NES regions (colours denote mutated residues in mutant forms generated; ). (C) Example images of HeLa cells treated with Leptomycin B fixed and stained for endogenous fascin. Scale bars are 10 µm. (D) Example images of WT and fascin KD HeLa cells fixed and stained using an antibody detecting endogenous nuclear actin (green) and DAPI (blue). Scale bars are 10 µm. (E) Example confocal images of HeLa cells expressing actin-NLS-FLAG fixed and stained for endogenous fascin (green) and FLAG (magenta). Scale bars are 10 µm. (F) Example confocal image of mixed population of fascin KD cells and those re-expressing GFP-fascin (green), transfected with actin-NLS-FLAG, fixed and stained for FLAG (Magenta). Scale bars are 10 µm.

Article Snippet: The following antibodies were used: fascin (55K2; MAB3582), FLAG (M2; F3165), GFP (11814460001) and pT3 Histone H3 (07-424) were from Merck; Actin (ab6276) and Lamin A/C (ab8984) were from Abcam; Histone H3 (2128A; MAB9448) and ɣH2AX (NB100-384) were from Novus; cleaved Caspase-3 (5A1E; 9664) and pS10 Histone H3 (9701) were from Cell Signaling Technology; GAPDH (6C5; GTX28245) was from GeneTex; and HSC70 (sc-7298) was from Santa Cruz.

Techniques: Staining, Mutagenesis, Generated, Expressing, Transfection

Journal: bioRxiv

Article Title: Nuclear fascin regulates cancer cell survival

doi: 10.1101/2022.06.17.496538

Figure Lengend Snippet: (A) Representative western blot of fascin KD HeLa cells expressing specified GFP-fascin constructs subjected to biochemical fractionation. Nuclear and cytoplasmic compartments probed for specified proteins. Representative of 3 independent experiments. (B) Representative confocal images of nuclei of fascin KD HeLa cells co-expressing specified GFP-fascin constructs (green) and actin-NLS-FLAG construct, fixed and stained for FLAG (magenta) and F-actin (phalloidin). Scale bars are 10 µm. (C) Representative stills from time-lapse confocal movies of fascin KD HeLa cells co-expressing GFP or GFP-fascin (top panels) and iRFP-nAC nuclear F-actin probe (bottom panels) pre- and post-cytokinesis. Arrowheads point to dividing or daughter cells. Scale bars are 10 µm. (D) Quantification of duration of nuclear F-actin filaments in cells as in (C). (E) Organisation of nuclear F-actin in synchronised cells, 10 h after release. For (D) and (E), N=89-100 cells/condition, pooled from 3 independent experiments. Graphs shows mix/max and mean of dataset. ***=p<0.001, ****=p<0.0001.

Article Snippet: The following antibodies were used: fascin (55K2; MAB3582), FLAG (M2; F3165), GFP (11814460001) and pT3 Histone H3 (07-424) were from Merck; Actin (ab6276) and Lamin A/C (ab8984) were from Abcam; Histone H3 (2128A; MAB9448) and ɣH2AX (NB100-384) were from Novus; cleaved Caspase-3 (5A1E; 9664) and pS10 Histone H3 (9701) were from Cell Signaling Technology; GAPDH (6C5; GTX28245) was from GeneTex; and HSC70 (sc-7298) was from Santa Cruz.

Techniques: Western Blot, Expressing, Construct, Fractionation, Staining

Journal: bioRxiv

Article Title: Nuclear fascin regulates cancer cell survival

doi: 10.1101/2022.06.17.496538

Figure Lengend Snippet: (A) Representative confocal images of fascin KD MDA-MB-231 cells expressing GFP-fascin (green) and specified mCherry-Nb2 constructs (magenta) fixed and stained with DAPI (blue). Scale bars are 10 µm. (B) Representative western blot of fascin KD MDA-MB-231 cells expressing GFP or GFP-fascin subjected to biochemical fractionation. Nuclear and cytoplasmic compartments probed for specified proteins. (C) Quantification of data from (B) from 4 independent experiments. (D) Representative western blot of HeLa cells expressing GFP or GFP-Nb2-NLS/NES, subjected to GFP-Trap and probed for specified proteins. Input shown on right. Representative of 4 independent experiments. (E) Representative confocal images of HeLa nuclei fixed and stained for fascin (green) and Histone H3 (magenta). Scale bar is 10 µm. (F) Representative images of HeLa cells expressing GFP-Histone H3 (top panels) or GFP-Histone H3 and mCherry-Nb2-NLS (bottom panels). Donor and acceptor channels shown; lifetime shown in far right panels. Scale bars are 10 µm. (G) Quantification of FRET efficiency from data as in (E). N=35 cells, from 4 independent experiments. Graph shows mix/max and mean of dataset; each point represents a single cell.

Article Snippet: The following antibodies were used: fascin (55K2; MAB3582), FLAG (M2; F3165), GFP (11814460001) and pT3 Histone H3 (07-424) were from Merck; Actin (ab6276) and Lamin A/C (ab8984) were from Abcam; Histone H3 (2128A; MAB9448) and ɣH2AX (NB100-384) were from Novus; cleaved Caspase-3 (5A1E; 9664) and pS10 Histone H3 (9701) were from Cell Signaling Technology; GAPDH (6C5; GTX28245) was from GeneTex; and HSC70 (sc-7298) was from Santa Cruz.

Techniques: Expressing, Construct, Staining, Western Blot, Fractionation

Journal: bioRxiv

Article Title: Nuclear fascin regulates cancer cell survival

doi: 10.1101/2022.06.17.496538

Figure Lengend Snippet: (A) Representative confocal images of fascin KD HeLa cells expressing GFP or GFP-fascin (green) fixed and stained for ɣH2AX (magenta) before (0 min) or after (30 min) treatment with neocarzinostatin (NCS). Scale bars are 10 µm. (B) Quantification of ɣH2AX levels in cells from data as in (A). N=300-350 cells/condition, pooled from 3 independent experiments. (C) Representative western blot of fascin KD HeLa cells expressing GFP or GFP-fascin (FSN) subjected to biochemical fractionation. Nuclear and cytoplasmic compartments probed for specified proteins. Representative of 3 independent experiments. (D) Representative confocal images of fascin KD HeLa cells expressing GFP-fascin (green) fixed and stained for ɣH2AX (cyan) and phalloidin (magenta) before (0 min) or after (60 min) treatment with NCS. Scale bars are 10 µm. (E) Representative images of WT or fascin KD HeLa cells expressing GFP-Histone H2B (left panels) or GFP-Histone H2B and mCherry-Histone H2B, with or without 30 mins NCS treatment. Donor and acceptor channels shown; lifetime shown in bottom panels. Scale bars are 10 µm. (F) Quantification of FRET efficiency from data as in (E). N=35 cells, pooled from 3 independent experiments. All graphs show mix/max and mean of dataset. **=p<0.01, ***=p<0.001, ****=p<0.0001.

Article Snippet: The following antibodies were used: fascin (55K2; MAB3582), FLAG (M2; F3165), GFP (11814460001) and pT3 Histone H3 (07-424) were from Merck; Actin (ab6276) and Lamin A/C (ab8984) were from Abcam; Histone H3 (2128A; MAB9448) and ɣH2AX (NB100-384) were from Novus; cleaved Caspase-3 (5A1E; 9664) and pS10 Histone H3 (9701) were from Cell Signaling Technology; GAPDH (6C5; GTX28245) was from GeneTex; and HSC70 (sc-7298) was from Santa Cruz.

Techniques: Expressing, Staining, Western Blot, Fractionation

Journal: bioRxiv

Article Title: Nuclear fascin regulates cancer cell survival

doi: 10.1101/2022.06.17.496538

Figure Lengend Snippet: (A) Representative confocal images of fascin KD MDA-MB-231 cells expressing GFP-fascin (green) and Nb2 constructs (magenta). Zoom region of filopodia shown below each. Scale bars are 10 µm. (B) Quantification of filopodia number/cell from data as in (A) from 30 cells, pooled from 3 independent experiments. (C) Quantification of 2D migration speed of cells as in (A) from 16 h time-lapse movies. (D) Representative images of confocal Z-stacks from inverted invasion assays from cells as in (A), fixed after 48 h invasion and stained for DAPI (shown). Fascin KD cells additionally shown (bottom row). (E) Quantification of invasion from data as in (D). 3 wells imaged per experiment (3 fields of view/well), pooled from 3 independent experiments. All graphs show mix/max and mean of dataset. **=p<0.01, ***=p<0.001, ****=p<0.0001.

Article Snippet: The following antibodies were used: fascin (55K2; MAB3582), FLAG (M2; F3165), GFP (11814460001) and pT3 Histone H3 (07-424) were from Merck; Actin (ab6276) and Lamin A/C (ab8984) were from Abcam; Histone H3 (2128A; MAB9448) and ɣH2AX (NB100-384) were from Novus; cleaved Caspase-3 (5A1E; 9664) and pS10 Histone H3 (9701) were from Cell Signaling Technology; GAPDH (6C5; GTX28245) was from GeneTex; and HSC70 (sc-7298) was from Santa Cruz.

Techniques: Expressing, Construct, Migration, Staining

Journal: bioRxiv

Article Title: Nuclear fascin regulates cancer cell survival

doi: 10.1101/2022.06.17.496538

Figure Lengend Snippet: (A) Example images from live cell time-lapse apoptosis screen in fascin KD HeLa cells expressing mScarlet-fascin or mScarlet, showing caspase reporter activity (images shown in fire LUT for clarity) after treatment with compound SN1151669528 (1.25 µM) for 4 or 72 h. Scale bars are 150 µm. (B) Plot showing AC50 of specified compounds based on apoptosis screen data. (C) Correlation plot from apoptosis screen data showing % difference in apoptosis AC50 between fascin KD HeLa cells expressing mScarlet-fascin (WT) or mScarlet (KD), plotted as a function of mean % nuclear fascin/cell/well from original screen (as in Fig. 6B), after treatment with hit compounds. Magenta data points denote hits selected with significantly lower death in KD cells vs WT. (D) Representative western blot of HeLa cells treated with target inhibitors (1.25 µM) probed for specified proteins. Representative of 3 independent experiments. (E) Representative western blots of HeLa cells expressing GFP or GFP-Nb2-NLS treated with specified compounds (1.25 µM, 4 h) followed by GFP-Trap and probing for Histone H3. Representative of 3 independent experiments. (F) Example confocal images of HeLa cells expressing iRFP-nAC nuclear actin probe, stained for fascin and pT3-Histone H3 (magenta). Scale bars are 10 µm. (G) ɣH2AX levels in cells treated with DMSO or 1.25 µM haspin inhibitor for 4 h. Each point represents a single cell; representative of 3 independent experiments. ***=p<0.001. (H) Correlation plot between nuclear GFP-fascin and ɣH2AX intensity from data as in (G). Values represent linear regression slope.

Article Snippet: The following antibodies were used: fascin (55K2; MAB3582), FLAG (M2; F3165), GFP (11814460001) and pT3 Histone H3 (07-424) were from Merck; Actin (ab6276) and Lamin A/C (ab8984) were from Abcam; Histone H3 (2128A; MAB9448) and ɣH2AX (NB100-384) were from Novus; cleaved Caspase-3 (5A1E; 9664) and pS10 Histone H3 (9701) were from Cell Signaling Technology; GAPDH (6C5; GTX28245) was from GeneTex; and HSC70 (sc-7298) was from Santa Cruz.

Techniques: Expressing, Activity Assay, Western Blot, Staining

Journal: bioRxiv

Article Title: Nuclear fascin regulates cancer cell survival

doi: 10.1101/2022.06.17.496538

Figure Lengend Snippet: Representative western blots of fascin KD HeLa cells expressing GFP-fascin treated with specified inhibitors subjected to GFP-Trap then probed for total phosphorylated protein (top lanes) and GFP. Input shown on right. Representative of 3 independent experiments.

Article Snippet: The following antibodies were used: fascin (55K2; MAB3582), FLAG (M2; F3165), GFP (11814460001) and pT3 Histone H3 (07-424) were from Merck; Actin (ab6276) and Lamin A/C (ab8984) were from Abcam; Histone H3 (2128A; MAB9448) and ɣH2AX (NB100-384) were from Novus; cleaved Caspase-3 (5A1E; 9664) and pS10 Histone H3 (9701) were from Cell Signaling Technology; GAPDH (6C5; GTX28245) was from GeneTex; and HSC70 (sc-7298) was from Santa Cruz.

Techniques: Western Blot, Expressing

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