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 expression is increased in melanoma and is associated with reduced overall survival of patients diagnosed with primary melanoma. A, Comparison of WISP1 mRNA expression in benign skin conditions (normal skin and benign melanocytic skin nevus) to primary melanoma. Original expression profiles were from . P-values calculated using ANOVA with post-hoc Tukey HSD test. B, Representative original and deconvoluted color images derived from human normal skin and melanoma tissue microarray probed using a WISP1 antibody (HPA007121) and imaged using 3,3’ diaminobenzidine and stained using hematoxylin for a normal skin (left) and two melanoma (right) tissue samples (original tissue microarray images were obtained from www.proteinatlas.org ) . Deconvoluted intensity of WISP1 staining is shown in red while cellular structures stained using hematoxylin are shown in blue. Arrows indicate melanocytes in epidermis and arrowheads indicate fibroblasts in dermis (stroma). C, The average WISP1 staining within normal skin and primary melanoma tissue samples. D, Distributions in non-zero pixel intensity values of WISP1 staining for normal skin (black curves) and primary melanoma (red curves) tissue samples. Numbers indicate the percentage of the distribution that have normalized pixel intensity values greater than 0.2. E, Kaplan-Meier estimate of overall survival of patients diagnosed with primary melanoma stratified by WISP1 transcript abundance (data from TCGA). Sample numbers and p-values calculated using the Peto & Peto modification of the Gehan-Wilcoxon test are indicated. F, Patient population characteristics of WISP1 high and WISP1 low groups. Statistical differences among categorical data and age were assessed using Fisher’s Exact test and Student’s t-Test, respectively (n.s. indicates p-value > 0.05).

Article Snippet: Recombinant mouse Wisp1 (rmWisp1, 1680-WS-050) was from R&D Systems and used at a final concentration of 5μg/ml following manufacturer’s instructions.

Techniques: Expressing, Comparison, Derivative Assay, Microarray, Staining, Modification

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: Recombinant mouse Wisp1 (rmWisp1, 1680-WS-050) was from R&D Systems and used at a final concentration of 5μg/ml following manufacturer’s instructions.

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: Real time genomic qPCR revealed that Wisp1 knockout repressed the spontaneous metastasis of melanoma cell line B16F10 in C57BL/6Ncrl mice. Growth of tumors derived from B16F10 or its knockout cell (-KO2) were monitored following subcutaneous injection in NSG mice (A: B16F10 (n = 5) or WISP1 KO cell (n = 5)) or in C57BL/6Ncrl mice (B: B16F10 (n=6) or WISP1 KO cell (n=6)). After 21 days, remaining C57BL/6crl mice (n = 4 in each group) were euthanized and lungs and livers were assayed for B16F10 tumor cells using real time genomic qPCR, as described in Materials and Methods. (C) Representative lungs and livers from C57BL/6Ncrl mice with B16F10 or knockout cell at day 21. (D) Real time genomic qPCR results showed quantitative tumor lung and liver metastatic burden in spontaneous metastasis assays, n.d., not detected.

Article Snippet: Recombinant mouse Wisp1 (rmWisp1, 1680-WS-050) was from R&D Systems and used at a final concentration of 5μg/ml following manufacturer’s instructions.

Techniques: Knock-Out, Derivative Assay, Injection

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 repressed the experimental metastasis of melanoma cell line B16F10 in immunodeficient NSG mice and immunocompetent C57BL/6Ncrl mice. Experimental metastasis assays were performed in NSG mice ( A-F ) and C57BL/6Ncrl mice ( G-I ) using B16F10 and indicated knockout cells with injection through mouse tail veins. Each group contained five duplicates (N=5) and three representative images were shown. These experiments were repeated and similar results were achieved. A, Bioluminescence imaging performed one day before NSG mice were euthanized. All animals were compared with the same bioluminescence scale. B-C, Tumor lung metastases (black colonies) of NSG mice as captured by photography ( B ) and real time genomic qPCR ( C ). Quantitative tumor lung metastatic burden was assayed and presented as tumor cell number within 10,000 mouse tissue cells. D-E , Tumor liver metastases (black and white nodules) of NSG mice as captured by photography ( D ) and real time genomic qPCR ( E ). Quantitative tumor liver metastatic burden was assayed and presented as tumor cell number within 10,000 mouse tissue cells. F, Tumor kidney metastases (black colonies) of NSG mice as captured by photography. G, Bioluminescence imaging performed one day before C57BL/6Ncrl mice were euthanized. All animals were compared with the same bioluminescence scale. H-I, Tumor lung metastases of C57BL/6Ncrl mice as captured by photography ( H ) and real time genomic qPCR ( I ). *: p-value < 0.05; **: p-value < 0.01; ***: p-value < 0.001.

Article Snippet: Recombinant mouse Wisp1 (rmWisp1, 1680-WS-050) was from R&D Systems and used at a final concentration of 5μg/ml following manufacturer’s instructions.

Techniques: Knock-Out, Injection, Imaging

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 repressed the experimental metastasis of melanoma cell line YUMM1.7 in NSG and C57BL/6Ncrl mice. Experimental metastasis assays were performed in NSG ( A-D ) and C57BL/6Ncrl ( E-H ) mice using YUMM1.7 and indicated knockout cells with injection through mouse tail veins. Each group contained five duplicates (N=5) and two representative images were shown. A, Bioluminescence imaging performed one day before NSG mice were euthanized. All animals were compared with the same bioluminescence scale. B, Tumor lung metastases (white nodules) of NSG mice as captured by photography. C, Real time genomic qPCR quantitatively comparing tumor lung metastatic burdens (tumor cell number within 10,000 mouse tissue cells). D, The whole-body metastasis of tumor cells in NSG mice were plotted and compared using bioluminescence intensity detected in panel (A). Total flux is presented as photon/second (p/s). E, Bioluminescence imaging performed one day before C57BL/6Ncrl mice were euthanized. All animals were compared with the same bioluminescence scale. F, Tumor lung metastases (white nodules) of C57BL/6Ncrl mice as captured by photography. G, Real time genomic qPCR quantitatively comparing tumor lung metastatic burdens. H, The whole-body metastasis of tumor cells in C57BL/6Ncrl mice were plotted and compared using bioluminescence intensity detected in panel (E). *: p-value < 0.05; **: p-value < 0.01; ***: p-value < 0.001.

Article Snippet: Recombinant mouse Wisp1 (rmWisp1, 1680-WS-050) was from R&D Systems and used at a final concentration of 5μg/ml following manufacturer’s instructions.

Techniques: Knock-Out, Injection, Imaging

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: Recombinant mouse Wisp1 (rmWisp1, 1680-WS-050) was from R&D Systems and used at a final concentration of 5μg/ml following manufacturer’s instructions.

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: Recombinant mouse Wisp1 (rmWisp1, 1680-WS-050) was from R&D Systems and used at a final concentration of 5μg/ml following manufacturer’s instructions.

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 activated Akt/MAPK signaling and promoted EMT marker gene expression in mouse melanoma cells. Unless otherwise specified, cell treatment for kinase immunoblot analysis maintained for 30 minutes before cells were lysed for protein extraction while cells were treated for 3 hours prior to RNA extraction for comparing EMT marker gene expression. A, EMT marker gene expression after inhibiting Akt and/or MAPK signaling in B16F10 cells. Cells were treated with specific phospho-Akt inhibitor MK-2206 and/or phospho-MAPK inhibitor U0126. Immunoblot for phospho-Akt and phospho-Erk1/2 inhibition was shown on the right upper corner. Pan-Akt and total Erk1/2 were also probed as loading control. B, EMT marker gene expression after inhibiting Akt and/or MAPK signaling in YUMM1.7 cells, with DMSO as control. C, Immunoblot for phospho-Akt and phospho-Erk1/2 in indicated mouse melanoma cells with treatment of recombinant mouse Wisp1 protein (rmWisp1, final 5μg/ml). Cells grown on 6-well plates in complete DMEM for 48 hours and serum-free DMEM (SFM) for another 48 hours before rmWISP1 was added. Pan-Akt and total Erk1/2 were probed as loading control. D, Immunoblot analysis of Akt/MAPK activation in B16F10 knockout cell (-KO1) by rmWisp1 under different basal phospho-kinase levels. All cells were grown on 6-well plates in complete DMEM for 48 hours (0 hour point for SFM) and switched to SFM for 24 hour or 48 hours. Indicated cells were treated with rmWisp1 for 30 minutes following 0, 24, or 48 hours in SFM before analyzed for kinase activation. The first lane loaded with YUMM 1.7 at 0 hour to compare the relative kinase level between B16F10 and YUMM1.7 cells. E, Immunoblot for Akt/MAPK activation in YUMM1.7 knockout cell (-KO1) by rmWisp1 under different basal phospho-kinase levels. All cells were treated similarly as described in panel (D). The first lane loaded with B16F10 at 0 hour to compare the relative kinase level between B16F10 and YUMM1.7 cells. F Snai1 activation and E-cadherin repression in B16F10 knockout cell (-KO1) by rmWisp1 under different basal phospho-kinase levels. All cells were treated similarly as described in panel (D) except that rmWisp1 treatment at each point maintained for 3 hours. G-H, EMT marker gene expression after Akt/MAPK activation in B16F10-KO1 (G) or YUMM1.7-KO1 (H) by rmWisp1 was blocked by Akt/MAPK inhibitors. rmWisp1 with DMSO or inhibitors was added after indicated cells were grown on 6-well plates in complete DMEM for 48 hours and in SFM for 24 hours. *: p-value < 0.05; **: p-value < 0.01; ***: p-value < 0.001; ns: not significant.

Article Snippet: Recombinant mouse Wisp1 (rmWisp1, 1680-WS-050) was from R&D Systems and used at a final concentration of 5μg/ml following manufacturer’s instructions.

Techniques: Marker, Expressing, Western Blot, Protein Extraction, RNA Extraction, Inhibition, Recombinant, Activation Assay, Knock-Out

Journal: Scientific Reports

Article Title: WISP1/CCN4 inhibits adipocyte differentiation through repression of PPARγ activity

doi: 10.1038/s41598-017-01866-2

Figure Lengend Snippet: WISP1 gene expression is decreased during adipocyte differentiation. ( a ) The expression of WISP1 mRNA was measured in subcutaneous (SAT) and epididymal adipose tissues (VAT) harvested from 12 weeks old C57BL6 mice (n = 4–5 per group). ( b ) Preadipocytes isolated from VAT and SAT were induced to differentiate. Total RNA was isolated and the expression of WISP1 and ADIPOQ mRNA were measured by real-time PCR. The values indicate the changes for the indicated samples compared to VAT on Day 0. ( c ) 3T3-F442A cells were induced to differentiate and the gene expression of WISP1 , and ADIPOQ was measured by real-time PCR. The values indicate the changes for the indicated samples compared to day 0. ( d ) Secreted and intracellular protein levels of WISP1 were analyzed during adipocyte differentiation by ELISA. ( e ) Expression of secreted and intracellular protein levels of WISP1 and adiponectin were measured by Western Blotting. Adiponectin serves as a positive control for adipocyte differentiation while actin was used as a loading control. All values are representative of data from 3 independent experiments each performed in duplicate. Results are presented as means ± SEM *p < 0.05; **p < 0.01; ***p < 0.005.

Article Snippet: After transfer onto nitrocellulose membrane, blots were incubated overnight at 4 °C with anti-WISP1 antibody (1/250, Santa Cruz Biotechnology), anti-β-catenin antibody (1/1000, Santa Cruz Biotechnology), anti-actin-HRP antibody (1/2000, Santa Cruz Biotechnology), anti-adiponectin antibody (1/1000, Pierce), anti-active-β-catenin antibody (1/1000, phospho-β-Catenin (Ser675), Cell Signaling) or anti-Flag-M2 (1/1000, Sigma) and followed by incubation with a horseradish peroxidase-conjugated secondary antibody (1/2000, Cell Signaling).

Techniques: Gene Expression, Expressing, Isolation, Real-time Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay, Western Blot, Positive Control, Control

Journal: Scientific Reports

Article Title: WISP1/CCN4 inhibits adipocyte differentiation through repression of PPARγ activity

doi: 10.1038/s41598-017-01866-2

Figure Lengend Snippet: Stimulation of Wnt-signaling increases the expression of WISP1 and inhibits adipocyte differentiation. 3T3-F442A preadipocytes were exposed to MDI media supplemented with 25 mM Lithium Chloride (LiCl) or, as negative control, 25 mM Sodium Chloride (NaCl). ( a ) Gene expression of WISP1, PPARG , and ADIPOQ were measured by real-time PCR. The values indicate the changes in the indicated sample compared to day 0. ( b ) The secretion of WISP1 following activation of Wnt signaling was determined by ELISA. The concentration of WISP1 is indicated as the ratio of LiCl-treated compared to the corresponding NaCl-treated sample. ( c ) 3T3-F442A preadipocytes at two days post confluence were treated with or without TNF-α at the indicated concentrations. After 24 hours, total RNA was isolated and the expression of WISP1 and ADIPOQ was analyzed by real-time PCR. The values indicate the changes for the indicated sample compared to the vehicle. All values are representative of data from 3 independent experiments each performed in duplicate. Results are presented as means ± SEM *p < 0.05; **p < 0.01; ***p < 0.005.

Article Snippet: After transfer onto nitrocellulose membrane, blots were incubated overnight at 4 °C with anti-WISP1 antibody (1/250, Santa Cruz Biotechnology), anti-β-catenin antibody (1/1000, Santa Cruz Biotechnology), anti-actin-HRP antibody (1/2000, Santa Cruz Biotechnology), anti-adiponectin antibody (1/1000, Pierce), anti-active-β-catenin antibody (1/1000, phospho-β-Catenin (Ser675), Cell Signaling) or anti-Flag-M2 (1/1000, Sigma) and followed by incubation with a horseradish peroxidase-conjugated secondary antibody (1/2000, Cell Signaling).

Techniques: Expressing, Negative Control, Gene Expression, Real-time Polymerase Chain Reaction, Activation Assay, Enzyme-linked Immunosorbent Assay, Concentration Assay, Isolation

Journal: Scientific Reports

Article Title: WISP1/CCN4 inhibits adipocyte differentiation through repression of PPARγ activity

doi: 10.1038/s41598-017-01866-2

Figure Lengend Snippet: WISP1 inhibits adipogenesis. ( a ) 3T3-F442A preadipocytes were transfected or not with 2.5 µg of empty (vector) or WISP1-encoding vector (WISP1) in the presence of MDI media for 72 hours. Alternatively, cells were incubated for 72 hours in MDI media (control) in the presence of recombinant WISP1 (Rec WISP1) or conditioned media from L cells expressing (Wnt3a-CM) or not (CM) Wnt3a. The differentiation process was monitored by microscopic imaging after Oil Red O-staining. Subsequently, Oil Red O-stained areas were quantified by ImageJ analysis (right). ( b ) 3T3-F442A preadipocytes were transfected with 2.5 µg of empty vector or human WISP1-encoding vector. Three, 5 and 8 days after transfection, endogenous murine WISP1, PPARG, ADIPOQ, LPL, FABP4, CD36, DLK1 and CEBPD mRNA levels were quantified by real-time PCR. The values indicate the changes for the indicated sample compared to day 0. All the values are averages of data from 3 independent experiments performed in duplicate. Results are presented as means ± SEM *p < 0.05; **p < 0.01; ***p < 0.005.

Article Snippet: After transfer onto nitrocellulose membrane, blots were incubated overnight at 4 °C with anti-WISP1 antibody (1/250, Santa Cruz Biotechnology), anti-β-catenin antibody (1/1000, Santa Cruz Biotechnology), anti-actin-HRP antibody (1/2000, Santa Cruz Biotechnology), anti-adiponectin antibody (1/1000, Pierce), anti-active-β-catenin antibody (1/1000, phospho-β-Catenin (Ser675), Cell Signaling) or anti-Flag-M2 (1/1000, Sigma) and followed by incubation with a horseradish peroxidase-conjugated secondary antibody (1/2000, Cell Signaling).

Techniques: Transfection, Plasmid Preparation, Incubation, Control, Recombinant, Expressing, Imaging, Staining, Real-time Polymerase Chain Reaction

Journal: Scientific Reports

Article Title: WISP1/CCN4 inhibits adipocyte differentiation through repression of PPARγ activity

doi: 10.1038/s41598-017-01866-2

Figure Lengend Snippet: WISP1 silencing induces adipogenic differentiation. 3T3-F442A preadipocytes were transfected for 48 hours with siRNA control or siWISP1 (30 pmoles). Then, the influence of WISP1 knockdown on ( a ) endogenous WISP1 mRNA levels and ( b ) secreted WISP1 protein levels in the culture media was determined by real-time PCR and ELISA. ( c ) The effect of WISP1 knockdown on lipid accumulation was monitored by microscopic imaging after Oil Red O-staining. mRNA levels of PPARG, ADIPOQ, LPL, FABP4, CD36, DLK1 and CEBPD were measured by real-time PCR. The values indicate the changes for the indicated sample compared to the siRNA-control. ( d ) 3T3-F442A preadipocytes were incubated in MDI media or alternatively transfected with si-WISP1 (30 pmoles) in the presence or the absence of conditioned media from L cells expressing Wnt3a (Wnt3a-CM) for 96 hours. The differentiation process was monitored by microscopic imaging after Oil Red O-staining. Red-stained areas were quantified by ImageJ analysis. All values are averages of data from 3 independent experiments each performed in duplicate. Results are presented as means ± SEM; *p < 0.05; **p < 0.01; ***p < 0.005.

Article Snippet: After transfer onto nitrocellulose membrane, blots were incubated overnight at 4 °C with anti-WISP1 antibody (1/250, Santa Cruz Biotechnology), anti-β-catenin antibody (1/1000, Santa Cruz Biotechnology), anti-actin-HRP antibody (1/2000, Santa Cruz Biotechnology), anti-adiponectin antibody (1/1000, Pierce), anti-active-β-catenin antibody (1/1000, phospho-β-Catenin (Ser675), Cell Signaling) or anti-Flag-M2 (1/1000, Sigma) and followed by incubation with a horseradish peroxidase-conjugated secondary antibody (1/2000, Cell Signaling).

Techniques: Transfection, Control, Knockdown, Real-time Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay, Imaging, Staining, Incubation, Expressing

Journal: Scientific Reports

Article Title: WISP1/CCN4 inhibits adipocyte differentiation through repression of PPARγ activity

doi: 10.1038/s41598-017-01866-2

Figure Lengend Snippet: WISP1 decreases PPARγ transcriptional activity. ( a ) Increasing amounts of expression vector for WISP1 (0.2, 0.4, 0.8 µg) were transfected into HEK or MEF cells along with 0.1 µg of PPRE-TK-Luc and 0.02 µg of an RSV-β-galactosidase construct as an internal control, alone or in combinations with 0.1 µg of PPARγ expressing vector. ( b ) Increasing amounts of WISP1 (0.2, 0.4, 0.8 µg) were transfected into HEK and MEF cells along with 0.1 µg of UAS-TK-Luc and 0.02 µg of an RSV-β-galactosidase construct as an internal control, alone or in combination with 0.1 µg of an expression vector for the Gal4-PPARγ-LBD fusion protein. Five hours after transfection, cells were treated (black bars) or not (open bars) with 1 µM rosiglitazone for 24 hours and assayed for luciferase and β-galactosidase activities. The results represent the average of at least three independent experiments each done in triplicate. Results are presented as means ± SEM; *p < 0.05; **p < 0.01; ***p < 0.005.

Article Snippet: After transfer onto nitrocellulose membrane, blots were incubated overnight at 4 °C with anti-WISP1 antibody (1/250, Santa Cruz Biotechnology), anti-β-catenin antibody (1/1000, Santa Cruz Biotechnology), anti-actin-HRP antibody (1/2000, Santa Cruz Biotechnology), anti-adiponectin antibody (1/1000, Pierce), anti-active-β-catenin antibody (1/1000, phospho-β-Catenin (Ser675), Cell Signaling) or anti-Flag-M2 (1/1000, Sigma) and followed by incubation with a horseradish peroxidase-conjugated secondary antibody (1/2000, Cell Signaling).

Techniques: Activity Assay, Expressing, Plasmid Preparation, Transfection, Construct, Control, Luciferase

Journal: Scientific Reports

Article Title: WISP1/CCN4 inhibits adipocyte differentiation through repression of PPARγ activity

doi: 10.1038/s41598-017-01866-2

Figure Lengend Snippet: WISP1 interacts with and down-regulates PPARγ ( a ) MEF cells were transfected with increasing amounts of Flag-WISP1 expression vector (0.65, 1.30 and 2.60 µg) in combination with 1 µg of PPARγ expression vector. After 4 hours, cells were treated with 1 µM of rosiglitazone and harvested 24 hours later. Lysates were subjected to immunoblotting with anti-PPARγ, anti-Flag, anti-active β-catenin or anti-β-catenin antibodies. Immunoblot signals were quantified by densitometry, and normalized with β-actin. Data were expressed as means ± SEM; *p < 0.05; **p < 0.01; ***p < 0.005. Data are representative of 3 independent experiments ( b ) MEF cells were transfected with 2 µg of Flag-WISP1 expression vector in combination with 2 µg of PPARγ expression vector. After 24 hours, cells were treated with 10 µM MG132 for 6 hours and harvested. Lysates were subjected to immunoblotting with anti-PPARγ and anti-Flag antibodies. Immunoblot signals were quantified by densitometry, and normalized with β-actin. Data were expressed as means ± SEM; *p < 0.05; **p < 0.01; ***p < 0.005. Data are representative of 3 independent experiments ( c ) MEF cells were transfected with 2 µg of Flag-WISP1expression vector in combination with 2 µg of PPARγ expression vector for 24 hours. Lysates were subjected to anti-Flag or anti-PPARγ immunoprecipitation (IP) and followed by immunoblotting (IB) with anti-PPARγ or anti-Flag antibodies respectively. Protein expressions were determined by direct immunoblotting (Input). Two independent experiments were performed. ( d ) MEF cells lysates were subjected to anti-WISP1 or anti-PPARγ immunoprecipitation (IP) and followed by immunoblotting (IB) with anti-PPARγ or anti-WISP1 antibodies, respectively. Immunoprecipitations with IgG were used as controls. Protein expressions were determined by direct immunoblotting (Input). Two independent experiments were performed.

Article Snippet: After transfer onto nitrocellulose membrane, blots were incubated overnight at 4 °C with anti-WISP1 antibody (1/250, Santa Cruz Biotechnology), anti-β-catenin antibody (1/1000, Santa Cruz Biotechnology), anti-actin-HRP antibody (1/2000, Santa Cruz Biotechnology), anti-adiponectin antibody (1/1000, Pierce), anti-active-β-catenin antibody (1/1000, phospho-β-Catenin (Ser675), Cell Signaling) or anti-Flag-M2 (1/1000, Sigma) and followed by incubation with a horseradish peroxidase-conjugated secondary antibody (1/2000, Cell Signaling).

Techniques: Transfection, Expressing, Plasmid Preparation, Western Blot, Immunoprecipitation

Journal: Scientific Reports

Article Title: WISP1/CCN4 inhibits adipocyte differentiation through repression of PPARγ activity

doi: 10.1038/s41598-017-01866-2

Figure Lengend Snippet: WISP1 expression is up-regulated in adipose tissues from obese mice.( a ) WISP1 and A DIPOQ mRNA levels were quantified by RT-qPCR in visceral (epididymal) adipose tissue (VAT) from mice fed with a standard diet (SD) and a high fat diet (HFD) for 12 weeks (n = 7 animals per group). ( b ) WISP1 and ADIPOQ mRNA levels were quantified in VAT from ob/ob mice (n = 7 animals per group). Results are presented as means ± SEM *p < 0.05; ***p < 0.005 versus SD fed mice in panel (a) and versus WT mice in panel (b).

Article Snippet: After transfer onto nitrocellulose membrane, blots were incubated overnight at 4 °C with anti-WISP1 antibody (1/250, Santa Cruz Biotechnology), anti-β-catenin antibody (1/1000, Santa Cruz Biotechnology), anti-actin-HRP antibody (1/2000, Santa Cruz Biotechnology), anti-adiponectin antibody (1/1000, Pierce), anti-active-β-catenin antibody (1/1000, phospho-β-Catenin (Ser675), Cell Signaling) or anti-Flag-M2 (1/1000, Sigma) and followed by incubation with a horseradish peroxidase-conjugated secondary antibody (1/2000, Cell Signaling).

Techniques: Expressing, Quantitative RT-PCR

Journal: Journal of Biological Chemistry

Article Title: Overexpression of WISP-1 Down-regulated Motility and Invasion of Lung Cancer Cells through Inhibition of Rac Activation

doi: 10.1074/jbc.m210945200

Figure Lengend Snippet: FIG. 3. WISP-1 down-regulates the activation of Rac in H460 and H1299 cells. PAK-agarose conjugates were used to precipitate GTP-bound Rac (activated Rac), and the pulled-down proteins were electrophoresed, transferred to a polyvinylidene difluoride membrane, and immunoblotted with anti-Rac antibody. The assay showed a strong reduction in the precipitation of activated Rac in WISP-1-transfected H460 and H1299 cells compared with vector controls. Cell lysate con- trols demonstrated equal amounts of total Rac.

Article Snippet: In Vivo Lung Metastasis Assay—In an in vivo lung metastasis experiment, vector control or WISP-1-expressing H460 cells (106 cells/0.1 ml of DPBS) were injected into the lateral tail veins of nude mice (Taconic, Germantown, NY).

Techniques: Activation Assay, Membrane, Transfection, Plasmid Preparation

Journal: Journal of Biological Chemistry

Article Title: Overexpression of WISP-1 Down-regulated Motility and Invasion of Lung Cancer Cells through Inhibition of Rac Activation

doi: 10.1074/jbc.m210945200

Figure Lengend Snippet: FIG. 1. Overexpression of WISP-1 inhibits in vivo metastasis and in vitro invasion of H460 lung cancer cells. A, H460/vector control formed large and extensive colonies of tumors in the lungs of nude mice. In contrast, tumor size was reduced in WISP-1 expressors. Arrows indicate tumor masses, which stained darker than the normal lung parenchyma. B, in an in vitro invasion assay, H460/WISP-1 cells demonstrated reduced invasiveness across Matrigel compared with con- trol cells. C, H460 cells were less invasive in the presence of WISP-1- conditioned medium (WISP-1CM) compared with vector control condi- tioned medium (vectorCM).

Article Snippet: In Vivo Lung Metastasis Assay—In an in vivo lung metastasis experiment, vector control or WISP-1-expressing H460 cells (106 cells/0.1 ml of DPBS) were injected into the lateral tail veins of nude mice (Taconic, Germantown, NY).

Techniques: Over Expression, In Vivo, In Vitro, Plasmid Preparation, Control, Staining, Invasion Assay

Journal: Journal of Biological Chemistry

Article Title: Overexpression of WISP-1 Down-regulated Motility and Invasion of Lung Cancer Cells through Inhibition of Rac Activation

doi: 10.1074/jbc.m210945200

Figure Lengend Snippet: FIG. 2. WISP-1 down-regulates motility of H460 and H1299 lung cancer cells. Vector (Vec)- and WISP-1 (W1)-expressing cells were evaluated for migration in the presence of 1.5% serum in Boyden Chambers. WISP-1 transfectants demonstrated reduced levels of mo- tility compared with vector control for both H460 and H1299 cell lines.

Article Snippet: In Vivo Lung Metastasis Assay—In an in vivo lung metastasis experiment, vector control or WISP-1-expressing H460 cells (106 cells/0.1 ml of DPBS) were injected into the lateral tail veins of nude mice (Taconic, Germantown, NY).

Techniques: Plasmid Preparation, Expressing, Migration, Control

Journal: Journal of Biological Chemistry

Article Title: Overexpression of WISP-1 Down-regulated Motility and Invasion of Lung Cancer Cells through Inhibition of Rac Activation

doi: 10.1074/jbc.m210945200

Figure Lengend Snippet: FIG. 6. MMP-1 expression is down-regulated by WISP-1 but up-regulated by RacG12V. Real-time PCR was conducted to verify data obtained from microarray analyses. CT values were used to calcu- late fold change of MMP-1 levels from each sample over that of the vector control. The data showed that the expression of MMP-1 was reduced in WISP-1-expressing H460 cells (W1) by 3-fold over control cells (Vec). In WISP-1/RacG12V (W1/Rac) co-transfectants, however, the MMP-1 transcript was increased by about 3-fold over the levels of control cells.

Article Snippet: In Vivo Lung Metastasis Assay—In an in vivo lung metastasis experiment, vector control or WISP-1-expressing H460 cells (106 cells/0.1 ml of DPBS) were injected into the lateral tail veins of nude mice (Taconic, Germantown, NY).

Techniques: Expressing, Real-time Polymerase Chain Reaction, Microarray, Plasmid Preparation, Control

Journal: Journal of Biological Chemistry

Article Title: Overexpression of WISP-1 Down-regulated Motility and Invasion of Lung Cancer Cells through Inhibition of Rac Activation

doi: 10.1074/jbc.m210945200

Figure Lengend Snippet: FIG. 7. Expression of RacG12V induces motility and invasion of H460/WISP-1 cells across both Matrigel and collagen I. In a modified Boyden Chamber assay, reconstitution of Rac activity in H460/ WISP-1/RacG12V cells increased both cell motility (A) and invasion across Matrigel (B). C, similarly, when collagen I was used as a barrier, WISP-1-expressing cells (W1) were less invasive compared with control cells (I). Transfection of activated Rac restored cell invasion in WISP- 1/RacG12V cells (W1/Rac) to the level in control cells.

Article Snippet: In Vivo Lung Metastasis Assay—In an in vivo lung metastasis experiment, vector control or WISP-1-expressing H460 cells (106 cells/0.1 ml of DPBS) were injected into the lateral tail veins of nude mice (Taconic, Germantown, NY).

Techniques: Expressing, Modification, Boyden Chamber Assay, Activity Assay, Control, Transfection