Structured Review

Agilent technologies fascin 1
<t>Fascin-1</t> overexpression in 143B cells promotes intratibial primary tumor growth and lung metastasis in SCID mice. (a) Representative X-ray images of tumor-bearing hind limbs of mice injected with 143B/EV cells (upper panel) or with 143B/Fascin-1 (lower panel). The images show primary tumor appearance on indicated days after tumor cell injection. (b) Representative X-ray images of tumor-bearing hind limbs of mice injected with 143B/Ctrl ShRNA cells (upper panel) or with 143B/ShFascin-1 cells (lower panel). (c) Mean primary tumor growth over time in mice intratibially injected with 143B/EV cells (black), with 143B/Fascin-1 cells (red), with 143B/Ctrl ShRNA (grey) or with 143B/ShFascin-1 blue). (d) Quantification of the number of metastatic lesions in lungs
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Images

1) Product Images from "Fascin-1 enhances experimental osteosarcoma tumor formation and metastasis and is related to poor patient outcome"

Article Title: Fascin-1 enhances experimental osteosarcoma tumor formation and metastasis and is related to poor patient outcome

Journal: BMC Cancer

doi: 10.1186/s12885-019-5303-3

Fascin-1 overexpression in 143B cells promotes intratibial primary tumor growth and lung metastasis in SCID mice. (a) Representative X-ray images of tumor-bearing hind limbs of mice injected with 143B/EV cells (upper panel) or with 143B/Fascin-1 (lower panel). The images show primary tumor appearance on indicated days after tumor cell injection. (b) Representative X-ray images of tumor-bearing hind limbs of mice injected with 143B/Ctrl ShRNA cells (upper panel) or with 143B/ShFascin-1 cells (lower panel). (c) Mean primary tumor growth over time in mice intratibially injected with 143B/EV cells (black), with 143B/Fascin-1 cells (red), with 143B/Ctrl ShRNA (grey) or with 143B/ShFascin-1 blue). (d) Quantification of the number of metastatic lesions in lungs
Figure Legend Snippet: Fascin-1 overexpression in 143B cells promotes intratibial primary tumor growth and lung metastasis in SCID mice. (a) Representative X-ray images of tumor-bearing hind limbs of mice injected with 143B/EV cells (upper panel) or with 143B/Fascin-1 (lower panel). The images show primary tumor appearance on indicated days after tumor cell injection. (b) Representative X-ray images of tumor-bearing hind limbs of mice injected with 143B/Ctrl ShRNA cells (upper panel) or with 143B/ShFascin-1 cells (lower panel). (c) Mean primary tumor growth over time in mice intratibially injected with 143B/EV cells (black), with 143B/Fascin-1 cells (red), with 143B/Ctrl ShRNA (grey) or with 143B/ShFascin-1 blue). (d) Quantification of the number of metastatic lesions in lungs

Techniques Used: Over Expression, Mouse Assay, Injection, shRNA

Kaplan-Meier analysis correlating immunohistochemical staining of Fascin-1 in human OS tissues with overall survival of the patients. ( A ) Representative images of TMA sections showing entire spots (upper panel) and higher magnification (lower panel) with non-detectable ( a ), weak ( b ), moderate ( c ), and intense ( d ) Fascin-1 immunostaining. ( B ) Overall survival of OS patients with non-detectable (Fascin-1 neg) or detectable (Fascin-1 pos) immunostaining of tumor tissues. ( C ) Overall survival of patients without (Mets neg) or with (Mets pos) metastases and Fascin-1 neg or Fascin-1 pos tumors
Figure Legend Snippet: Kaplan-Meier analysis correlating immunohistochemical staining of Fascin-1 in human OS tissues with overall survival of the patients. ( A ) Representative images of TMA sections showing entire spots (upper panel) and higher magnification (lower panel) with non-detectable ( a ), weak ( b ), moderate ( c ), and intense ( d ) Fascin-1 immunostaining. ( B ) Overall survival of OS patients with non-detectable (Fascin-1 neg) or detectable (Fascin-1 pos) immunostaining of tumor tissues. ( C ) Overall survival of patients without (Mets neg) or with (Mets pos) metastases and Fascin-1 neg or Fascin-1 pos tumors

Techniques Used: Immunohistochemistry, Staining, Immunostaining

Validation and characterization of OS cell lines with altered Fascin-1 expression. ( a ) Western blot analysis with antibodies to Fascin-1 (top panel), to V5 (middle panel), and to GAPDH as a loading control (lower panel) of protein extracts from SaOS-2 (left panel) and 143B (right panel) cells stably transduced with a scrambled control ShRNA (Ctrl ShRNA), a Fascin-1-specific ShRNA (ShFascin-1), a pLenti6/V5-DEST empty vector (EV), or pLenti6/V5-DEST-Fascin-1 (Fascin-1). ( b ) SaOS-2/WT (upper row), SaOS-2/Fascin-1 (middle row), and SaOS-2/ShFascin-1 (bottom row) cells stained with anti-Fascin-1 (green), with Alexa-633-phalloidin (filamentous actin, red), and with NucBlue (nuclei in blue). ( c ) While silencing Fascin-1 reduces the perimeter of the cells in both cases, overexpression has little impact ( n = 34–57)
Figure Legend Snippet: Validation and characterization of OS cell lines with altered Fascin-1 expression. ( a ) Western blot analysis with antibodies to Fascin-1 (top panel), to V5 (middle panel), and to GAPDH as a loading control (lower panel) of protein extracts from SaOS-2 (left panel) and 143B (right panel) cells stably transduced with a scrambled control ShRNA (Ctrl ShRNA), a Fascin-1-specific ShRNA (ShFascin-1), a pLenti6/V5-DEST empty vector (EV), or pLenti6/V5-DEST-Fascin-1 (Fascin-1). ( b ) SaOS-2/WT (upper row), SaOS-2/Fascin-1 (middle row), and SaOS-2/ShFascin-1 (bottom row) cells stained with anti-Fascin-1 (green), with Alexa-633-phalloidin (filamentous actin, red), and with NucBlue (nuclei in blue). ( c ) While silencing Fascin-1 reduces the perimeter of the cells in both cases, overexpression has little impact ( n = 34–57)

Techniques Used: Expressing, Western Blot, Stable Transfection, Transduction, shRNA, Plasmid Preparation, Staining, Over Expression

Fascin-1 overexpression in SaOS-2 cells promotes intratibial primary tumor growth and lung metastasis in SCID mice. ( a ) Representative X-ray images of tumor-bearing hind limbs of mice injected with SaOS-2/EV cells (upper panel) or with SaOS-2/Fascin-1 (lower panel). The images show primary tumor appearance on indicated days after tumor cell injection. ( b ) Representative X-ray images of tumor-bearing hind limbs of mice injected with SaOS-2/Ctrl ShRNA cells (upper panel) or with SaOS-2/ShFascin-1 cells (lower panel). ( c ) Mean primary tumor growth over time in mice intratibially injected with SaOS-2/EV cells (black), with SaOS-2/Fascin-1 cells (red), with SaOS-2/Ctrl ShRNA (grey) or with SaOS-2/ShFascin-1 blue). ( d ) Mean number ± SEM of metastatic lesions in the lungs
Figure Legend Snippet: Fascin-1 overexpression in SaOS-2 cells promotes intratibial primary tumor growth and lung metastasis in SCID mice. ( a ) Representative X-ray images of tumor-bearing hind limbs of mice injected with SaOS-2/EV cells (upper panel) or with SaOS-2/Fascin-1 (lower panel). The images show primary tumor appearance on indicated days after tumor cell injection. ( b ) Representative X-ray images of tumor-bearing hind limbs of mice injected with SaOS-2/Ctrl ShRNA cells (upper panel) or with SaOS-2/ShFascin-1 cells (lower panel). ( c ) Mean primary tumor growth over time in mice intratibially injected with SaOS-2/EV cells (black), with SaOS-2/Fascin-1 cells (red), with SaOS-2/Ctrl ShRNA (grey) or with SaOS-2/ShFascin-1 blue). ( d ) Mean number ± SEM of metastatic lesions in the lungs

Techniques Used: Over Expression, Mouse Assay, Injection, shRNA

Migration assessed by wound healing . Overexpression of Fascin-1 in SaOS-2 ( a ) or in 143B (b) OS cells increases significantly the migration ability. Similarly, silencing of Fascin-1 slightly decreased the migratory rate of SaOS-2 ( a ) and 143B ( b ) cells. Results are the mean ± SEM of at least three independent experiments. ( c ) Zymography analysis showing increased MMP-9 activity in SaOS-2/Fascin-1 (left panel) and in 143B/Fascin-1 (right panel) in comparison to control SaOS-1/EV and 143B/EV cells respectively. Results are the mean ± SEM of three independent experiments
Figure Legend Snippet: Migration assessed by wound healing . Overexpression of Fascin-1 in SaOS-2 ( a ) or in 143B (b) OS cells increases significantly the migration ability. Similarly, silencing of Fascin-1 slightly decreased the migratory rate of SaOS-2 ( a ) and 143B ( b ) cells. Results are the mean ± SEM of at least three independent experiments. ( c ) Zymography analysis showing increased MMP-9 activity in SaOS-2/Fascin-1 (left panel) and in 143B/Fascin-1 (right panel) in comparison to control SaOS-1/EV and 143B/EV cells respectively. Results are the mean ± SEM of three independent experiments

Techniques Used: Migration, Over Expression, Zymography, Activity Assay

2) Product Images from "Fascin-1 enhances experimental osteosarcoma tumor formation and metastasis and is related to poor patient outcome"

Article Title: Fascin-1 enhances experimental osteosarcoma tumor formation and metastasis and is related to poor patient outcome

Journal: BMC Cancer

doi: 10.1186/s12885-019-5303-3

Validation and characterization of OS cell lines with altered Fascin-1 expression. ( a ) Western blot analysis with antibodies to Fascin-1 (top panel), to V5 (middle panel), and to GAPDH as a loading control (lower panel) of protein extracts from SaOS-2 (left panel) and 143B (right panel) cells stably transduced with a scrambled control ShRNA (Ctrl ShRNA), a Fascin-1-specific ShRNA (ShFascin-1), a pLenti6/V5-DEST empty vector (EV), or pLenti6/V5-DEST-Fascin-1 (Fascin-1). ( b ) SaOS-2/WT (upper row), SaOS-2/Fascin-1 (middle row), and SaOS-2/ShFascin-1 (bottom row) cells stained with anti-Fascin-1 (green), with Alexa-633-phalloidin (filamentous actin, red), and with NucBlue (nuclei in blue). ( c ) While silencing Fascin-1 reduces the perimeter of the cells in both cases, overexpression has little impact ( n = 34–57)
Figure Legend Snippet: Validation and characterization of OS cell lines with altered Fascin-1 expression. ( a ) Western blot analysis with antibodies to Fascin-1 (top panel), to V5 (middle panel), and to GAPDH as a loading control (lower panel) of protein extracts from SaOS-2 (left panel) and 143B (right panel) cells stably transduced with a scrambled control ShRNA (Ctrl ShRNA), a Fascin-1-specific ShRNA (ShFascin-1), a pLenti6/V5-DEST empty vector (EV), or pLenti6/V5-DEST-Fascin-1 (Fascin-1). ( b ) SaOS-2/WT (upper row), SaOS-2/Fascin-1 (middle row), and SaOS-2/ShFascin-1 (bottom row) cells stained with anti-Fascin-1 (green), with Alexa-633-phalloidin (filamentous actin, red), and with NucBlue (nuclei in blue). ( c ) While silencing Fascin-1 reduces the perimeter of the cells in both cases, overexpression has little impact ( n = 34–57)

Techniques Used: Expressing, Western Blot, Stable Transfection, Transduction, shRNA, Plasmid Preparation, Staining, Over Expression

Fascin-1 overexpression in SaOS-2 cells promotes intratibial primary tumor growth and lung metastasis in SCID mice. ( a ) Representative X-ray images of tumor-bearing hind limbs of mice injected with SaOS-2/EV cells (upper panel) or with SaOS-2/Fascin-1 (lower panel). The images show primary tumor appearance on indicated days after tumor cell injection. ( b ) Representative X-ray images of tumor-bearing hind limbs of mice injected with SaOS-2/Ctrl ShRNA cells (upper panel) or with SaOS-2/ShFascin-1 cells (lower panel). ( c ) Mean primary tumor growth over time in mice intratibially injected with SaOS-2/EV cells (black), with SaOS-2/Fascin-1 cells (red), with SaOS-2/Ctrl ShRNA (grey) or with SaOS-2/ShFascin-1 blue). ( d ) Mean number ± SEM of metastatic lesions in the lungs
Figure Legend Snippet: Fascin-1 overexpression in SaOS-2 cells promotes intratibial primary tumor growth and lung metastasis in SCID mice. ( a ) Representative X-ray images of tumor-bearing hind limbs of mice injected with SaOS-2/EV cells (upper panel) or with SaOS-2/Fascin-1 (lower panel). The images show primary tumor appearance on indicated days after tumor cell injection. ( b ) Representative X-ray images of tumor-bearing hind limbs of mice injected with SaOS-2/Ctrl ShRNA cells (upper panel) or with SaOS-2/ShFascin-1 cells (lower panel). ( c ) Mean primary tumor growth over time in mice intratibially injected with SaOS-2/EV cells (black), with SaOS-2/Fascin-1 cells (red), with SaOS-2/Ctrl ShRNA (grey) or with SaOS-2/ShFascin-1 blue). ( d ) Mean number ± SEM of metastatic lesions in the lungs

Techniques Used: Over Expression, Mouse Assay, Injection, shRNA

Migration assessed by wound healing . Overexpression of Fascin-1 in SaOS-2 ( a ) or in 143B (b) OS cells increases significantly the migration ability. Similarly, silencing of Fascin-1 slightly decreased the migratory rate of SaOS-2 ( a ) and 143B ( b ) cells. Results are the mean ± SEM of at least three independent experiments. ( c ) Zymography analysis showing increased MMP-9 activity in SaOS-2/Fascin-1 (left panel) and in 143B/Fascin-1 (right panel) in comparison to control SaOS-1/EV and 143B/EV cells respectively. Results are the mean ± SEM of three independent experiments
Figure Legend Snippet: Migration assessed by wound healing . Overexpression of Fascin-1 in SaOS-2 ( a ) or in 143B (b) OS cells increases significantly the migration ability. Similarly, silencing of Fascin-1 slightly decreased the migratory rate of SaOS-2 ( a ) and 143B ( b ) cells. Results are the mean ± SEM of at least three independent experiments. ( c ) Zymography analysis showing increased MMP-9 activity in SaOS-2/Fascin-1 (left panel) and in 143B/Fascin-1 (right panel) in comparison to control SaOS-1/EV and 143B/EV cells respectively. Results are the mean ± SEM of three independent experiments

Techniques Used: Migration, Over Expression, Zymography, Activity Assay

Kaplan-Meier analysis correlating immunohistochemical staining of Fascin-1 in human OS tissues with overall survival of the patients. ( A ) Representative images of TMA sections showing entire spots (upper panel) and higher magnification (lower panel) with non-detectable ( a ), weak ( b ), moderate ( c ), and intense ( d ) Fascin-1 immunostaining. ( B ) Overall survival of OS patients with non-detectable (Fascin-1 neg) or detectable (Fascin-1 pos) immunostaining of tumor tissues. ( C ) Overall survival of patients without (Mets neg) or with (Mets pos) metastases and Fascin-1 neg or Fascin-1 pos tumors
Figure Legend Snippet: Kaplan-Meier analysis correlating immunohistochemical staining of Fascin-1 in human OS tissues with overall survival of the patients. ( A ) Representative images of TMA sections showing entire spots (upper panel) and higher magnification (lower panel) with non-detectable ( a ), weak ( b ), moderate ( c ), and intense ( d ) Fascin-1 immunostaining. ( B ) Overall survival of OS patients with non-detectable (Fascin-1 neg) or detectable (Fascin-1 pos) immunostaining of tumor tissues. ( C ) Overall survival of patients without (Mets neg) or with (Mets pos) metastases and Fascin-1 neg or Fascin-1 pos tumors

Techniques Used: Immunohistochemistry, Staining, Immunostaining

Fascin-1 overexpression in 143B cells promotes intratibial primary tumor growth and lung metastasis in SCID mice. (a) Representative X-ray images of tumor-bearing hind limbs of mice injected with 143B/EV cells (upper panel) or with 143B/Fascin-1 (lower panel). The images show primary tumor appearance on indicated days after tumor cell injection. (b) Representative X-ray images of tumor-bearing hind limbs of mice injected with 143B/Ctrl ShRNA cells (upper panel) or with 143B/ShFascin-1 cells (lower panel). (c) Mean primary tumor growth over time in mice intratibially injected with 143B/EV cells (black), with 143B/Fascin-1 cells (red), with 143B/Ctrl ShRNA (grey) or with 143B/ShFascin-1 blue). (d) Quantification of the number of metastatic lesions in lungs
Figure Legend Snippet: Fascin-1 overexpression in 143B cells promotes intratibial primary tumor growth and lung metastasis in SCID mice. (a) Representative X-ray images of tumor-bearing hind limbs of mice injected with 143B/EV cells (upper panel) or with 143B/Fascin-1 (lower panel). The images show primary tumor appearance on indicated days after tumor cell injection. (b) Representative X-ray images of tumor-bearing hind limbs of mice injected with 143B/Ctrl ShRNA cells (upper panel) or with 143B/ShFascin-1 cells (lower panel). (c) Mean primary tumor growth over time in mice intratibially injected with 143B/EV cells (black), with 143B/Fascin-1 cells (red), with 143B/Ctrl ShRNA (grey) or with 143B/ShFascin-1 blue). (d) Quantification of the number of metastatic lesions in lungs

Techniques Used: Over Expression, Mouse Assay, Injection, shRNA

3) Product Images from "A novel Rho-dependent pathway that drives interaction of fascin-1 with p-Lin-11/Isl-1/Mec-3 kinase (LIMK) 1/2 to promote fascin-1/actin binding and filopodia stability"

Article Title: A novel Rho-dependent pathway that drives interaction of fascin-1 with p-Lin-11/Isl-1/Mec-3 kinase (LIMK) 1/2 to promote fascin-1/actin binding and filopodia stability

Journal: BMC Biology

doi: 10.1186/1741-7007-10-72

Rho kinase activity promotes peripheral fascin-containing protrusions via a myosin-independent process . (A) Confocal images of C2C12 cells after 1 hour of adhesion to 50 nmol/l fibronectin (FN), either untreated or pretreated with specified inhibitors, fixed and stained for fascin-1. Arrowheads indicate examples of peripheral fascin-actin bundles in Y27632-treated cells. Scale bars, 10 μm. (B) Confocal images of C2C12 cells transiently expressing green fluorescent protein (GFP)-caldesmon or an inactive GFP-caldesmon-445 mutant after 1 hour of adhesion to 50 nmol/l FN. Cells were fixed and stained either for F-actin (left panels) or fascin-1 (right panels). In the anti-fascin-1 stained samples, arrowheads indicate the transfected cells. Scale bars, 10 μm. (C) Quantification of peripheral fascin-1 bundles/cell under the conditions shown in (A) and (B ). Data are from 75 to 125 cells/condition and 3 independent experiments. * P
Figure Legend Snippet: Rho kinase activity promotes peripheral fascin-containing protrusions via a myosin-independent process . (A) Confocal images of C2C12 cells after 1 hour of adhesion to 50 nmol/l fibronectin (FN), either untreated or pretreated with specified inhibitors, fixed and stained for fascin-1. Arrowheads indicate examples of peripheral fascin-actin bundles in Y27632-treated cells. Scale bars, 10 μm. (B) Confocal images of C2C12 cells transiently expressing green fluorescent protein (GFP)-caldesmon or an inactive GFP-caldesmon-445 mutant after 1 hour of adhesion to 50 nmol/l FN. Cells were fixed and stained either for F-actin (left panels) or fascin-1 (right panels). In the anti-fascin-1 stained samples, arrowheads indicate the transfected cells. Scale bars, 10 μm. (C) Quantification of peripheral fascin-1 bundles/cell under the conditions shown in (A) and (B ). Data are from 75 to 125 cells/condition and 3 independent experiments. * P

Techniques Used: Activity Assay, Staining, Expressing, Mutagenesis, Transfection

The fascin-1/p-Lin-11/Isl-1/Mec-3 kinase (LIMK)1 interaction promotes stabilization of filopodia . (A-D) Images from confocal time-lapse movies of cells expressing mRFP-fascin-1 with (A) green fluorescent protein (GFP), (B) GFP-LIMK1 (B), (C) GFP-LIMK1T508A or (D) GFP-LIMK1D460A. see Additional files 7 to 10 (movies 4 to 7). There were 15 to 25 cells per condition analyzed in 4 independent experiments. and cells from representative movies are shown. (A-D) Boxed 15 × 15 mm regions in the lefthand panels are enlarged in the images from a series of time points in the right panels. See Additional file 3 , Figure S3, for single-channel images from (A) and (B) . Scale bars, 10 μm. (E) Kymographs of representative filopodia from cells co-expressing GFP or GFP-LIMK1 and mRFP-fascin-1. Displacement of filopodia was measured in accordance with the maximum change in position of filopodial tips over 2 minutes, as indicated by the dotted yellow lines. (F) Quantification of the maximum displacement of filopodia from cells expressing GFP, or wild-type or mutant GFP-LIMK1 and mRFP-fascin-1. Each column represents the mean from five filopodia from at least five cells per condition in four independent experiments; bars indicate SEM. * P
Figure Legend Snippet: The fascin-1/p-Lin-11/Isl-1/Mec-3 kinase (LIMK)1 interaction promotes stabilization of filopodia . (A-D) Images from confocal time-lapse movies of cells expressing mRFP-fascin-1 with (A) green fluorescent protein (GFP), (B) GFP-LIMK1 (B), (C) GFP-LIMK1T508A or (D) GFP-LIMK1D460A. see Additional files 7 to 10 (movies 4 to 7). There were 15 to 25 cells per condition analyzed in 4 independent experiments. and cells from representative movies are shown. (A-D) Boxed 15 × 15 mm regions in the lefthand panels are enlarged in the images from a series of time points in the right panels. See Additional file 3 , Figure S3, for single-channel images from (A) and (B) . Scale bars, 10 μm. (E) Kymographs of representative filopodia from cells co-expressing GFP or GFP-LIMK1 and mRFP-fascin-1. Displacement of filopodia was measured in accordance with the maximum change in position of filopodial tips over 2 minutes, as indicated by the dotted yellow lines. (F) Quantification of the maximum displacement of filopodia from cells expressing GFP, or wild-type or mutant GFP-LIMK1 and mRFP-fascin-1. Each column represents the mean from five filopodia from at least five cells per condition in four independent experiments; bars indicate SEM. * P

Techniques Used: Expressing, Mutagenesis

Activation of p-Lin-11/Isl-1/Mec-3 kinase (LIMK)1 leads to its interaction with fascin-1 and affects formation of filopodia . (A) Measurement of the interaction of wild-type or mutant forms of green fluorescent protein (GFP)-LIMK1 with monomeric red fluorescent protein (mRFP)-fascin-1 in SW480 cells on laminin (LN). Cells transiently transfected with the indicated plasmids were plated on LN for 2 hours, then fixed, mounted, and imaged using fluorescence lifetime imaging microscopy (FLIM) to measure fluorescence resonance energy transfer (FRET). Intensity multiphoton GFP (donor) images are shown with the corresponding epifluorescence image for mRFP (acceptor). Lifetime images are presented in a blue-to-red pseudocolor scale with red as short lifetime. (B) Percentage FRET efficiency under each experimental condition. Each column represents the mean from fourteen to seventeen cells per condition and three independent experiments; bars indicate SEM. * P = 0.001 versus wildtype. (C) Role of LIMK1 activity in organization of filopodia. Live SW480 cells transiently transfected with GFP alone or GFP-LIMK1 (wild-type (WT), D460A or T508A) and mRFP-fascin-1, and protein localizations and cell edges were imaged using confocal microscopy. Arrowheads indicate points where GFP-LIMK1 and mRFP-fascin-1 colocalize in filopodia. Scale bars, 10 μm. (D) The number/cell and (E) length of filopodia were counted from images obtained as in (C) , from 12 to 20 cells per condition and 4 independent experiments. * P
Figure Legend Snippet: Activation of p-Lin-11/Isl-1/Mec-3 kinase (LIMK)1 leads to its interaction with fascin-1 and affects formation of filopodia . (A) Measurement of the interaction of wild-type or mutant forms of green fluorescent protein (GFP)-LIMK1 with monomeric red fluorescent protein (mRFP)-fascin-1 in SW480 cells on laminin (LN). Cells transiently transfected with the indicated plasmids were plated on LN for 2 hours, then fixed, mounted, and imaged using fluorescence lifetime imaging microscopy (FLIM) to measure fluorescence resonance energy transfer (FRET). Intensity multiphoton GFP (donor) images are shown with the corresponding epifluorescence image for mRFP (acceptor). Lifetime images are presented in a blue-to-red pseudocolor scale with red as short lifetime. (B) Percentage FRET efficiency under each experimental condition. Each column represents the mean from fourteen to seventeen cells per condition and three independent experiments; bars indicate SEM. * P = 0.001 versus wildtype. (C) Role of LIMK1 activity in organization of filopodia. Live SW480 cells transiently transfected with GFP alone or GFP-LIMK1 (wild-type (WT), D460A or T508A) and mRFP-fascin-1, and protein localizations and cell edges were imaged using confocal microscopy. Arrowheads indicate points where GFP-LIMK1 and mRFP-fascin-1 colocalize in filopodia. Scale bars, 10 μm. (D) The number/cell and (E) length of filopodia were counted from images obtained as in (C) , from 12 to 20 cells per condition and 4 independent experiments. * P

Techniques Used: Activation Assay, Mutagenesis, Transfection, Fluorescence, Imaging, Microscopy, Förster Resonance Energy Transfer, Activity Assay, Confocal Microscopy

Model for the novel pathway that regulates fascin-1/actin interaction and filopodia stability . See Discussion for details. As shown in Figure 6, active p-Lin-11/Isl-1/Mec-3 kinase (LIMK)1/2 bound to both S39-phosphorylated and non-phosphorylated fascin-1.
Figure Legend Snippet: Model for the novel pathway that regulates fascin-1/actin interaction and filopodia stability . See Discussion for details. As shown in Figure 6, active p-Lin-11/Isl-1/Mec-3 kinase (LIMK)1/2 bound to both S39-phosphorylated and non-phosphorylated fascin-1.

Techniques Used:

Rho activity promotes the interaction of fascin-1 with actin: detection by a novel fascin-1/lifeact fluorescence resonance energy transfer (FRET) system . (A,B) Measurement of the interaction of monomeric red fluorescent protein (mRFP)-fascin-1S39A with green fluorescent protein (GFP)-lifeact in (A) C2C12 cells on fibronectin (FN) or (B) SW480 cells on laminin (LN). Cells transiently transfected with the indicated plasmids were plated on (A) FN for 1 hour, or (B) LN for 2 hours, without or with pre-treatment with the indicated inhibitors, then fixed, mounted, and imaged using fluorescence lifetime imaging microscopy (FLIM) to measure FRET. In each panel, intensity multiphoton GFP (donor) images are shown with the corresponding epifluorescence image for mRFP (acceptor). (B) Representative images of GFP and lifetime plot in the absence of an acceptor, or in presence of mRFP-fascin-1S39D, which does not bundle F-actin. In each panel, lifetime images are presented in a blue-to-red pseudocolor scale with red as short lifetime. (C) ,Percentage FRET efficiency under each experimental condition. Each column represents the mean from eight to twelve cells per condition and three independent experiments; bars indicate SEM. * P
Figure Legend Snippet: Rho activity promotes the interaction of fascin-1 with actin: detection by a novel fascin-1/lifeact fluorescence resonance energy transfer (FRET) system . (A,B) Measurement of the interaction of monomeric red fluorescent protein (mRFP)-fascin-1S39A with green fluorescent protein (GFP)-lifeact in (A) C2C12 cells on fibronectin (FN) or (B) SW480 cells on laminin (LN). Cells transiently transfected with the indicated plasmids were plated on (A) FN for 1 hour, or (B) LN for 2 hours, without or with pre-treatment with the indicated inhibitors, then fixed, mounted, and imaged using fluorescence lifetime imaging microscopy (FLIM) to measure FRET. In each panel, intensity multiphoton GFP (donor) images are shown with the corresponding epifluorescence image for mRFP (acceptor). (B) Representative images of GFP and lifetime plot in the absence of an acceptor, or in presence of mRFP-fascin-1S39D, which does not bundle F-actin. In each panel, lifetime images are presented in a blue-to-red pseudocolor scale with red as short lifetime. (C) ,Percentage FRET efficiency under each experimental condition. Each column represents the mean from eight to twelve cells per condition and three independent experiments; bars indicate SEM. * P

Techniques Used: Activity Assay, Fluorescence, Förster Resonance Energy Transfer, Transfection, Imaging, Microscopy

Rho activity does not modulate the interaction of fascin-1 with conventional protein kinase C (cPKC) . (A) Measurement of the interaction of green fluorescent protein (GFP)-fascin-1 with PKCα- monomeric red fluorescent protein (mRFP) in C2C12 cells on fibronectin (FN). (B) Measurement of the interaction of green fluorescent protein (GFP)-fascin-1 with PKCγ-mRFP in SW480 cells on laminin (LN). Cells transiently transfected with the indicated plasmids were plated on (A) FN for 1 hour, or (B) LN for 2 hours, without or with pre-treatment with the indicated inhibitors, then fixed, mounted, and imaged using fluorescence lifetime imaging microscopy (FLIM) to measure fluorescence resonance energy transfer (FRET). In each panel, intensity multiphoton GFP (donor) images are shown with the corresponding epifluorescence image for mRFP (acceptor). Lifetime images are presented in a blue-to-red pseudocolor scale with red as short lifetime. (C) , Percentage FRET efficiency under each experimental condition. Each column represents the mean from eight to twelve cells per condition and three independent experiments; bars indicate SEM.* P
Figure Legend Snippet: Rho activity does not modulate the interaction of fascin-1 with conventional protein kinase C (cPKC) . (A) Measurement of the interaction of green fluorescent protein (GFP)-fascin-1 with PKCα- monomeric red fluorescent protein (mRFP) in C2C12 cells on fibronectin (FN). (B) Measurement of the interaction of green fluorescent protein (GFP)-fascin-1 with PKCγ-mRFP in SW480 cells on laminin (LN). Cells transiently transfected with the indicated plasmids were plated on (A) FN for 1 hour, or (B) LN for 2 hours, without or with pre-treatment with the indicated inhibitors, then fixed, mounted, and imaged using fluorescence lifetime imaging microscopy (FLIM) to measure fluorescence resonance energy transfer (FRET). In each panel, intensity multiphoton GFP (donor) images are shown with the corresponding epifluorescence image for mRFP (acceptor). Lifetime images are presented in a blue-to-red pseudocolor scale with red as short lifetime. (C) , Percentage FRET efficiency under each experimental condition. Each column represents the mean from eight to twelve cells per condition and three independent experiments; bars indicate SEM.* P

Techniques Used: Activity Assay, Transfection, Fluorescence, Imaging, Microscopy, Förster Resonance Energy Transfer

Rho kinase activity promotes the interaction of fascin-1 with actin . (A) Percentage FRET efficiency of the interaction of monomeric red fluorescent protein (mRFP)-fascin-1S39A with green fluorescent protein (GFP)-lifeact in SW480 cells on laminin (LN) under control conditions or after inhibition of Rho kinases by Y27632. Each column represents the mean from eight to twelve cells per condition and three independent experiments; bars indicate SEM. * P
Figure Legend Snippet: Rho kinase activity promotes the interaction of fascin-1 with actin . (A) Percentage FRET efficiency of the interaction of monomeric red fluorescent protein (mRFP)-fascin-1S39A with green fluorescent protein (GFP)-lifeact in SW480 cells on laminin (LN) under control conditions or after inhibition of Rho kinases by Y27632. Each column represents the mean from eight to twelve cells per condition and three independent experiments; bars indicate SEM. * P

Techniques Used: Activity Assay, Inhibition

Rho-dependent and Rho kinase-dependent interaction of fascin-1 with p-Lin-11/Isl-1/Mec-3 kinase (LIMK) (A,B) . Measurement of the interaction of (A) green fluorescent protein (GFP)-LIMK1 (A), or (B) GFP-LIMK2, with monomeric red fluorescent protein (mRFP)-fascin-1S39A in SW480 cells on laminin (LN). Cells transiently transfected with the indicated plasmids were plated on LN for 2 hours, without or with pre-treatment with the indicated inhibitors, then fixed, mounted, and imaged using fluorescence lifetime imaging microscopy (FLIM) to measure fluorescence resonance energy transfer (FRET). In each panel, intensity multiphoton GFP (donor) images are shown with the corresponding epifluorescence image for monomeric red fluorescent protein (mRFP) (acceptor). Lifetime images are presented in a blue-to-red pseudocolor scale with red as short lifetime. (C) , Percentage FRET efficiency under each experimental condition. Each column represents the mean from nine to sixteen cells per condition and three independent experiments; bars indicate SEM. * P
Figure Legend Snippet: Rho-dependent and Rho kinase-dependent interaction of fascin-1 with p-Lin-11/Isl-1/Mec-3 kinase (LIMK) (A,B) . Measurement of the interaction of (A) green fluorescent protein (GFP)-LIMK1 (A), or (B) GFP-LIMK2, with monomeric red fluorescent protein (mRFP)-fascin-1S39A in SW480 cells on laminin (LN). Cells transiently transfected with the indicated plasmids were plated on LN for 2 hours, without or with pre-treatment with the indicated inhibitors, then fixed, mounted, and imaged using fluorescence lifetime imaging microscopy (FLIM) to measure fluorescence resonance energy transfer (FRET). In each panel, intensity multiphoton GFP (donor) images are shown with the corresponding epifluorescence image for monomeric red fluorescent protein (mRFP) (acceptor). Lifetime images are presented in a blue-to-red pseudocolor scale with red as short lifetime. (C) , Percentage FRET efficiency under each experimental condition. Each column represents the mean from nine to sixteen cells per condition and three independent experiments; bars indicate SEM. * P

Techniques Used: Transfection, Fluorescence, Imaging, Microscopy, Förster Resonance Energy Transfer

Rho inhibition modulates peripheral fascin-containing protrusions . (A) C2C12 cells (control or treated with the indicated pharmacological inhibitors), were plated onto 50 nmol/l fibronectin (FN) for 1 hour, then fixed and stained for fascin-1. Arrowheads indicate examples of fascin-containing protrusions, dotted arrow indicates fascin in association with stress fibers. Boxed areas are enlarged below. Scale bars, 10 μm. (B) Representative results of rhotekin-Rho-binding domain (RBD) pull-down of Rho-guanine triphosphate (GTP) from C2C12 cells adherent on 30 nmol/l FN or thrombospondin-1 for 1 hour, or suspended for 90 minutes over BSA-coated plastic. (C,D) Quantification of (C) numbers and (D) length of peripheral fascin bundles in C2C12 cells adherent for 1 hour on 50 nnmol/l FN after each treatment. Each column represents the mean from 70 to 100 cells from 3 independent experiments; bars indicate SEM. * P
Figure Legend Snippet: Rho inhibition modulates peripheral fascin-containing protrusions . (A) C2C12 cells (control or treated with the indicated pharmacological inhibitors), were plated onto 50 nmol/l fibronectin (FN) for 1 hour, then fixed and stained for fascin-1. Arrowheads indicate examples of fascin-containing protrusions, dotted arrow indicates fascin in association with stress fibers. Boxed areas are enlarged below. Scale bars, 10 μm. (B) Representative results of rhotekin-Rho-binding domain (RBD) pull-down of Rho-guanine triphosphate (GTP) from C2C12 cells adherent on 30 nmol/l FN or thrombospondin-1 for 1 hour, or suspended for 90 minutes over BSA-coated plastic. (C,D) Quantification of (C) numbers and (D) length of peripheral fascin bundles in C2C12 cells adherent for 1 hour on 50 nnmol/l FN after each treatment. Each column represents the mean from 70 to 100 cells from 3 independent experiments; bars indicate SEM. * P

Techniques Used: Inhibition, Staining, Binding Assay

4) Product Images from "Analysis of Fascin-1 in Relation to Gleason Risk Classification and Nuclear ETS-Related Gene Status of Human Prostate Carcinomas: An Immunohistochemical Study of Clinically Annotated Tumours From the Wales Cancer Bank"

Article Title: Analysis of Fascin-1 in Relation to Gleason Risk Classification and Nuclear ETS-Related Gene Status of Human Prostate Carcinomas: An Immunohistochemical Study of Clinically Annotated Tumours From the Wales Cancer Bank

Journal: Biomarkers in Cancer

doi: 10.1177/1179299X17710944

Fascin-1 staining patterns in human prostate carcinomas. Examples of fascin-1 staining in conventional sections of prostate carcinomas. (A) Uninvolved tissue; (B, C), low/intermediate grade, Gleason score 7 tumours; (D)-(F), high grade, Gleason score 9 tumours. Focal areas of fascin-1–positive tumour cells are indicated by asterisks in (D). All sections were counter-stained with hematoxylin-eosin.
Figure Legend Snippet: Fascin-1 staining patterns in human prostate carcinomas. Examples of fascin-1 staining in conventional sections of prostate carcinomas. (A) Uninvolved tissue; (B, C), low/intermediate grade, Gleason score 7 tumours; (D)-(F), high grade, Gleason score 9 tumours. Focal areas of fascin-1–positive tumour cells are indicated by asterisks in (D). All sections were counter-stained with hematoxylin-eosin.

Techniques Used: Staining

Assessment of fascin-1 status against clinico-pathological characteristics of human prostate carcinomas from the tissue microarray. (A) Relationship between fascin-1 score and Gleason risk classification of the TMA tumour specimens. (B) Relationship between fascin-1 score and tumour stage (stage T1 = 12 tumours, T2 = 93 tumours, T3/4 = 106 tumours). (C) Relationship between fascin-1 score and serum PSA levels of the patients prior to prostate biopsy. Serum PSA was between 0 and 10 ng/mL for 108 patients; between 10 and 20 ng/mL for 61 patients, and > 20 ng/mL for 42 patients. (D) Relationship between fascin-1 score and occurrence of biochemical relapse after surgery. The data set includes 19 samples from 18 patients with biochemical relapse and 160 from patients who did not undergo biochemical relapse. (E) Relationship between elevated stromal fascin-1 (scored as yes or no, see Supplementary Datafile) and Gleason risk classification. Data in (A) to (D) are presented as box and whisker plots, and statistical analyses were carried out by Mann-Whitney U test. CI indicates confidence interval; PSA, prostate-specific antigen; TMA, tissue microarray.
Figure Legend Snippet: Assessment of fascin-1 status against clinico-pathological characteristics of human prostate carcinomas from the tissue microarray. (A) Relationship between fascin-1 score and Gleason risk classification of the TMA tumour specimens. (B) Relationship between fascin-1 score and tumour stage (stage T1 = 12 tumours, T2 = 93 tumours, T3/4 = 106 tumours). (C) Relationship between fascin-1 score and serum PSA levels of the patients prior to prostate biopsy. Serum PSA was between 0 and 10 ng/mL for 108 patients; between 10 and 20 ng/mL for 61 patients, and > 20 ng/mL for 42 patients. (D) Relationship between fascin-1 score and occurrence of biochemical relapse after surgery. The data set includes 19 samples from 18 patients with biochemical relapse and 160 from patients who did not undergo biochemical relapse. (E) Relationship between elevated stromal fascin-1 (scored as yes or no, see Supplementary Datafile) and Gleason risk classification. Data in (A) to (D) are presented as box and whisker plots, and statistical analyses were carried out by Mann-Whitney U test. CI indicates confidence interval; PSA, prostate-specific antigen; TMA, tissue microarray.

Techniques Used: Microarray, Whisker Assay, MANN-WHITNEY

Analysis of fascin-1 in relation to nuclear ERG status in human prostate carcinomas from the tissue microarray. (A) Correlation of increased nuclear ERG score with Gleason risk classification. (B) Relationship between fascin-1 score and ERG score. (C) Relationship between fascin-1 score and ERG-positive or ERG-negative status, where ERG negativity is taken as tumours containing
Figure Legend Snippet: Analysis of fascin-1 in relation to nuclear ERG status in human prostate carcinomas from the tissue microarray. (A) Correlation of increased nuclear ERG score with Gleason risk classification. (B) Relationship between fascin-1 score and ERG score. (C) Relationship between fascin-1 score and ERG-positive or ERG-negative status, where ERG negativity is taken as tumours containing

Techniques Used: Microarray

Examples of fascin-1 and ETS-related gene (ERG) staining patterns in human prostate carcinomas from the tissue microarray. (A, B) Staining for fascin-1; (C, D), staining for ERG. (A) and (C) are from Gleason grade 6 tumours with fascin-1 (A) or ERG (C), staining that is indistinguishable from the normal tissue. (B) Example of a Gleason grade 8 tumour with elevated fascin-1 in the stroma and de-differentiated glands. Arrow indicates a predominantly fascin-1–negative gland adjacent to 2 fascin-1–positive glands. (D) Example of a Gleason grade 7 tumour with elevated nuclear ERG in most cells.
Figure Legend Snippet: Examples of fascin-1 and ETS-related gene (ERG) staining patterns in human prostate carcinomas from the tissue microarray. (A, B) Staining for fascin-1; (C, D), staining for ERG. (A) and (C) are from Gleason grade 6 tumours with fascin-1 (A) or ERG (C), staining that is indistinguishable from the normal tissue. (B) Example of a Gleason grade 8 tumour with elevated fascin-1 in the stroma and de-differentiated glands. Arrow indicates a predominantly fascin-1–negative gland adjacent to 2 fascin-1–positive glands. (D) Example of a Gleason grade 7 tumour with elevated nuclear ERG in most cells.

Techniques Used: Staining, Microarray

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Incubation:

Article Title: Geldanamycin-mediated inhibition of heat shock protein 90 partially activates dendritic cells, but interferes with their full maturation, accompanied by impaired upregulation of RelB
Article Snippet: .. For intracellular detection of Fascin 1 (Fscn1), MO-DCs were permeabilized with methanol (10 min on ice), washed with pre-cooled PBS, and incubated with FITC-conjugated anti-Fscn1 (55 K-2; Dako, Glostrup, Denmark) or isotype control antibody. .. All samples were analysed at the same fluorescence detector settings in order to allow for direct comparison of mean fluorescence intensities (MFIs).

Immunohistochemistry:

Article Title: Analysis of Fascin-1 in Relation to Gleason Risk Classification and Nuclear ETS-Related Gene Status of Human Prostate Carcinomas: An Immunohistochemical Study of Clinically Annotated Tumours From the Wales Cancer Bank
Article Snippet: .. Immunohistochemistry was performed with a mouse monoclonal antibody to fascin-1 (clone 55k2; Dako, Denmark) at 1:50 dilution for 30 minutes, followed by Vectastain Universal Elite ABC immunohistochemistry kit (with 1:100 dilution of secondary antibody) and ImmPACT DAB peroxidase substrate detection reagent (all from Vector Labs, Peterborough, UK). .. Slides were then washed in cold running water for 5 minutes and counter-stained in Gill’s hematoxylin (Sigma-Aldrich, Gillingham, UK).