Journal: Cells

Article Title: Stage-Dependent Fibrotic Gene Profiling of WISP1-Mediated Fibrogenesis in Human Fibroblasts.

doi: 10.3390/cells13232005

Figure Lengend Snippet: Figure 1. WISP1 is endogenously upregulated in primary human DHLF. WISP1 transcript and secreted protein are expressed in NHLF and DHLF. DHLF exhibit significantly higher (A) WISP1 mRNA transcript and (B) secreted WISP1 protein in the condition media, detected by RT-qPCR and ELISA, respectively. Each data point represents one donor-derived primary cell line for both NHLF (n = 5 donors) and DHLF (n = 5 donors), with each experiment performed in triplicates and represented as mean ± SD. Statistical analysis was performed using paired t-test and significance denoted as * p < 0.5 or ** p < 0.01 versus healthy NHLF, as shown.

Article Snippet: WISP1 (DY1627), IL-6 (D6050), and CCL-2 (DCP00) protein content was assessed using an enzyme-linked immunosorbent assay (ELISA) kit from R&D systems as per the manufacturer’s recommendations.

Techniques: Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Derivative Assay

Journal: Cells

Article Title: Stage-Dependent Fibrotic Gene Profiling of WISP1-Mediated Fibrogenesis in Human Fibroblasts.

doi: 10.3390/cells13232005

Figure Lengend Snippet: Figure 2. TGFβ induces WISP1 expression in primary lung and dermal fibroblasts. Stimulation with TGFβ (1 ng/mL) for 24 h significantly increases COL1A1, COL3A1, IL6, ACTA2, FN1, and WISP1 mRNA as compared to control in (A) NHLF, (B) DHLF, and (C) NHDF for 3 respective donors (n = 3/each) analyzed by RT-qPCR. Notably, TGFβ decreased CCL2 in NHLF and DHLF, except in NHDF. Each experiment was independently performed in triplicates with three biological replicates (n = 3) for each donor and represented as mean ± SD. Statistical analysis was performed using one-way ANOVA with Tukey’s post hoc analysis vs. vehicle control for respective donors. * denotes p < 0.5, ** denotes p < 0.01, *** denotes p < 0.001, **** denotes p < 0.0001 versus the unstimulated control, as shown.

Article Snippet: WISP1 (DY1627), IL-6 (D6050), and CCL-2 (DCP00) protein content was assessed using an enzyme-linked immunosorbent assay (ELISA) kit from R&D systems as per the manufacturer’s recommendations.

Techniques: Expressing, Control, Quantitative RT-PCR

Journal: Cells

Article Title: Stage-Dependent Fibrotic Gene Profiling of WISP1-Mediated Fibrogenesis in Human Fibroblasts.

doi: 10.3390/cells13232005

Figure Lengend Snippet: Figure 3. WISP1 promotes a striking increase in primary human dermal fibroblast cell proliferation. (A) Treatment with WISP1 (1000 nM) shows a striking increase in cell proliferation (p < 0.0001 via one- way ANOVA with Tukey’s post hoc analysis) detected by increased pRb positive cells as compared to control in NHDF. Representative 10× images were captured with the top image representing Hoechst nuclear stain, while the bottom panel represents pRb green fluorescence. PDGF-BB (100 nM) was used as an internal positive control to ensure assay reliability. The PDGF-BB dose response curve is shown in the Supplementary Materials. (B) % pRb positive cells were calculated by dividing pRb positive cells over total number of cells calculated by nuclear Hoechst staining. Three independent experiments (n = 3) were performed in triplicates and represented as mean ± SD. Statistical analysis was performed using one-way ANOVA with Tukey’s post hoc analysis versus the vehicle-control condition and significance denoted as * p < 0.5, **** p < 0.0001.

Article Snippet: WISP1 (DY1627), IL-6 (D6050), and CCL-2 (DCP00) protein content was assessed using an enzyme-linked immunosorbent assay (ELISA) kit from R&D systems as per the manufacturer’s recommendations.

Techniques: Control, Staining, Fluorescence, Positive Control

Journal: Cells

Article Title: Stage-Dependent Fibrotic Gene Profiling of WISP1-Mediated Fibrogenesis in Human Fibroblasts.

doi: 10.3390/cells13232005

Figure Lengend Snippet: Figure 4. WISP1 increases CCL2 and IL6 in primary fibroblasts. Stimulation with WISP1 (1000 nM) for 24 h significantly increases CCL2 and IL6 gene expression in (A) NHLF, (B) DHLF, and (C) NHDF as compared to control. TGFβ (1 ng/mL) for 24 h served as a positive control and significantly increases

Article Snippet: WISP1 (DY1627), IL-6 (D6050), and CCL-2 (DCP00) protein content was assessed using an enzyme-linked immunosorbent assay (ELISA) kit from R&D systems as per the manufacturer’s recommendations.

Techniques: Gene Expression, Control, Positive Control

Journal: Cells

Article Title: Stage-Dependent Fibrotic Gene Profiling of WISP1-Mediated Fibrogenesis in Human Fibroblasts.

doi: 10.3390/cells13232005

Figure Lengend Snippet: Figure 5. Recombinant WISP1 synergistically induces inflammatory markers along with TGFβ. (A) Primary human fibroblasts were stimulated with TGFβ (1 ng/mL) for 24 h for initiation, followed by WISP1 (1000 nM) for another 24 h as per the experimental layout. CCL2 and IL6 mRNA expression was significantly increased in (B) non-diseased lung and (D) dermal fibroblasts, but not in (C) IPF- diseased lung fibroblasts. Statistical analysis was performed using one-way ANOVA with Tukey’s post hoc analysis vs. vehicle control for respective donors. * denotes p < 0.5, ** denotes p < 0.01, *** denotes p < 0.001, **** denotes p < 0.0001 versus the unstimulated control, for three biological replicates (n = 3) and represented as mean ± SD.

Article Snippet: WISP1 (DY1627), IL-6 (D6050), and CCL-2 (DCP00) protein content was assessed using an enzyme-linked immunosorbent assay (ELISA) kit from R&D systems as per the manufacturer’s recommendations.

Techniques: Recombinant, Expressing, Control

Journal: Cells

Article Title: Stage-Dependent Fibrotic Gene Profiling of WISP1-Mediated Fibrogenesis in Human Fibroblasts.

doi: 10.3390/cells13232005

Figure Lengend Snippet: Figure 6. WISP1 fibrotic gene signature in dermal fibroblasts. The volcano plot illustrates fibroblast genes upregulated (red) and downregulated (blue) upon stimulation with either (A) WISP1 alone or (B) in conjugation with TGFβ as compared to vehicle control (PBS) in NHDF (n = 3). The plots representing statistically significant gene with |FC| > 1.5 and adj. p-value < 0.1 are plotted with the x-axis representing log2 fold change (FC) versus the y-axis −log10 p-value. (C) The heatmap represents pathways significantly associated with the corresponding treatment condition as percent of genes influenced and the list of genes used in the pathway analysis is highlighted in Table S8. (D) Scatterplot showing log2 fold change for the differentially expressed genes when comparing WISP1 + TGFβ treatment with WISP1 (x-axis) versus TGFβ (y-axis). (E) Venn diagram representing unique and overlapping set of genes between the WISP1, TGFβ, and WISP1 + TGFβ treatment paradigms. A comprehensive lists of genes is provided in the Supplementary Materials.

Article Snippet: WISP1 (DY1627), IL-6 (D6050), and CCL-2 (DCP00) protein content was assessed using an enzyme-linked immunosorbent assay (ELISA) kit from R&D systems as per the manufacturer’s recommendations.

Techniques: Conjugation Assay, Control

Journal: Cells

Article Title: Stage-Dependent Fibrotic Gene Profiling of WISP1-Mediated Fibrogenesis in Human Fibroblasts.

doi: 10.3390/cells13232005

Figure Lengend Snippet: Figure 7. WISP1 fibrotic gene signature in lung fibroblasts. The volcano plot illustrates fibroblast genes upregulated (red) and downregulated (blue) upon stimulation with either (A) WISP1 alone or (B) in conjugation with TGFβ as compared to vehicle control (PBS) in NHLF (n = 3). The plots representing statistically significant gene with |FC| > 1.5 and adj p-value < 0.1 are plotted with the x-axis representing log2 fold change (FC) versus the y-axis −log10 p-value. (C) The heatmap represents pathways significantly associated with the corresponding treatment condition as percent of genes influenced and the list of genes used in the pathway analysis is highlighted in Table S8. (D) Scatterplot showing log2 fold change for the differentially expressed genes when comparing WISP1 + TGFβ treatment with WISP1 (x-axis) versus TGFβ (y-axis). (E) Venn diagram representing unique and overlapping set of genes between the WISP1, TGFβ, and WISP1 + TGFβ treatment paradigms. A comprehensive lists of genes is provided in the Supplementary Materials.

Article Snippet: WISP1 (DY1627), IL-6 (D6050), and CCL-2 (DCP00) protein content was assessed using an enzyme-linked immunosorbent assay (ELISA) kit from R&D systems as per the manufacturer’s recommendations.

Techniques: Conjugation Assay, Control

Journal: Cells

Article Title: Stage-Dependent Fibrotic Gene Profiling of WISP1-Mediated Fibrogenesis in Human Fibroblasts.

doi: 10.3390/cells13232005

Figure Lengend Snippet: Figure 8. WISP1 fibrotic gene signature in IPF-diseased fibroblast. The volcano plot illustrates fibroblast genes upregulated (red) and downregulated (blue) upon stimulation with either (A) WISP1 alone or (B) in conjugation with TGFβ as compared to vehicle control (PBS) in DHLF (n = 3). The plots representing statistically significant gene with |FC| > 1.5 and adj p-value < 0.1 are plotted with the x-axis representing log2 fold change (FC) versus the y-axis −log10 p-value. (C) The heatmap represents pathways significantly associated with the corresponding treatment condition as percent of genes influenced and the list of genes used in the pathway analysis is highlighted in Table S8. (D) Scatterplot showing log2 fold change for the differentially expressed genes when comparing WISP1 + TGFβ treatment with WISP1 (x-axis) versus TGFβ (y-axis). (E) Venn diagram represents unique and overlapping set of genes between the WISP1, TGFβ, and WISP1 + TGFβ treatment paradigms. A comprehensive list of genes is provided in the Supplementary Materials.

Article Snippet: WISP1 (DY1627), IL-6 (D6050), and CCL-2 (DCP00) protein content was assessed using an enzyme-linked immunosorbent assay (ELISA) kit from R&D systems as per the manufacturer’s recommendations.

Techniques: Conjugation Assay, Control

Journal: Cells

Article Title: Stage-Dependent Fibrotic Gene Profiling of WISP1-Mediated Fibrogenesis in Human Fibroblasts.

doi: 10.3390/cells13232005

Figure Lengend Snippet: Figure 9. Schematic summary of WISP1-TGFβ crosstalk in regulation of fibrosis progression.

Article Snippet: WISP1 (DY1627), IL-6 (D6050), and CCL-2 (DCP00) protein content was assessed using an enzyme-linked immunosorbent assay (ELISA) kit from R&D systems as per the manufacturer’s recommendations.

Techniques:

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 expression is increased in melanoma and is associated with reduced overall survival of patients diagnosed with primary melanoma. A, WISP1 mRNA expression in benign skin conditions (normal skin and benign melanocytic skin nevus) compared with primary melanoma. The original expression data set (GSE3189) was deposited by Talantov et al. (55). p values were calculated using analysis of variance with the post hoc Tukey honest significant difference 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 normal skin (left) and two melanoma (right) tissue samples. Original tissue microarray images were obtained from www.proteinatlas.org3 (77). Deconvoluted intensity of WISP1 staining is shown in red, whereas the cellular structures stained using hematoxylin are shown in blue. Arrows, melanocytes in epidermis; arrowheads, fibroblasts in dermis (stroma). C, average WISP1 staining within normal skin and primary melanoma tissue samples. D, distributions in nonzero pixel intensity values of WISP1 staining for normal skin (black curves) and primary melanoma (red curves) tissue samples. Numbers, percentage of the distribution that has pixel intensity values greater than a normalized pixel intensity of 0.2. E, Kaplan–Meier estimate of overall survival of melanoma patients stratified by WISP1 transcript abundance. The original data set was from the Cancer Genome Atlas. Sample numbers and p values calculated using the Peto and 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., p > 0.05).

Article Snippet: Recombinant mouse WISP1 (rmWISP1, 1680-WS-050), produced in mouse myeloma NS0 cells, was from R&D Systems and was used at a final concentration of 5 μg/ml following the manufacturer's instructions.

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

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: Recombinant mouse WISP1 (rmWISP1, 1680-WS-050), produced in mouse myeloma NS0 cells, was from R&D Systems and was used at a final concentration of 5 μg/ml following the manufacturer's instructions.

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 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 the indicated knockout cells with injection through mouse tail veins. Each group contained five duplicates (n = 5), and only mice surviving the whole experiments were analyzed at the same time for imaging, photography, and qPCR (final n ≥ 3). These experiments were repeated, and similar results were achieved. A, bioluminescence imaging performed 1 day before NSG mice were euthanized. All animals were compared with the same bioluminescence scale. B and 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 1 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). Four high-resolution images for B, D, F, and H are provided as Figs. S1–S4. *, p < 0.05; **, p < 0.01; ***, p < 0.001. Error bars, S.D.

Article Snippet: Recombinant mouse WISP1 (rmWISP1, 1680-WS-050), produced in mouse myeloma NS0 cells, was from R&D Systems and was used at a final concentration of 5 μg/ml following the manufacturer's instructions.

Techniques: Knock-Out, Injection, Imaging

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 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 the indicated knockout cells with injection through mouse tail veins. Each group contained five duplicates (n = 5), and two representative images are shown. A, bioluminescence imaging performed 1 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 was plotted and compared using bioluminescence intensity detected in A. Total flux is presented as photons/s (p/s). E, bioluminescence imaging performed 1 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, whole-body metastasis of tumor cells in C57BL/6Ncrl mice was plotted and compared using bioluminescence intensity detected in E. Two high-resolution images for B and F are provided as Figs. S7 and S8. *, p < 0.05; **, p < 0.01; ***, p < 0.001. Error bars, S.D.

Article Snippet: Recombinant mouse WISP1 (rmWISP1, 1680-WS-050), produced in mouse myeloma NS0 cells, was from R&D Systems and was used at a final concentration of 5 μg/ml following the manufacturer's instructions.

Techniques: Knock-Out, Injection, Imaging

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: Recombinant mouse WISP1 (rmWISP1, 1680-WS-050), produced in mouse myeloma NS0 cells, was from R&D Systems and was used at a final concentration of 5 μg/ml following the manufacturer's instructions.

Techniques: Recombinant, Expressing, Quantitative RT-PCR, Marker, Transwell Invasion Assay, Western Blot, Disruption, Knock-Out, Control, Retroviral, Transduction, Positive Control, Comparison, Gene Expression, 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: Recombinant mouse WISP1 (rmWISP1, 1680-WS-050), produced in mouse myeloma NS0 cells, was from R&D Systems and was used at a final concentration of 5 μg/ml following the manufacturer's instructions.

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

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 activated AKT and MEK/ERK signaling and promoted EMT marker gene expression in mouse melanoma cells. Unless otherwise specified, cell treatment for kinase immunoblot analysis was maintained for 30 min before cells were lysed for protein extraction, whereas cell treatment for comparison of EMT marker gene expression was maintained for 3 h before cells were harvested for RNA extraction. A, comparison of EMT marker gene expression after inhibition of AKT and/or MEK/ERK signaling in B16F10 cells. DMSO was used for control cells. Immunoblotting for phospho-AKT (pAKT) and phospho-ERK1/2 (pERK1/2) is shown in the top right corner. Pan-AKT and total ERK1/2 were probed as loading control. B, comparison of EMT marker gene expression after inhibition of AKT and/or MEK/ERK signaling in YUMM1.7 cells. C, immunoblot analysis of AKT and ERK1/2 activation in the indicated mouse melanoma cells with treatment of recombinant mouse WISP1 protein (rmWISP1; final concentration 5 μg/ml). All cells were grown on 6-well plates in complete DMEM for 48 h and SFM for another 48 h before rmWISP1 was added. D, immunoblot analysis of AKT and ERK1/2 activation in B16F10 knockout cell (-KO1) by rmWISP1 under different basal phosphokinase levels. All cells were grown on 6-well plates in complete DMEM for 48 h (0-h point for SFM) and switched to SFM for 24 or 48 h. The indicated cells were treated with rmWISP1 at the 0-, 24-, and 48-h time points (of SFM) for 30 min before they were lysed for kinase analysis. The first lane on the gels was loaded with YUMM1.7 at the 0-h point to compare the relative kinase level between B16F10 and YUMM1.7 cells. E, immunoblot analysis of AKT and ERK1/2 activation in YUMM1.7 knockout cells (-KO1) by rmWISP1 under different basal phosphokinase levels. All cells were treated similarly as described in D. The first lane on the gels was loaded with B16F10 at the 0-h point to compare the relative kinase level between B16F10 and YUMM1.7 cells. F, comparison of SNAI11 activation and E-cadherin repression in B16F10 knockout cells (-KO1) by rmWISP1 under different basal phosphokinase levels. All cells were treated similarly as described in D except that rmWISP1 treatment at each point was maintained for 3 h. G and H, comparison of EMT marker gene expression after AKT/ERK1/2 activation in B16F10-KO1 (G) or YUMM1.7-KO1 (H) by rmWISP1 was blocked. rmWISP1 with DMSO or inhibitors was added after the indicated cells were grown on 6-well plates in complete DMEM for 48 h and in SFM for 24 h. The relative protein levels of pAKT and AKT and of pERK1/2 and ERK1/2 in C–E were measured, and they are listed in Table S3. *, p < 0.05; **, p < 0.01; ***, p < 0.001; ns, not significant. Error bars, S.D.

Article Snippet: Recombinant mouse WISP1 (rmWISP1, 1680-WS-050), produced in mouse myeloma NS0 cells, was from R&D Systems and was used at a final concentration of 5 μg/ml following the manufacturer's instructions.

Techniques: Marker, Gene Expression, Western Blot, Protein Extraction, Comparison, RNA Extraction, Inhibition, Control, Activation Assay, Recombinant, Concentration Assay, Knock-Out