Journal: bioRxiv

Article Title: Endocytic control of cell-autonomous and non-cell-autonomous functions of p53

doi: 10.1101/2025.08.16.670648

Figure Lengend Snippet: A. Domain structure of NUMB isoforms. The positions of the two alternatively spliced exons (Ex3 and Ex9) and the PTB domain are shown. B. HEK-293 cells were transfected with vectors encoding the indicated FLAG-tagged NUMB isoforms (N1-N4) or empty vector (Ctrl). FLAG-NUMB was immunoprecipitated (IP) and immunoblotted (IB) as indicated on the right. Molecular weight markers are in kDa (here and all figures). C. Endogenous NUMB was IP from HEK-293 cells and IB as indicated. A peptide corresponding to the NUMB antibody epitope was used as negative control to compete antibody binding during the IP. D. HEK-293 cell lysates were IP using an anti-SNX9 antibody or irrelevant IgGs and IB as indicated. E. HEK-293 cells were silenced with SNX9 siRNA (+) or Ctrl siRNA (-), followed by IP with anti-NUMB or irrelevant IgGs and IB as indicated. F. HEK-293 cells were transfected with the indicated amounts of SNX9-HA or NUMB-1-FLAG and IB as shown (right). G. HEK-293 cells were silenced with SNX9 siRNA (+) or Ctrl siRNA (-) and transfected with NUMB-1-FLAG and MDM2-HA. Anti-FLAG IP was performed followed by IB as shown. H. HEK-293 cells transfected with NUMB-1-FLAG were IP using an anti-FLAG antibody and IB as shown. A FLAG peptide was used as negative control to compete antibody binding during the IP in G and H . In all panels, l.e., long exposure. Here and in subsequent figures, EPS15 and AP2B1 (AP2 complex subunit beta-1) are known NUMB (all isoforms) interactors, and CLTC (clathrin heavy chain 1) is a known SNX9 interactor.

Article Snippet: Ab for immunoprecipitation (IP) were directed against: anti-FLAG magnetic Agarose (A36797, Life Technologies); anti-FLAG M2-agarose affinity gel (A2220, Merck Life Science); anti-HA (A2095, Merck Life Science); NUMB (AB21 ); SNX9 (15721-1-AP, Proteintech).

Techniques: Transfection, Plasmid Preparation, Immunoprecipitation, Molecular Weight, Negative Control, Binding Assay

Journal: bioRxiv

Article Title: Endocytic control of cell-autonomous and non-cell-autonomous functions of p53

doi: 10.1101/2025.08.16.670648

Figure Lengend Snippet: A. HEK-293 cells were transfected (TFX) with the indicated NUMB/PTB FLAG-tagged constructs or empty vector (Ctrl), followed by IP with anti-FLAG antibody and IB as shown (right). B. Structure of PTB-L with the Ex3-encoded sequence indicated in yellow. The positions of key amino acids involved in protein-protein interactions are shown. In red, amino acids critical for MDM2 binding . In purple, the F162 residue in the PTB binding groove. C. In vitro binding assay with purified GST-SNX9 or GST alone as a negative control (top), immobilized on GSH beads, and purified NUMB-PTB proteins at increasing concentrations as indicated (bottom). After recovery, bound proteins were detected by Coomassie staining. D. HEK-293 cells were TFX with vectors encoding FLAG-tagged WT NUMB or the F162V mutant, or with EV (Ctrl). Anti-FLAG IP was performed followed by IB as shown (l.e., long exposure). E. Left, schematic of the SNX9 protein domain structure, with amino acid boundaries and binding sites of known interactors (dynamin and clathrin) indicated (top). The different SNX9 constructs tested are shown below (asterisks indicate positions of point mutations). Right, summary of the binding properties of SNX9 constructs to PTB-L and NUMB-1 (data are shown in Fig. S4 and S5).

Article Snippet: Ab for immunoprecipitation (IP) were directed against: anti-FLAG magnetic Agarose (A36797, Life Technologies); anti-FLAG M2-agarose affinity gel (A2220, Merck Life Science); anti-HA (A2095, Merck Life Science); NUMB (AB21 ); SNX9 (15721-1-AP, Proteintech).

Techniques: Transfection, Construct, Plasmid Preparation, Sequencing, Protein-Protein interactions, Binding Assay, Residue, In Vitro, Purification, Negative Control, Staining, Mutagenesis

Journal: bioRxiv

Article Title: Endocytic control of cell-autonomous and non-cell-autonomous functions of p53

doi: 10.1101/2025.08.16.670648

Figure Lengend Snippet: A. MCF10A cells were transfected with the indicated FLAG-tagged NUMB constructs. Transfected NUMB was visualized by immunofluorescence (IF) imaging with an anti-FLAG antibody (purple). Blue, DAPI counterstain. Bar, 20 μm. B . MCF10A cells were silenced with the indicated siRNAs and endogenous NUMB was visualized by IF imaging with anti-NUMB antibody (red). Blue, DAPI. Bar, 20 μm. C. HEK-293 cells were transfected with NUMB-1-FLAG and SNX9-HA WT or PXmut constructs (top). Anti-HA IP was performed followed by IB as indicated (right; l.e., long exposure). An HA peptide was used as negative control to compete with antibody binding during the IP. D. HEK-293 cells stably expressing the GFP 1-9 fragment and KO for SNX9 and NUMB, were transfected with the SNX9-WT/PXmut-GFP10 and NUMB-1-GFP11 constructs as indicated (right). Images of a representative IF analysis of SNX9 (red) and NUMB (purple) expression, GFP fluorescence and DAPI counterstain are shown. Bar, 20 μm. Quantification of the percentage of GFP-positive cells is shown below.

Article Snippet: Ab for immunoprecipitation (IP) were directed against: anti-FLAG magnetic Agarose (A36797, Life Technologies); anti-FLAG M2-agarose affinity gel (A2220, Merck Life Science); anti-HA (A2095, Merck Life Science); NUMB (AB21 ); SNX9 (15721-1-AP, Proteintech).

Techniques: Transfection, Construct, Immunofluorescence, Imaging, Negative Control, Binding Assay, Stable Transfection, Expressing, Fluorescence

Journal: bioRxiv

Article Title: Endocytic control of cell-autonomous and non-cell-autonomous functions of p53

doi: 10.1101/2025.08.16.670648

Figure Lengend Snippet: A. Liposome binding assay with purified PTB-L, PTB-S, and SNX9-WT proteins (6 μM) and liposomes composed of PC/PS (No PIP) or PC/PS and either PI(4)P or PI(4,5)P2 as indicated. After centrifugation, the pellet (P) is enriched with liposomes and bound proteins. S: supernatant; T: total uncentrifuged sample. Bound proteins were detected by Coomassie staining. B . PIP 2 -liposome binding assay performed with purified PTB-L (10 μM), and increasing concentrations of SNX9 or MDM2 (216–302) (20 and 60 μM) as indicated. MDM2 (216-302) corresponds to aa 216-302 fragment harboring phosphomimetic mutations that increase binding to NUMB . Bound proteins were detected by Coomassie staining. The low staining intensity observed for MDM2 is attributable to the intrinsically low affinity of MDM2 for the Coomassie dye C. PIP 2 -liposome binding assay performed with purified SNX9 (10 μM), PTB-L (10 μM) and p53 (5 μM) as indicated. Triton-X100 was used to solubilize liposomes in negative controls. Bound proteins were detected by Coomassie staining. D. In vitro binding assay with purified GST-SNX9 or GST proteins immobilized on GSH beads and recombinant purified PTB-L (10 μM) and p53 WT (5 μM) proteins as indicated. Bound proteins were detected by Coomassie staining.

Article Snippet: Ab for immunoprecipitation (IP) were directed against: anti-FLAG magnetic Agarose (A36797, Life Technologies); anti-FLAG M2-agarose affinity gel (A2220, Merck Life Science); anti-HA (A2095, Merck Life Science); NUMB (AB21 ); SNX9 (15721-1-AP, Proteintech).

Techniques: Binding Assay, Purification, Liposomes, Centrifugation, Staining, In Vitro, Recombinant

Journal: bioRxiv

Article Title: Endocytic control of cell-autonomous and non-cell-autonomous functions of p53

doi: 10.1101/2025.08.16.670648

Figure Lengend Snippet: A. Dual color dSTORM images with anti-SNX9 (red) or anti-p53 (green) antibodies in HBL-100 cells. Bars, 20 μm. The percentage colocalization of the two channels in the vicinity of the PM (defined as the region within 2 μm from the cell edge) and in the cytoplasm is shown on the right as the mean ± SD (N = 10 cells). B. A.M.I.CO image analysis of NUMB, SNX9 and p53 spot distribution at the basal membrane of representative TIRF images of HBL-100 cells (top panels). Colocalization is represented by white dots in the bottom panels. Bars, 20 μm. C . Summary of the percentage colocalization of the indicated proteins in the TIRF experiments shown in B . In each row the % of colocalization of the indicated protein 1 (or protein pair 1) with protein 2 is shown. Results are expressed as the mean ± SD (N = 10 cells). D. Cartoon depicting the NUMB:SNX9:p53 tripartite complex assembled at the PM (only the relevant portions of the proteins are shown). Binding to the PM is mediated through the SNX9-PX domain and the Ex3-encoded sequence of NUMB-PTB-L. SNX9 and PTB-L interact through a bidentate interaction involving two surfaces in the low-complexity (LC) domain of SNX9, and the Ex3-coding sequence and the canonical PTB-binding groove on NUMB (lower and higher affinity surfaces, respectively). Upon binding to the PM, conformational changes in the SNX9 LC domain allow it to bind p53.

Article Snippet: Ab for immunoprecipitation (IP) were directed against: anti-FLAG magnetic Agarose (A36797, Life Technologies); anti-FLAG M2-agarose affinity gel (A2220, Merck Life Science); anti-HA (A2095, Merck Life Science); NUMB (AB21 ); SNX9 (15721-1-AP, Proteintech).

Techniques: Membrane, Binding Assay, Sequencing

Journal: bioRxiv

Article Title: Endocytic control of cell-autonomous and non-cell-autonomous functions of p53

doi: 10.1101/2025.08.16.670648

Figure Lengend Snippet: A. Extracellular vesicles (EV) were purified from the conditioned medium of HEK-293 cells and IB as shown (right). Lys, total cellular lysate (here and in all relevant panels). The quality of the EV purification was assessed using positive EV markers TSG101 and Flotilin-1 (FLOT1) and negative EV markers (BCL2; cytochrome c, CYCS; HSP90B1 also known as GRP94; GOLGA2 also known as GM130). B. HEK-293 cells were transfected with SNX9-FLAG and EVs were purified from conditioned medium. Anti-FLAG IP of isolated EVs was performed, followed by IB with the indicated antibodies (right; l.e., long exposure). A FLAG peptide was used as negative control to compete antibody binding during the IP. C. HEK-293 cells were treated with GW4869 (20 μM for 24 h). EVs were purified from conditioned medium and analyzed by IB as indicated (right). PDCD6IP (Alix) and TSG101 are enriched in exosomes. ANXA1 (annexin 1) and ITGA3 (integrin α3) are enriched in microvesicles. D. MCF10A cells stably expressing p53-GFP (green) were treated with chloroquine (50 μM for 24 h) or vehicle control. Anti-RAB7 (red) IF analysis was performed to visualize late endosomes. Blue. DAPI counterstain. Merged and magnified images are shown on the right. Bar, 20 μm. E. Quantification of RAB7 and p53-GFP colocalization, using Mander’s coefficient, in the experiment shown in panel D. A total of 8 fields from untreated cells and 12 fields from chloroquine-treated cells were analyzed by confocal microscopy. F. EVs were purified from the conditioned medium of the cells in panel D and IB as indicated. G. EVs were purified from the conditioned medium of Ctrl-KO, SNX9-KO or NUMB-KO HEK-293 cells as indicated on top and IB as shown (right).

Article Snippet: Ab for immunoprecipitation (IP) were directed against: anti-FLAG magnetic Agarose (A36797, Life Technologies); anti-FLAG M2-agarose affinity gel (A2220, Merck Life Science); anti-HA (A2095, Merck Life Science); NUMB (AB21 ); SNX9 (15721-1-AP, Proteintech).

Techniques: Purification, Transfection, Isolation, Negative Control, Binding Assay, Stable Transfection, Expressing, Control, Confocal Microscopy

Journal: bioRxiv

Article Title: Endocytic control of cell-autonomous and non-cell-autonomous functions of p53

doi: 10.1101/2025.08.16.670648

Figure Lengend Snippet: A. Ctrl-KO and SNX9-KO MCF10A cells were treated with etoposide at 25 and 50 μM (indicated by triangles) and analyzed by IB for total and phosphorylated (p53 ser15 ) p53, and the p53 target genes MDM2 and CDKN1A. Vinculin (VCL), loading control. B . RT-qPCR analysis of MDM2 and CDKN1A expression in the samples described in “A”. Data are from three independent experiments and expressed as mean ± SD. *, p<0.05; **, p<0.01. C. 3D reconstruction of MCF10A-p53-KO recipient cells treated for 8 h with EVs purified from the conditioned medium of HEK-293 cells transfected with p53-HA. Recipient cells were treated with etoposide (50 μM for 8 h) and analyzed by IF using an anti-p53 antibody (green) and DAPI (blue). Bar, 20 μm. D. MCF10A-p53-KO recipient cells were treated for 8 h with etoposide (50 μM) and EVs purified from HEK-293 WT (EV) or HEK-293 p53-HA (EV p53-HA) cells as indicated. After treatment cells were harvested and analyzed by RT-qPCR for CDKN1A levels. Data are from three independent experiments and expressed as mean ± SD. * p<0.05, ** p<0.01 and *** p<0.001 vs . same condition in cells not treated with EVs (only the most relevant statistical comparisons are shown). E. Scheme of the co-culture experiment shown in in panel F. a) MCF10A-p53-KO-H2B-Cherry cells were plated onto coverslips. b) After 24 h, coverslips were harvested and seeded (c) in plates in which the indicated cell lines had been previously seeded. Cells were then treated with etoposide (50 μM) or mock-treatment for 8 h. d) Coverslips were harvested and analyzed for purity of H2B-Cherry labeled cells (Fig. S10D) and for the levels of CDKN1A mRNA (panel F). Details are in Materials and Methods. F . RT-qPCR analysis of CDKN1A levels in harvested H2B-Cherry labeled MCF10A-p53-KO cells, co-cultured as described in panel “ E ”. Data are from three independent experiments and expressed as mean ± SD. **, p<0.01; n.s., not significant (only the most relevant statistical comparisons are shown). G . SAOS2 cells were treated with EVs purified from the indicated MCF10A cell lines (see experimental scheme in Fig. S10F), and cell viability/growth was assessed indirectly by quantifying intracellular ATP levels using a luminescence-based assay. Data are from ten samples/condition from two independent experiments and expressed as mean ± SD. One-way ANOVA test: *, and ***, p < 0.05 and < 0.001, respectively, vs . SAOS2 treated with EVs derived from MCF10A-p53-KO cells.

Article Snippet: Ab for immunoprecipitation (IP) were directed against: anti-FLAG magnetic Agarose (A36797, Life Technologies); anti-FLAG M2-agarose affinity gel (A2220, Merck Life Science); anti-HA (A2095, Merck Life Science); NUMB (AB21 ); SNX9 (15721-1-AP, Proteintech).

Techniques: Control, Quantitative RT-PCR, Expressing, Purification, Transfection, Co-Culture Assay, Labeling, Cell Culture, Luminescence 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 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: Frontiers in Physiology

Article Title: Atlas of human dental pulp cells at multiple spatial and temporal levels based on single-cell sequencing analysis

doi: 10.3389/fphys.2022.993478

Figure Lengend Snippet: The top 20 genes in odontoblasts.

Article Snippet: The following antibodies were used in our study: CD163 (1:500, Abcam, United States), SLC12A2 (1:100, Proteintech, China), ST8SIA1 (1:100, Proteintech), CD24 (1: 50, Santa Cruz, United States), WISP1 (1:100, Proteintech), CD146/MCAM (1: 250, Abcam), and CD90 (1:200, Abcam).

Techniques:

Journal: The Journal of biological chemistry

Article Title: Acetylation stabilizes the signaling protein WISP2 by preventing its degradation to suppress the progression of acute myeloid leukemia.

doi: 10.1016/j.jbc.2023.102971

Figure Lengend Snippet: Figure 1. Low WISP2 expression predicts reduced survival of AML patients. A, correlation of WISP2 expression with the survival probability of AML patients via the UALCAN cancer database. B, analysis of WISP2 expression in AML patients with different subtypes via the UALCAN cancer database. C, Western blot analysis of WISP2 expression in bone marrow mononuclear cells from six healthy donors and 16 AML patients. D, Co-IP assay to detect the acetylation of WISP2 in bone marrow mononuclear cells from healthy donors and AML patients. E, the protein expression and acetylation level of WISP2 in normal bone marrow mononuclear cells and AML cells. Ac, acetylated lysine; AML, acute myeloid leukemia; Co-IP, co-immunoprecipitation; WISP2, Wnt-1- induced signaling protein-2.

Article Snippet: The lysates were incubated with 1 μg of antibodies against WISP2 (Santa Cruz; sc514070), Flag-tag (Abcam; ab125243) or IgG (Solarbio, SP031) overnight at 4 C with gentle rotation, followed by incubation with 60 μl of Protein A Affinity Chromatography Media (Aladdin; P5362–01) for 2 h. The immunocomplex was washed, denatured, and subjected to Western blotting using primary antibodies against WISP2 (ABclonal, A7456), acetylated-Lysine (ABclonal, A2391), HDAC1 (ABclonal, A19571), HDAC3 (ABclonal, A19537), HDAC6 (ABclonal, A1732), HDAC7 (ABclonal, A13008), Flag-tag (Abcam, ab205606), and NEDD4 (ABclonal, A4385).

Techniques: Expressing, Western Blot, Co-Immunoprecipitation Assay, Immunoprecipitation

Journal: The Journal of biological chemistry

Article Title: Acetylation stabilizes the signaling protein WISP2 by preventing its degradation to suppress the progression of acute myeloid leukemia.

doi: 10.1016/j.jbc.2023.102971

Figure Lengend Snippet: Figure 2. WISP2 overexpression inhibits the proliferation and promotes the apoptosis of AML cells. A and B, WISP2 lentivirus or negative control (NC) was infected into HL-60 cells (A) or Kasumi-1 cells (B) for 24, 48, 72 h, 96 h, and 120 h, and cell viability was determined by CCK8 assay. Data represent the mean ± SD (n = 5 for each group). C and D, apoptosis of HL-60 cells (C) or Kasumi-1 cells (D) was measured by flow cytometry after 72 h of infection with LV-WISP2 or LV-NC. Data represent the mean ± SD of three independent experiments. E and F, FACS analysis of the mitochondrial membrane potential of HL-60 cells (E) or Kasumi-1 cells (F) after 72 h of infection with lentivirus. Data represent the mean ± SD of three independent experiments. G and H, Western blot for WISP2 and apoptosis-related markers in whole cell lysates, as well as cytochrome C in mitochondrial and cytoplasmic fractions of HL-60 cells (G) or Kasumi-1 cells (H). p values in Panels A and B were determined with two-way ANOVA followed by Tukey’s multiple comparisons test, and the p values in Panels C–F were determined with one-way ANOVA followed by Tukey’s test (**p < 0.01, ****p < 0.0001). AML, acute myeloid leukemia; cl cas-3, cleaved caspase-3; cl cas-9, cleaved caspase-9; cl PARP, cleaved PARP; cyt c, cytochrome C; cyto, cytoplasmic; LV, lentivirus; LV-WISP2, lentivirus plasmids containing full-length WISP2; mito, mitochondrial; WISP2, Wnt-1-induced signaling protein-2.

Article Snippet: The lysates were incubated with 1 μg of antibodies against WISP2 (Santa Cruz; sc514070), Flag-tag (Abcam; ab125243) or IgG (Solarbio, SP031) overnight at 4 C with gentle rotation, followed by incubation with 60 μl of Protein A Affinity Chromatography Media (Aladdin; P5362–01) for 2 h. The immunocomplex was washed, denatured, and subjected to Western blotting using primary antibodies against WISP2 (ABclonal, A7456), acetylated-Lysine (ABclonal, A2391), HDAC1 (ABclonal, A19571), HDAC3 (ABclonal, A19537), HDAC6 (ABclonal, A1732), HDAC7 (ABclonal, A13008), Flag-tag (Abcam, ab205606), and NEDD4 (ABclonal, A4385).

Techniques: Over Expression, Negative Control, Infection, CCK-8 Assay, Cytometry, Membrane, Western Blot

Journal: The Journal of biological chemistry

Article Title: Acetylation stabilizes the signaling protein WISP2 by preventing its degradation to suppress the progression of acute myeloid leukemia.

doi: 10.1016/j.jbc.2023.102971

Figure Lengend Snippet: Figure 3. WISP2 overexpression exerts antileukemic effects in the in vivo model of AML. A, percentage of CD45+ cells in the bone marrow of mice transplanted with HL-60 cells. B, Leishman–Wright–Giemsa staining of mouse bone marrow. Black arrows indicate ring-like differentiated cells. The p value in Panel A was determined with an unpaired t test (****p < 0.0001). AML, acute myeloid leukemia; WISP2, Wnt-1-induced signaling protein-2.

Article Snippet: The lysates were incubated with 1 μg of antibodies against WISP2 (Santa Cruz; sc514070), Flag-tag (Abcam; ab125243) or IgG (Solarbio, SP031) overnight at 4 C with gentle rotation, followed by incubation with 60 μl of Protein A Affinity Chromatography Media (Aladdin; P5362–01) for 2 h. The immunocomplex was washed, denatured, and subjected to Western blotting using primary antibodies against WISP2 (ABclonal, A7456), acetylated-Lysine (ABclonal, A2391), HDAC1 (ABclonal, A19571), HDAC3 (ABclonal, A19537), HDAC6 (ABclonal, A1732), HDAC7 (ABclonal, A13008), Flag-tag (Abcam, ab205606), and NEDD4 (ABclonal, A4385).

Techniques: Over Expression, In Vivo, Staining

Journal: The Journal of biological chemistry

Article Title: Acetylation stabilizes the signaling protein WISP2 by preventing its degradation to suppress the progression of acute myeloid leukemia.

doi: 10.1016/j.jbc.2023.102971

Figure Lengend Snippet: Figure 4. Pan-HDAC inhibitors induce apoptosis and efficiently prevent WISP2 degradation in AML cells. Two pan-HDAC inhibitors, VPA and TSA, were used to treat AML cells. A and B, HL-60 cells and Kasumi-1 cells were treated with VPA or TSA for 24 h and then subjected to CCK8 assay. Data represent the mean ± SD (n = 5 for each group). C and D, apoptosis of AML cells was measured by flow cytometry after 24 h of VPA or TSA treatment. Data represent the mean ± SD of three independent experiments. E and F, quantitative analysis of the percentage of apoptosis. G and H, Western blot for WISP2 in VPA- or TSA-treated AML cells. I and J, the acetylation of WISP2 in VPA- or TSA-treated AML cells was assessed by co-IP assay. K and L, AML cells were treated with VPA or TSA for 12 h and treated with CHX (25 μg/ml) for the indicated times, followed by Western blot analysis of WISP2 expression. p values were determined with one-way ANOVA followed by Dunnett’s multiple comparisons test (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001). Ac, acetylated lysine; AML, acute myeloid leukemia; CHX, cycloheximide; co-IP, co-immunoprecipitation; HDAC, histone deacetylase; TSA, trichostatin A; VPA, valproic acid; WISP2, Wnt-1-induced signaling protein-2.

Article Snippet: The lysates were incubated with 1 μg of antibodies against WISP2 (Santa Cruz; sc514070), Flag-tag (Abcam; ab125243) or IgG (Solarbio, SP031) overnight at 4 C with gentle rotation, followed by incubation with 60 μl of Protein A Affinity Chromatography Media (Aladdin; P5362–01) for 2 h. The immunocomplex was washed, denatured, and subjected to Western blotting using primary antibodies against WISP2 (ABclonal, A7456), acetylated-Lysine (ABclonal, A2391), HDAC1 (ABclonal, A19571), HDAC3 (ABclonal, A19537), HDAC6 (ABclonal, A1732), HDAC7 (ABclonal, A13008), Flag-tag (Abcam, ab205606), and NEDD4 (ABclonal, A4385).

Techniques: CCK-8 Assay, Cytometry, Western Blot, Co-Immunoprecipitation Assay, Expressing, Immunoprecipitation, Histone Deacetylase Assay

Journal: The Journal of biological chemistry

Article Title: Acetylation stabilizes the signaling protein WISP2 by preventing its degradation to suppress the progression of acute myeloid leukemia.

doi: 10.1016/j.jbc.2023.102971

Figure Lengend Snippet: Figure 5. HDAC3 interacts with and deacetylates WISP2 in AML cells. A and B, co-IP was performed with anti-WISP2 antibody and IgG as a control, followed by immunoblotting with anti-HDAC1, anti-HDAC3, anti-HDAC6, and anti-HDAC7 antibodies in AML cells. C, the correlation between HDAC3 or HDAC1 and WISP2 expression levels from 151 AML samples analyzed by StarBase. D, Western blot analysis of HDAC3 expression in bone marrow mononuclear cells from healthy donors and AML patients. E and F, the protein and acetylation levels of WISP2 in RGFP966-treated AML cells. G and H, the protein and acetylation levels of WISP2 and the protein expression of HDAC3 in AML cells infected with HDAC3 lentivirus. Ac, acetylated lysine; AML, acute myeloid leukemia; co-IP, co-immunoprecipitation; HDAC, histone deacetylase; WISP2, Wnt-1-induced signaling protein-2.

Article Snippet: The lysates were incubated with 1 μg of antibodies against WISP2 (Santa Cruz; sc514070), Flag-tag (Abcam; ab125243) or IgG (Solarbio, SP031) overnight at 4 C with gentle rotation, followed by incubation with 60 μl of Protein A Affinity Chromatography Media (Aladdin; P5362–01) for 2 h. The immunocomplex was washed, denatured, and subjected to Western blotting using primary antibodies against WISP2 (ABclonal, A7456), acetylated-Lysine (ABclonal, A2391), HDAC1 (ABclonal, A19571), HDAC3 (ABclonal, A19537), HDAC6 (ABclonal, A1732), HDAC7 (ABclonal, A13008), Flag-tag (Abcam, ab205606), and NEDD4 (ABclonal, A4385).

Techniques: Co-Immunoprecipitation Assay, Control, Western Blot, Expressing, Infection, Immunoprecipitation, Histone Deacetylase Assay

Journal: The Journal of biological chemistry

Article Title: Acetylation stabilizes the signaling protein WISP2 by preventing its degradation to suppress the progression of acute myeloid leukemia.

doi: 10.1016/j.jbc.2023.102971

Figure Lengend Snippet: Figure 6. The lysine K6 residue is essential for WISP2 acetylation. A, prediction of acetylation on internal lysine residues indicated two potential acetylated lysine (K) residues, K6 and K20, within WISP2. B, HEK293 cells were transfected with wildtype WISP2 (wt-WISP2-flag), mutant WISP2_K6R (WISP2_K6R-flag), or mutant WISP2_K20R (WISP2_K20R-flag) for 48 h, and the transfection efficiency was determined by immunoblotting with an anti-Flag antibody. C and D, HEK293 cells were transfected with wt-WISP2-flag, WISP2_K6R-flag, or WISP2_K20R-flag for 48 h and then treated with VPA or RGFP966 for 24 h. Whole-cell lysates were subjected to detection with anti-acetylated-lysine antibody. Ac, acetylated lysine; VPA, valproic acid; WISP2, Wnt-1-induced signaling protein-2.

Article Snippet: The lysates were incubated with 1 μg of antibodies against WISP2 (Santa Cruz; sc514070), Flag-tag (Abcam; ab125243) or IgG (Solarbio, SP031) overnight at 4 C with gentle rotation, followed by incubation with 60 μl of Protein A Affinity Chromatography Media (Aladdin; P5362–01) for 2 h. The immunocomplex was washed, denatured, and subjected to Western blotting using primary antibodies against WISP2 (ABclonal, A7456), acetylated-Lysine (ABclonal, A2391), HDAC1 (ABclonal, A19571), HDAC3 (ABclonal, A19537), HDAC6 (ABclonal, A1732), HDAC7 (ABclonal, A13008), Flag-tag (Abcam, ab205606), and NEDD4 (ABclonal, A4385).

Techniques: Residue, Transfection, Mutagenesis, Western Blot

Journal: The Journal of biological chemistry

Article Title: Acetylation stabilizes the signaling protein WISP2 by preventing its degradation to suppress the progression of acute myeloid leukemia.

doi: 10.1016/j.jbc.2023.102971

Figure Lengend Snippet: Figure 7. Acetylation of WISP2 prevents NEDD4-mediated ubiquitination. A, HL-60 cells were treated with or without RGFP966 for 24 h, and MG132 was added 6 h before harvesting cells. Proteins were co-IP with anti-WISP2 antibody, followed by immunoblotting with anti-ubiquitin or anti-WISP2 antibodies. B, HDAC3 lentivirus was infected into HL-60 cells for 72 h, and MG132 was added 6 h before harvesting cells. The ubiquitination of WISP2 was detected by co-IP with an anti-WISP2 antibody, followed by immunoblotting with the indicated antibodies. C, HL-60 cells were infected with HDAC3 lentivirus. After 72 h, the cells were treated with MG132 for 6 h. Total lysates were analyzed by Western blotting using an anti-WISP2 antibody. D, prediction of Ubibrowser indicated that NEDD4 was the top E3 ubiquitin ligase that probably targets WISP2. E and F, endogenous interaction between WISP2 and NEDD4 in HL-60 cells and Kasumi-1 cells was examined by co-IP assay. G and H, the protein levels of NEDD4 and WISP2 were confirmed by Western blotting in NEDD4- overexpressing AML cells. I, co-IP assay to assess WISP2 ubiquitination in HL-60 cells infected with NEDD4 lentivirus. J, co-IP assay to assess WISP2 ubiquitination in HL-60 cells transfected with NEDD4 overexpression plasmid and wt-WISP2-flag or WISP2_K6R-flag. K, HL-60 cells were infected with LV-oeHDAC3 and LV-shNEDD4 as indicated. Western blot analysis was performed to determine WISP2 expression. L, schematic diagram for the antileukemic effect of WISP2: HDAC3-induced deacetylation disrupts WISP2 stability by augmenting ubiquitination mediated by NEDD4, which contributes to the in- crease in tumor cell proliferation and the suppression of apoptosis. The blockade of histone deacetylation by HDAC inhibitors restores WISP2 expression in AML cells, thereby allowing WISP2 to exert its antileukemia effect. AML, acute myeloid leukemia; co-IP, coimmunoprecipitation; LV, lentivirus; ub, ubiquitin; WISP2, Wnt-1-induced signaling protein-2.

Article Snippet: The lysates were incubated with 1 μg of antibodies against WISP2 (Santa Cruz; sc514070), Flag-tag (Abcam; ab125243) or IgG (Solarbio, SP031) overnight at 4 C with gentle rotation, followed by incubation with 60 μl of Protein A Affinity Chromatography Media (Aladdin; P5362–01) for 2 h. The immunocomplex was washed, denatured, and subjected to Western blotting using primary antibodies against WISP2 (ABclonal, A7456), acetylated-Lysine (ABclonal, A2391), HDAC1 (ABclonal, A19571), HDAC3 (ABclonal, A19537), HDAC6 (ABclonal, A1732), HDAC7 (ABclonal, A13008), Flag-tag (Abcam, ab205606), and NEDD4 (ABclonal, A4385).

Techniques: Ubiquitin Proteomics, Co-Immunoprecipitation Assay, Western Blot, Infection, Transfection, Over Expression, Plasmid Preparation, Expressing

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: 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: 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: 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: 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: 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, 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: 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, 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: 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, 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: 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

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