nih3t3 mwisp1 cells  (R&D Systems)

Journal: The Journal of Biological Chemistry

Article Title: WNT1-inducible signaling pathway protein 1 (WISP1/CCN4) stimulates melanoma invasion and metastasis by promoting the epithelial–mesenchymal transition

doi: 10.1074/jbc.RA118.006122

Figure Lengend Snippet: WISP1 knockout in mouse and human melanoma cells inhibited tumor cell migration and invasion. A, 48-h 2D growth of mouse metastatic melanoma cell line B16F10 and two B16F10 Wisp1-knockout cells (-KO1 and -KO2). B, anchorage-independent growth assay of B16F10 and the two knockout cells in soft agar. Colonies were fixed and counted after 14 days. A representative staining image for each sample is shown on the left, and colony counts are plotted on the right. C, wound healing assay of B16F10 and the two knockout cells. Scratches were created on 6-well plates in biological triplicate, and the healing rate was calculated after 24 h. D, Boyden transwell migration assay of B16F10 and the two knockout cells. A representative staining image for each sample is shown on the left, and relative migration efficiency is graphed on the right. E, Boyden transwell invasion assay of B16F10 and the two knockout cells. F, Boyden transwell invasion assay of human metastatic melanoma cell line RPMI-7951 and its two WISP1-knockout cells (-KO1 and -KO2). G, transwell migration assay of B16F10 and its knockout cell (-KO1) using conditioned media with different concentrations of WISP1 as chemoattractant. B16F10 migrated cells with conditioned medium from NIH3T3-Babe were set up as 100% of relative migration efficiency and compared with other cells. H, transwell invasion assay of B16F10 and the two knockout cells using conditioned media with different concentrations of WISP1 as chemoattractant. B16F10 invaded cells with conditioned medium from NIH3T3-Babe were set up as 100% of relative invasion efficiency and compared with other cells. Statistical significance was determined by Student's t test, where p < 0.05 was considered significant, and asterisks were used to indicate calculated range in p values. *, p < 0.05; **, p < 0.01; ***, p < 0.001; ns, not significant. Error bars, S.D.

Article Snippet: The third group was conditioned media from WISP1-overexpressed NIH3T3-mWisp1 cells, with rat anti-Wisp1 (MAB1680, R&D Systems) at a final concentration of 20 μg/ml.

Techniques: Knock-Out, Migration, Growth Assay, Staining, Wound Healing Assay, Transwell Migration Assay, Transwell Invasion Assay

Journal: The Journal of Biological Chemistry

Article Title: WNT1-inducible signaling pathway protein 1 (WISP1/CCN4) stimulates melanoma invasion and metastasis by promoting the epithelial–mesenchymal transition

doi: 10.1074/jbc.RA118.006122

Figure Lengend Snippet: WISP1 induced an EMT gene signature in mouse/human melanoma cells. Unless otherwise specified, all cells were plated on 6-well plates in complete growth medium for 48 h before they were harvested for RNA analysis or treated with the indicated conditioned medium or recombinant protein. A, mRNA expression, revealed by real-time quantitative RT-PCR, of select EMT marker genes and Mitf in uninvaded and invaded B16F10 cells from a Boyden transwell invasion assay. B, immunoblot analysis of WISP1 protein to confirm the disruption of Wisp1 gene in B16F10 and YUMM1.7 knockout cells. 20 μg of whole-cell lysate was loaded in each lane, and β-actin was used as an internal loading control. B16F10-KO1-mWisp1 cells, in which mouse WISP1 expression was resumed with retroviral transduction, were used as a positive control. C, immunoblot analysis of certain EMT marker proteins in B16F10 and YUMM1.7 knockout cells. 20 μg of whole-cell lysate was loaded in each lane, and all cells were compared on the same gel to reveal the relative intensity of each protein. D, comparison of EMT marker gene expression in mouse melanoma B16F10 and its two Wisp1-knockout cells (-KO1 and -KO2). E, comparison of EMT marker gene expression in mouse melanoma YUMM1.7 and its two Wisp1-knockout cells (-KO1 and -KO2). F, comparison of EMT marker gene expression in human melanoma RPMI-7951 and its two WISP1-knockout cells (-KO1 and -KO2). G, stimulation of EMT marker gene expression with recombinant mouse WISP1 protein (rmWISP1). B16F10-KO1 cells were treated with rmWISP1 (final concentration 5 μg/ml) and harvested at the indicated time point for real-time quantitative RT-PCR analysis. H, stimulation of EMT marker gene expression with WISP1-overexpressed or WISP1-immunodepleted conditioned medium (CM). The conditioned media were pretreated with the indicated antibodies for 30 min before they were used on Wisp1-knockout B16F10 cells (-KO1). The cells were collected for real-time qRT-PCR after 3 h of treatment. *, p < 0.05; **, p < 0.01; ***, p < 0.001; ns, not significant. Error bars, S.D.

Article Snippet: The third group was conditioned media from WISP1-overexpressed NIH3T3-mWisp1 cells, with rat anti-Wisp1 (MAB1680, R&D Systems) at a final concentration of 20 μg/ml.

Techniques: Recombinant, Expressing, Quantitative RT-PCR, Marker, Transwell Invasion Assay, Western Blot, Disruption, Knock-Out, Transduction, Positive Control, Comparison, Concentration Assay

Journal: The Journal of Biological Chemistry

Article Title: WNT1-inducible signaling pathway protein 1 (WISP1/CCN4) stimulates melanoma invasion and metastasis by promoting the epithelial–mesenchymal transition

doi: 10.1074/jbc.RA118.006122

Figure Lengend Snippet: SNAI1 overexpression in B16F10 Wisp1-knockout cell rescued the repression of tumor invasion in vitro and metastasis in vivo. A, immunoblot analysis of WISP1 and SNAI1 using B16F10-KO1 cell that were transduced with retroviral vector control (-pBabe) or retrovirus expressing either mouse WISP1 (-mWisp1) or human SNAI1 (-hSnai1). B, comparison of EMT marker gene expression after overexpression of SNAI1 or reintroduction of WISP1 in B16F10-KO1 cells. Cells were plated on 6-well plates in complete growth medium for 48 h before they were harvested for RNA analysis. C, Boyden transwell invasion assay after overexpression of SNAI1 or reintroduction of WISP1 in B16F10-KO1 cells. A representative staining image for each sample is shown on the left, and relative invasion efficiency is graphed on the right. D, experimental metastasis assay in NSG mice using the indicated cells. Each group contained 3–4 mice. All mice were imaged 1 day before the end of the assay, and representative bioluminescence images are shown. E, representative lung and liver images from NSG mice in the experimental metastasis assay described in D. Metastatic tumor colonies on the lung surface from mice with -mWisp1 or -hSnai1 cells are indicated by arrows. F, real-time genomic qPCR for lungs and livers from the experimental metastasis assay in D. The quantitative tumor metastatic burdens were presented as tumor cell number within 10,000 mouse tissue cells. A high-resolution image for E is provided as Fig. S9. *, p < 0.05; **, p < 0.01; ***, p < 0.001; ns, not significant. Error bars, S.D.

Article Snippet: The third group was conditioned media from WISP1-overexpressed NIH3T3-mWisp1 cells, with rat anti-Wisp1 (MAB1680, R&D Systems) at a final concentration of 20 μg/ml.

Techniques: Over Expression, Knock-Out, In Vitro, In Vivo, Western Blot, Transduction, Plasmid Preparation, Expressing, Comparison, Marker, Transwell Invasion Assay, Staining

Journal: bioRxiv

Article Title: WNT1 Inducible Signaling Pathway Protein 1 (WISP1) stimulates melanoma cell invasion and metastasis by promoting epithelial – mesenchymal transition

doi: 10.1101/427088

Figure Lengend Snippet: WISP1 knockout in mouse and human melanoma cells inhibited tumor cell migration and invasion. A, 48-hour 2D growth of mouse metastatic melanoma cell line B16F10 and two B16F10 Wisp1-knockout cells (-KO1 and -KO2). B, Anchorage-independent growth assay of B16F10 and the two knockout cells in soft agar. Colonies were fixed and counted after 14 days. A representative staining image for each sample is shown on left, colony counts is plotted on the right. C, Wound healing assay of B16F10 and the two knockout cells. Scratches were created on 6-well plates in biological triplicate and the healing rate was calculated after 24 hours. D, Boyden transwell migration assay of B16F10 and the two knockout cells. A representative staining image for each sample is shown on left, relative migration efficiency is graphed on the right. E, Boyden transwell invasion assay of B16F10 and the two knockout cells. F, Boyden transwell invasion assay of human metastatic melanoma cell line RPMI-7951 and its two Wisp1-knockout cells (-KO1 and -KO2). G, Transwell migration assay of B16F10 and its knockout cell (-KO1) using conditioned media with different concentration of Wisp1 as chemoattractant. B16F10 migrated cells with conditioned medium from NIH3T3-Babe were set up as 100% of relative migration efficiency and compared with other cells. H, Transwell invasion assay of B16F10 and the two knockout cells using conditioned media with different concentration of Wisp1 as chemoattractant. B16F10 invaded cells with conditioned medium from NIH3T3-Babe were set up as 100% of relative invasion efficiency and compared with other cells. Statistical significance was determined by Student’s t test, where a p-value < 0.05 was considered significant and asterisks was used to indicate calculated range in p-values. *: p-value < 0.05; **: p-value < 0.01; ***: p-value < 0.001; and ns: not significant.

Article Snippet: The third group was conditioned media from Wisp1-overexpressed NIH3T3-mWisp1, with rat anti-Wisp1 (MAB1680, R&D Systems) at a final concentration of 20μg/ml.

Techniques: Knock-Out, Migration, Growth Assay, Staining, Wound Healing Assay, Transwell Migration Assay, Transwell Invasion Assay, Concentration Assay

Journal: bioRxiv

Article Title: WNT1 Inducible Signaling Pathway Protein 1 (WISP1) stimulates melanoma cell invasion and metastasis by promoting epithelial – mesenchymal transition

doi: 10.1101/427088

Figure Lengend Snippet: WISP1 induced an EMT gene signature in mouse/human melanoma cells. Unless otherwise specified, all cells were plated on 6-well plates in complete growth medium for 48 hours before harvested for RNA analysis or treated with indicated conditioned medium or recombinant protein. A, mRNA expression, revealed by real-time quantitative RT-PCR, of select EMT marker genes and Mitf in uninvaded and invaded B16F10 cells from Boyden transwell invasion assay. B, Immunoblot analysis of Wisp1 protein to confirm the disruption of Wisp1 gene in B16F10 and YUMM1.7 knockout cells. 20μg of whole cells lysate was load in each lane and P-actin was used as internal loading control. B16F10-KO1-mWisp1 cell, in which mouse Wisp1 expression was resumed with retroviral transduction, was used as a positive control. C, Immunoblot analysis of certain EMT marker proteins in B16F10 and YUMM1.7 knockout cells. 20μg of whole cells lysate was load in each lane and all cells were compared on the same gel to reveal the relative intensity of each protein. D, Comparison of EMT marker gene expression in mouse melanoma B16F10 and its two Wisp1-knockout cells (-KO1 and -KO2). E Comparison of EMT marker gene expression in mouse melanoma YUMM1.7 and its two Wisp1-knockout cells (-KO1 and - KO2). F, Comparison of EMT marker gene expression in human melanoma RPMI-7951 and its two Wisp1-knockout cells (-KO1 and -KO2). G, Stimulation of EMT marker gene expression with recombinant mouse Wisp1 protein (rmWisp1). B16F10-KO1 cells were treated with rmWisp1 (final 5μg/ml) and harvested at indicated time point for real-time quantitative RT-PCR analysis. H, Stimulation of EMT marker gene expression with Wisp1-overexpressed or Wisp1-immunodepleted conditioned medium. The conditioned media were pre-treated with indicated antibodies for 30 minutes before used on Wisp1-knockout B16F10 cells (-KO1). The cells were collected for real-time qRT-PCR after 3 hour treatment. *: p-value < 0.05; **: p-value < 0.01; ***: p-value < 0.001; ns: not significant.

Article Snippet: The third group was conditioned media from Wisp1-overexpressed NIH3T3-mWisp1, with rat anti-Wisp1 (MAB1680, R&D Systems) at a final concentration of 20μg/ml.

Techniques: Recombinant, Expressing, Quantitative RT-PCR, Marker, Transwell Invasion Assay, Western Blot, Disruption, Knock-Out, Transduction, Positive Control, Comparison

Journal: bioRxiv

Article Title: WNT1 Inducible Signaling Pathway Protein 1 (WISP1) stimulates melanoma cell invasion and metastasis by promoting epithelial – mesenchymal transition

doi: 10.1101/427088

Figure Lengend Snippet: Snai1 overexpression in B16F10 Wisp1-knockout cell rescued the repression on tumor invasion in vitro and metastasis in vivo. A, Immunoblot analysis of Wisp1 and Snai1 using B16F10-KO1 cell that were transduced with retroviral vector control (-pBabe), or retrovirus expressing either mouse Wisp1 (-mWisp1) or human Snai1 (-hSnai1). B, Comparison of EMT marker gene expression after overexpression of Snai1 or reintroduction of Wisp1 in B16F10-KO1 cells. Cells were plated on 6-well plates in complete growth medium for 48 hours before harvested for RNA analysis. C, Boyden transwell invasion assay after overexpression of Snai1 or reintroduction of Wisp1 in B16F10-KO1 cells. A representative staining image for each sample is shown on left, relative invasion efficiency is graphed on the right. D, Experimental metastasis assay in NSG mice using indicated cells. Each group contained 3-4 mice. All mice were imaged one day before the end of the assay and representative bioluminescence images were shown. E, Representative lung and liver images from NSG mice in experimental metastasis assay described in panel ( D ). Metastatic tumor colonies on lung surface from mice with (-mWisp1) or (-hSnai1) cells were pointed by arrows. F, Real time genomic qPCR for lungs and livers from experimental metastasis assay in panel ( D ). The quantitative tumor metastatic burdens were presented as tumor cell number within 10,000 mouse tissue cells. *: p-value < 0.05; **: p-value < 0.01; ***: p-value < 0.001; ns: not significant.

Article Snippet: The third group was conditioned media from Wisp1-overexpressed NIH3T3-mWisp1, with rat anti-Wisp1 (MAB1680, R&D Systems) at a final concentration of 20μg/ml.

Techniques: Over Expression, Knock-Out, In Vitro, In Vivo, Western Blot, Transduction, Plasmid Preparation, Expressing, Comparison, Marker, Transwell Invasion Assay, Staining