Journal: PLoS ONE

Article Title: Investigation of FOXM1 as a Potential New Target for Melanoma

doi: 10.1371/journal.pone.0144241

Figure Lengend Snippet: PANC1 cells were used as a positive control. (a) The results of the quantitative RT-PCR analyses of FOXM1 mRNA expression. The relative transcript levels of FOXM1 normalized to the level in PANC1 are shown. (b) The expression of FOXM1 proteins in the malignant melanoma cell lines and NHEM. (c) The results of the quantitative RT-PCR analyses of the miR-370 mRNA expression. (d) The results of the semiquantitative RT-PCR using primers that can detect three splicing variants: FOXM1a (472bp), FOXM1b (323bp) and FOXM1c (368bp).

Article Snippet: Sections of paraffin-embedded melanomas and nevus tissue samples were stained with a monoclonal mouse anti-FOXM1 antibody (clone 3A9; Abnova, Taipei, Taiwan), monoclonal mouse anti-BRAFV600E antibody (clone VE1; Spring Bioscience, Pleasanton, CA) and monoclonal rabbit anti-phospho-AKT (Ser473) antibody (Cell Signaling Technologies, Tokyo, Japan).

Techniques: Positive Control, Quantitative RT-PCR, Expressing, Reverse Transcription Polymerase Chain Reaction

Journal: PLoS ONE

Article Title: Investigation of FOXM1 as a Potential New Target for Melanoma

doi: 10.1371/journal.pone.0144241

Figure Lengend Snippet: The FOXM1 expression (normalized to GAPDH) in the patients with primary melanoma (n = 25), metastatic melanoma (n = 9) and nevi (n = 10) is shown. The bars indicate the median values.

Article Snippet: Sections of paraffin-embedded melanomas and nevus tissue samples were stained with a monoclonal mouse anti-FOXM1 antibody (clone 3A9; Abnova, Taipei, Taiwan), monoclonal mouse anti-BRAFV600E antibody (clone VE1; Spring Bioscience, Pleasanton, CA) and monoclonal rabbit anti-phospho-AKT (Ser473) antibody (Cell Signaling Technologies, Tokyo, Japan).

Techniques: Expressing

Journal: PLoS ONE

Article Title: Investigation of FOXM1 as a Potential New Target for Melanoma

doi: 10.1371/journal.pone.0144241

Figure Lengend Snippet: Representative images of the immunohistochemical staining for FOXM1 in primary malignant melanoma (a, b, c, d, e) and nevus tissues samples (f, g, h). Hematoxylin and eosin staining (a, f: × 40) and FOXM1 immunohistochemistry (b, g: × 40, c, h: × 400). Negative controls using an isotype monoclonal antibody were presented in d and e (d: × 40, e: × 400). Melanin granules are indicated by blue staining, although they did not exhibit FOXM1 expression. Bars: 500 μm (a, b, d, f, g), 50 μm (c, e, h).

Article Snippet: Sections of paraffin-embedded melanomas and nevus tissue samples were stained with a monoclonal mouse anti-FOXM1 antibody (clone 3A9; Abnova, Taipei, Taiwan), monoclonal mouse anti-BRAFV600E antibody (clone VE1; Spring Bioscience, Pleasanton, CA) and monoclonal rabbit anti-phospho-AKT (Ser473) antibody (Cell Signaling Technologies, Tokyo, Japan).

Techniques: Immunohistochemical staining, Staining, Immunohistochemistry, Expressing

Journal: PLoS ONE

Article Title: Investigation of FOXM1 as a Potential New Target for Melanoma

doi: 10.1371/journal.pone.0144241

Figure Lengend Snippet: The results of the immunohistochemical analysis of FOXM1.

Article Snippet: Sections of paraffin-embedded melanomas and nevus tissue samples were stained with a monoclonal mouse anti-FOXM1 antibody (clone 3A9; Abnova, Taipei, Taiwan), monoclonal mouse anti-BRAFV600E antibody (clone VE1; Spring Bioscience, Pleasanton, CA) and monoclonal rabbit anti-phospho-AKT (Ser473) antibody (Cell Signaling Technologies, Tokyo, Japan).

Techniques: Immunohistochemical staining

Journal: PLoS ONE

Article Title: Investigation of FOXM1 as a Potential New Target for Melanoma

doi: 10.1371/journal.pone.0144241

Figure Lengend Snippet: The results of the immunohistochemical analysis of FOXM1, BRAFV600E and p-AKT.

Article Snippet: Sections of paraffin-embedded melanomas and nevus tissue samples were stained with a monoclonal mouse anti-FOXM1 antibody (clone 3A9; Abnova, Taipei, Taiwan), monoclonal mouse anti-BRAFV600E antibody (clone VE1; Spring Bioscience, Pleasanton, CA) and monoclonal rabbit anti-phospho-AKT (Ser473) antibody (Cell Signaling Technologies, Tokyo, Japan).

Techniques: Immunohistochemical staining

Journal: PLoS ONE

Article Title: Investigation of FOXM1 as a Potential New Target for Melanoma

doi: 10.1371/journal.pone.0144241

Figure Lengend Snippet: The correlation between FOXM1 expression and the tumor thickness.

Article Snippet: Sections of paraffin-embedded melanomas and nevus tissue samples were stained with a monoclonal mouse anti-FOXM1 antibody (clone 3A9; Abnova, Taipei, Taiwan), monoclonal mouse anti-BRAFV600E antibody (clone VE1; Spring Bioscience, Pleasanton, CA) and monoclonal rabbit anti-phospho-AKT (Ser473) antibody (Cell Signaling Technologies, Tokyo, Japan).

Techniques: Expressing

Journal: PLoS ONE

Article Title: Investigation of FOXM1 as a Potential New Target for Melanoma

doi: 10.1371/journal.pone.0144241

Figure Lengend Snippet: A comparison of the overall survival between the patients positive for FOXM1 and those negative for expression, as determined using immunohistochemical staining. The p -values were determined using the log-rank test.

Article Snippet: Sections of paraffin-embedded melanomas and nevus tissue samples were stained with a monoclonal mouse anti-FOXM1 antibody (clone 3A9; Abnova, Taipei, Taiwan), monoclonal mouse anti-BRAFV600E antibody (clone VE1; Spring Bioscience, Pleasanton, CA) and monoclonal rabbit anti-phospho-AKT (Ser473) antibody (Cell Signaling Technologies, Tokyo, Japan).

Techniques: Comparison, Expressing, Immunohistochemical staining, Staining

Journal: PLoS ONE

Article Title: Investigation of FOXM1 as a Potential New Target for Melanoma

doi: 10.1371/journal.pone.0144241

Figure Lengend Snippet: The human melanoma cell lines, MeWo and SK-MEL28, were transfected with control and FOXM1 siRNA. Twenty-four hours after treatment, the quantitative RT-PCR analyses were carried out (a,). Seventy-two hours after treatment, a Western blotting analysis and the BrdU cell proliferation assay were performed (b, c). The p -values were determined using the Mann–Whitney U-test. * p < 0.05.

Article Snippet: Sections of paraffin-embedded melanomas and nevus tissue samples were stained with a monoclonal mouse anti-FOXM1 antibody (clone 3A9; Abnova, Taipei, Taiwan), monoclonal mouse anti-BRAFV600E antibody (clone VE1; Spring Bioscience, Pleasanton, CA) and monoclonal rabbit anti-phospho-AKT (Ser473) antibody (Cell Signaling Technologies, Tokyo, Japan).

Techniques: Transfection, Control, Quantitative RT-PCR, Western Blot, BrdU Cell Proliferation Assay, MANN-WHITNEY

Journal: PLoS ONE

Article Title: Investigation of FOXM1 as a Potential New Target for Melanoma

doi: 10.1371/journal.pone.0144241

Figure Lengend Snippet: The expression of FOXM1 was assessed using a Western blotting analysis. The human melanoma cell lines were treated with 10 μM of MEK1 siRNA for 72 hours.

Article Snippet: Sections of paraffin-embedded melanomas and nevus tissue samples were stained with a monoclonal mouse anti-FOXM1 antibody (clone 3A9; Abnova, Taipei, Taiwan), monoclonal mouse anti-BRAFV600E antibody (clone VE1; Spring Bioscience, Pleasanton, CA) and monoclonal rabbit anti-phospho-AKT (Ser473) antibody (Cell Signaling Technologies, Tokyo, Japan).

Techniques: Expressing, Western Blot

Journal: PLoS ONE

Article Title: Investigation of FOXM1 as a Potential New Target for Melanoma

doi: 10.1371/journal.pone.0144241

Figure Lengend Snippet: (a) The expression of activated AKT was assessed by a Western blotting analysis using phospho-specific anti-AKT antibodies. (b) The results of the Western blotting analysis of the cell lysates from the human melanoma cell lines treated with LY294002 (30 μM) and an AKT inhibitor (20 μM) for 24 hours. The levels of FOXM1, p-AKT (ser 473) and AKT were determined. (c) Melanoma cell lines were transfected with control or FOXM1 siRNA, and a Western blotting was carried out with p-AKT (ser 473), AKT and FOXM1 antibodies 72 hours after treatment.

Article Snippet: Sections of paraffin-embedded melanomas and nevus tissue samples were stained with a monoclonal mouse anti-FOXM1 antibody (clone 3A9; Abnova, Taipei, Taiwan), monoclonal mouse anti-BRAFV600E antibody (clone VE1; Spring Bioscience, Pleasanton, CA) and monoclonal rabbit anti-phospho-AKT (Ser473) antibody (Cell Signaling Technologies, Tokyo, Japan).

Techniques: Expressing, Western Blot, Transfection, Control

Journal: Nature Communications

Article Title: Senescent tumor cells lead the collective invasion in thyroid cancer

doi: 10.1038/ncomms15208

Figure Lengend Snippet: ( a ) Scheme of a leader-cell assay. PTC tissues were digested with collagenase to obtain small fragments of cancer tissues, and fragments were cultured in collagen I containing matrigel for 96 h, followed by SA-β-Gal staining (left panel). SA-β-Gal-positive migrating cells emerged from the tumour organoid (right panel). ‘1', ‘2' and ‘3' indicate the high-magnification field of the original figure. ( b ) CXCLs/CCLs and their receptor expression in cancer invasive region. Experimental scheme was same as . Raw data of mRNA expression is summarized in . ( c ) Expression of CXCLs and their receptors in BRAFV600E-expressing PTC. Expression of CXCLs and CXCRs was analysed in the normal region and PTC by real-time PCR and represented as a dot graph ( n =13, left panel). The values indicate the relative value compared to that of a normal follicle. Expression of CXCL12 and CXCR4 was analysed in the centre and invasive area of cancer by real-time PCR and represented as a bar graph ( n =9, right panel). ‘Cen' and ‘Inv' indicate the centre and invasive area of cancer, respectively. ( d ) Immunohistochemical analysis of CXCL12, CXCR4 and p16 INK4A expression in BRAFV600E-expressing PTC ( n =13). Normal, centre and collective invasive regions of cancer were serially sectioned, and CXCL12, CXCR4 and p16 INK4A expression was analysed by H score. ‘N.S' indicates not significant. ( e ) Expression of CXCLs/CXCRs in BRAFV600E -induced senescent thyrocytes ( n =2, average value). Experimental scheme was same as . Secreted CXCL12 protein was measured by ELISA ( n =3, right lower panel). The P value shown ( d ) was calculated by Wilcoxon signed rank test and the others were calculated by Student's t- test. Bars indicate 50 μm ( a ), 100 μm ( d ), respectively. Error bars, s.d.

Article Snippet: The primary antibodies used were as follows: anti-BRAFV600E (VE1), predilution (#790-4855,Ventana Medical Systems Inc); p16 INK4A , predilution (#705-4713, Ventana Medical Systems Inc); Anti-Human Ki67 antigen, clone MIB-1, 1:100 (M7240, Dako Denmark A/S, Glostrup, Denmark); TTF-1, clone 8G7G3/1, 1:50 (343M-95, Cell Marque, Rocklin, USA); D2-40 (Podoplanin), 1:100 (322M-15, Cell Marque); MMP1, 1:100 (GTX100534, GeneTex, Irvine, CA, USA); MMP3, 1:100 (GTX100723, GeneTex); MMP9, 1:100 (GTX100458,GeneTex); CXCR4, 1:100 (MAB172, R&D System, Minneapolis, MN, USA); CXCL12, 1:100 (MAB350, R&D System); E-cadherin, 1:100 (ab15148, Abcam, Cambridge, MA, USA); Twist1, 1;100 (ab50887, Abcam); Zeb1, 1;100 (NBP1-05987, Novus Biologicals, Littleton, CO, USA).

Techniques: Cell Culture, Staining, Expressing, Real-time Polymerase Chain Reaction, Immunohistochemical staining, Enzyme-linked Immunosorbent Assay

Journal: Nature Communications

Article Title: Senescent tumor cells lead the collective invasion in thyroid cancer

doi: 10.1038/ncomms15208

Figure Lengend Snippet: ( a ) In vitro cell-migration assay. Normal/SNU790- CXCR4 , BRAFV600E /SNU790- CXCR4 and BRAFV600E-shCXCL12 /SNU790- CXCR4 cells were seeded. After 24 h, cell migration was measured. One set of BRAFV600E /SNU790- CXCR4 cells was treated with 1μM of AMD3100. Bar graph indicates the average of independent measurements ( n =3). ( b ) Transwell assay. SNU790- CXCR4 cells suspended in medium were seeded in transwell. Control, BRAFV600E , BRAFV600E / shCXCL12 or BRAFV600E /AMD3100 treated cells were seeded at the bottom. After 24 h, cells that invaded the lower surface of the filters were counted. The number of migrated cells was counted in the 40-fold magnification field, and presented in the bar graph ( n =3, right panel). ( c ) Three-dimensional invasion assay. SNU790- CXCR4 cells were co-cultured with normal or BRAFV600E -induced thyrocytes on the top of collagen I containing matrigel for 48 h (upper panel). Cell invasion was assessed by HE staining (lower panel). Bar graph indicates the average of independent experiments ( n =3). ( d ) mCherry lentivirus-infected SNU790- CXCR4 cells were co-cultured with GFP lentivirus-infected normal (upper panel), BRAFV600E (middle panel) or BRAFV600E/shCXCL12 thyrocytes (lower panel) on the top of collagen I containing matrigel for 48 h. Independent experiments were performed and data are presented in the bar graph ( n =3). The P values were calculated by Student's t- test. Bars indicate 50 μm ( c , d ) and 100 μm ( a , b ), respectively. Error bars, s.d.

Article Snippet: The primary antibodies used were as follows: anti-BRAFV600E (VE1), predilution (#790-4855,Ventana Medical Systems Inc); p16 INK4A , predilution (#705-4713, Ventana Medical Systems Inc); Anti-Human Ki67 antigen, clone MIB-1, 1:100 (M7240, Dako Denmark A/S, Glostrup, Denmark); TTF-1, clone 8G7G3/1, 1:50 (343M-95, Cell Marque, Rocklin, USA); D2-40 (Podoplanin), 1:100 (322M-15, Cell Marque); MMP1, 1:100 (GTX100534, GeneTex, Irvine, CA, USA); MMP3, 1:100 (GTX100723, GeneTex); MMP9, 1:100 (GTX100458,GeneTex); CXCR4, 1:100 (MAB172, R&D System, Minneapolis, MN, USA); CXCL12, 1:100 (MAB350, R&D System); E-cadherin, 1:100 (ab15148, Abcam, Cambridge, MA, USA); Twist1, 1;100 (ab50887, Abcam); Zeb1, 1;100 (NBP1-05987, Novus Biologicals, Littleton, CO, USA).

Techniques: In Vitro, Cell Migration Assay, Migration, Transwell Assay, Invasion Assay, Cell Culture, Staining, Infection

Journal: Nature Communications

Article Title: Senescent tumor cells lead the collective invasion in thyroid cancer

doi: 10.1038/ncomms15208

Figure Lengend Snippet: ( a ) The epithelial marker E-cadherin is retained in cancer emboli in lymphatic channels. PTC specimens were serially immunostained with TTF-1 (brown colour in nuclei)/D2-40 (red colour in cytoplasm), E-cadherin, CXCR4, CXCL12 and p16 INK4A . White triangles indicated D2-40 stained lymphatic vessels. ( b ) Metastatic tumour cells at lymph nodes were stained with CXCR4 and CXCL12, respectively. ( c ) Anoikis inhibitory function of senescent cells. Control, BRAFV600E /shCon, BRAFV600E/shCXCL12 or BRAFV600E /AMD3100 treated cells were co-cultured with thyroid carcinoma cells (SNU790- CXCR4 ) in HEMA-coated plates for 12 h, and cell death was determined by Calcein AM and EthD-1 staining, ( d ) caspase activity and ( e ) apoptosis related proteins expression. Independent experiments were performed and data are presented in the bar graph ( n =3). Thick bars indicate 1 mm ( b ) and thin bars indicate 50 μm ( a – c ), respectively. The P values were calculated by Student's t- test. Error bars, s.d.

Article Snippet: The primary antibodies used were as follows: anti-BRAFV600E (VE1), predilution (#790-4855,Ventana Medical Systems Inc); p16 INK4A , predilution (#705-4713, Ventana Medical Systems Inc); Anti-Human Ki67 antigen, clone MIB-1, 1:100 (M7240, Dako Denmark A/S, Glostrup, Denmark); TTF-1, clone 8G7G3/1, 1:50 (343M-95, Cell Marque, Rocklin, USA); D2-40 (Podoplanin), 1:100 (322M-15, Cell Marque); MMP1, 1:100 (GTX100534, GeneTex, Irvine, CA, USA); MMP3, 1:100 (GTX100723, GeneTex); MMP9, 1:100 (GTX100458,GeneTex); CXCR4, 1:100 (MAB172, R&D System, Minneapolis, MN, USA); CXCL12, 1:100 (MAB350, R&D System); E-cadherin, 1:100 (ab15148, Abcam, Cambridge, MA, USA); Twist1, 1;100 (ab50887, Abcam); Zeb1, 1;100 (NBP1-05987, Novus Biologicals, Littleton, CO, USA).

Techniques: Marker, Staining, Cell Culture, Activity Assay, Expressing