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R&D Systems mouse rwisp 1 1680 ws
Mouse Rwisp 1 1680 Ws, supplied by R&D Systems, used in various techniques. Bioz Stars score: 92/100, based on 14 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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mouse rwisp 1 1680 ws - by Bioz Stars, 2026-03

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WISP-1 protein induced type I collagen processing in conditioned media of human cardiac fibroblasts (HCFs). HCFs were cultured in supplemented fibroblast growth medium for 24 h and then in serum-free medium (SFM) for 48 h. The medium was replaced with fresh SFM in the presence or absence of recombinant human WISP-1 protein (500 ng/mL) for 24 h, and conditioned media were collected and concentrated for Western blotting. Stain-free gel bands from corresponding cell lysate samples were used as the loading control. Representative Western blots of ( A ) type I procollagen and pC-collagen (tropocollagen with PICP), detected using anti-C-telo antibody (n = 16), ( B ) type I procollagen, pC-collagen (tropocollagen with PICP), and PICP, detected using anti-PICP antibody (n = 8), and ( C ) type I procollagen, detected using anti-PINP antibody (n = 8). Schematic molecular structures and approximate molecular weights in kDa are indicated adjacent to representative immunoblots.

Journal: Cells

Article Title: WISP-1 Regulates Cardiac Fibrosis by Promoting Cardiac Fibroblasts’ Activation and Collagen Processing

doi: 10.3390/cells13110989

Figure Lengend Snippet: WISP-1 protein induced type I collagen processing in conditioned media of human cardiac fibroblasts (HCFs). HCFs were cultured in supplemented fibroblast growth medium for 24 h and then in serum-free medium (SFM) for 48 h. The medium was replaced with fresh SFM in the presence or absence of recombinant human WISP-1 protein (500 ng/mL) for 24 h, and conditioned media were collected and concentrated for Western blotting. Stain-free gel bands from corresponding cell lysate samples were used as the loading control. Representative Western blots of ( A ) type I procollagen and pC-collagen (tropocollagen with PICP), detected using anti-C-telo antibody (n = 16), ( B ) type I procollagen, pC-collagen (tropocollagen with PICP), and PICP, detected using anti-PICP antibody (n = 8), and ( C ) type I procollagen, detected using anti-PINP antibody (n = 8). Schematic molecular structures and approximate molecular weights in kDa are indicated adjacent to representative immunoblots.

Article Snippet: Forty-eight hours later, SFM was replaced with fresh SFM in the presence or absence of recombinant human WISP-1 protein (500 ng/mL, Biotechne, Minneapolis, MN, USA, 1627-WS-050).

Techniques: Cell Culture, Recombinant, Western Blot, Staining

Silencing ADAMTS-2 inhibited WISP-1 protein-induced type I collagen processing in conditioned media of human cardiac fibroblasts (HCFs). HCFs were either transfected with control SiRNA (1.228 μM), ADAMTS SiRNAs (614 nM/target gene), or left untransfected prior to seeding on a 12-well plate. After culture in supplemented fibroblast growth medium for 24 h, HCFs were starved in serum-free medium (SFM) for 48 h. The medium was then replaced with fresh SFM in the presence or absence of recombinant human WISP-1 protein (500 ng/mL) and HCFs cultured for 15 h for qPCR analysis, and 24 h or 96 h for Western blotting analysis. ( A ) Quantification of ADAMTS-2 mRNA expression using qPCR analysis. Data were normalised to 36B4 housekeeping gene and expressed as the relative fold change to the untransfected HCFs (Control). ( B ) Quantification of ADAMTS-2 protein expression (168 h post-transfection) using Western blotting analysis. Data were normalised to stain-free gel bands and expressed as the relative fold change to the untransfected HCFs (Control). ( C ) Representative Western blots of type I procollagen and pC-collagen (tropocollagen with PICP) detected using anti-C-telo antibody. Stain-free gel bands from corresponding cell lysate samples were used as loading control. Quantification of pC-collagen I protein expression (96 h post-transfection) was expressed as the relative fold change to the WISP-1 protein treatment group. Data shown as mean ± SEM (n = 4–6). Statistical analysis was performed using Kruskal–Wallis H test. * indicates p < 0.05. Approximate molecular weights in kDa are indicated adjacent to representative immunoblots.

Journal: Cells

Article Title: WISP-1 Regulates Cardiac Fibrosis by Promoting Cardiac Fibroblasts’ Activation and Collagen Processing

doi: 10.3390/cells13110989

Figure Lengend Snippet: Silencing ADAMTS-2 inhibited WISP-1 protein-induced type I collagen processing in conditioned media of human cardiac fibroblasts (HCFs). HCFs were either transfected with control SiRNA (1.228 μM), ADAMTS SiRNAs (614 nM/target gene), or left untransfected prior to seeding on a 12-well plate. After culture in supplemented fibroblast growth medium for 24 h, HCFs were starved in serum-free medium (SFM) for 48 h. The medium was then replaced with fresh SFM in the presence or absence of recombinant human WISP-1 protein (500 ng/mL) and HCFs cultured for 15 h for qPCR analysis, and 24 h or 96 h for Western blotting analysis. ( A ) Quantification of ADAMTS-2 mRNA expression using qPCR analysis. Data were normalised to 36B4 housekeeping gene and expressed as the relative fold change to the untransfected HCFs (Control). ( B ) Quantification of ADAMTS-2 protein expression (168 h post-transfection) using Western blotting analysis. Data were normalised to stain-free gel bands and expressed as the relative fold change to the untransfected HCFs (Control). ( C ) Representative Western blots of type I procollagen and pC-collagen (tropocollagen with PICP) detected using anti-C-telo antibody. Stain-free gel bands from corresponding cell lysate samples were used as loading control. Quantification of pC-collagen I protein expression (96 h post-transfection) was expressed as the relative fold change to the WISP-1 protein treatment group. Data shown as mean ± SEM (n = 4–6). Statistical analysis was performed using Kruskal–Wallis H test. * indicates p < 0.05. Approximate molecular weights in kDa are indicated adjacent to representative immunoblots.

Article Snippet: Forty-eight hours later, SFM was replaced with fresh SFM in the presence or absence of recombinant human WISP-1 protein (500 ng/mL, Biotechne, Minneapolis, MN, USA, 1627-WS-050).

Techniques: Transfection, Recombinant, Cell Culture, Western Blot, Expressing, Staining

WISP-1 protein promoted Akt phosphorylation via integrin β1/FAK/ILK in human cardiac fibroblasts (HCFs). HCFs were cultured in supplemented fibroblast growth medium for 24 h and then starved in serum-free medium (SFM) for 48 h. The medium was replaced with fresh SFM in the presence or absence of recombinant human WISP-1 protein (500 ng/mL) for 30 min before cell lysis. Cell lysate samples were analysed by Western blotting using phosphorylated Akt (p-Akt) (Ser473) and total Akt (t-Akt) antibodies. ( A ) Representative Western blots of p-Akt (Ser473) and t-Akt protein expression. The ratio of p-Akt (Ser473) to t-Akt was calculated and expressed as the relative fold change to the control. Data shown as mean ± SEM (n = 9). Statistical analysis was performed using Mann–Whitney U test. * indicates p < 0.05. ( B ) Representative Western blots of p-Akt (Ser473) and t-Akt protein expression. The ratio of p-Akt (Ser473) to t-Akt was calculated and expressed as the relative fold change to WISP-1 + mouse non-immune IgG 1 control (mIgG) group. HCFs were pre-incubated with integrin β1-blocking antibodies (mouse IgG 1 clone) (β1 mAb, 10 μg/mL), integrin αVβ5-blocking antibodies (mouse IgG 1 clone) (αVβ5 mAb, 10 μg/mL), and mIgG control antibodies (10 μg/mL), respectively, for 30 min prior to WISP-1 protein treatment. Data shown as mean ± SEM (n = 5). Statistical analysis was performed using Kruskal–Wallis H test. * indicates p < 0.05. ( C ) Representative Western blots of p-Akt (Ser473) and t-Akt protein expression. The ratio of p-Akt (Ser473) to t-Akt was calculated and expressed as the relative fold change to WISP-1 group. HCFs were pre-incubated with defactinib (5 μM) or CPD22 (2.5 μM) for 30 min prior to WISP-1 protein treatment. Data shown as mean ± SEM (n = 4). Statistical analysis was performed using Mann–Whitney U test. * indicates p < 0.05. Approximate molecular weights in kDa are indicated adjacent to representative immunoblots.

Journal: Cells

Article Title: WISP-1 Regulates Cardiac Fibrosis by Promoting Cardiac Fibroblasts’ Activation and Collagen Processing

doi: 10.3390/cells13110989

Figure Lengend Snippet: WISP-1 protein promoted Akt phosphorylation via integrin β1/FAK/ILK in human cardiac fibroblasts (HCFs). HCFs were cultured in supplemented fibroblast growth medium for 24 h and then starved in serum-free medium (SFM) for 48 h. The medium was replaced with fresh SFM in the presence or absence of recombinant human WISP-1 protein (500 ng/mL) for 30 min before cell lysis. Cell lysate samples were analysed by Western blotting using phosphorylated Akt (p-Akt) (Ser473) and total Akt (t-Akt) antibodies. ( A ) Representative Western blots of p-Akt (Ser473) and t-Akt protein expression. The ratio of p-Akt (Ser473) to t-Akt was calculated and expressed as the relative fold change to the control. Data shown as mean ± SEM (n = 9). Statistical analysis was performed using Mann–Whitney U test. * indicates p < 0.05. ( B ) Representative Western blots of p-Akt (Ser473) and t-Akt protein expression. The ratio of p-Akt (Ser473) to t-Akt was calculated and expressed as the relative fold change to WISP-1 + mouse non-immune IgG 1 control (mIgG) group. HCFs were pre-incubated with integrin β1-blocking antibodies (mouse IgG 1 clone) (β1 mAb, 10 μg/mL), integrin αVβ5-blocking antibodies (mouse IgG 1 clone) (αVβ5 mAb, 10 μg/mL), and mIgG control antibodies (10 μg/mL), respectively, for 30 min prior to WISP-1 protein treatment. Data shown as mean ± SEM (n = 5). Statistical analysis was performed using Kruskal–Wallis H test. * indicates p < 0.05. ( C ) Representative Western blots of p-Akt (Ser473) and t-Akt protein expression. The ratio of p-Akt (Ser473) to t-Akt was calculated and expressed as the relative fold change to WISP-1 group. HCFs were pre-incubated with defactinib (5 μM) or CPD22 (2.5 μM) for 30 min prior to WISP-1 protein treatment. Data shown as mean ± SEM (n = 4). Statistical analysis was performed using Mann–Whitney U test. * indicates p < 0.05. Approximate molecular weights in kDa are indicated adjacent to representative immunoblots.

Article Snippet: Forty-eight hours later, SFM was replaced with fresh SFM in the presence or absence of recombinant human WISP-1 protein (500 ng/mL, Biotechne, Minneapolis, MN, USA, 1627-WS-050).

Techniques: Cell Culture, Recombinant, Lysis, Western Blot, Expressing, MANN-WHITNEY, Incubation, Blocking Assay

WISP-1 protein promoted human cardiac fibroblasts (HCFs) activation. HCFs were cultured on soft substrate plates (8 kPa) in supplemented fibroblast growth medium for 24 h. HCFs were starved in serum-free medium (SFM) for 48 h, then the medium was replaced with fresh SFM in the presence or absence of recombinant human WISP-1 protein (500 ng/mL) and cultured for 24 h. ( A ) HCFs were fixed for immunocytochemical staining with anti-α-SMA antibody. α-SMA positive cells are stained green, and nuclei are stained blue with DAPI (4′,6-diamidino-2-phenylindole). Some positive cells are indicated by white arrows. Scale bar represents 50 μm. Quantification of positive α-SMA staining was expressed as the relative fold change to the control of the percentage of positive α-SMA staining cells to total cells on soft substrate. Data shown as mean ± SEM (n = 8). Statistical analysis was performed using Mann–Whitney U test. * indicates p < 0.05. ( B ) Quantification of α-SMA protein expression and ( C ) quantification of PCNA protein expression using Western blotting analysis. Data were normalised to stain-free gel bands and expressed as the relative fold change to the control. Data shown as mean ± SEM (n = 9). Statistical analysis was performed using Mann–Whitney U test. * indicates p < 0.05. ( D ) Quantification of accumulated migration distance per cell over the duration of consecutive images (21 h 30 min). Data shown as mean ± SEM (n = 4). Statistical analysis was performed using Student’s t test. * indicates p < 0.05.

Journal: Cells

Article Title: WISP-1 Regulates Cardiac Fibrosis by Promoting Cardiac Fibroblasts’ Activation and Collagen Processing

doi: 10.3390/cells13110989

Figure Lengend Snippet: WISP-1 protein promoted human cardiac fibroblasts (HCFs) activation. HCFs were cultured on soft substrate plates (8 kPa) in supplemented fibroblast growth medium for 24 h. HCFs were starved in serum-free medium (SFM) for 48 h, then the medium was replaced with fresh SFM in the presence or absence of recombinant human WISP-1 protein (500 ng/mL) and cultured for 24 h. ( A ) HCFs were fixed for immunocytochemical staining with anti-α-SMA antibody. α-SMA positive cells are stained green, and nuclei are stained blue with DAPI (4′,6-diamidino-2-phenylindole). Some positive cells are indicated by white arrows. Scale bar represents 50 μm. Quantification of positive α-SMA staining was expressed as the relative fold change to the control of the percentage of positive α-SMA staining cells to total cells on soft substrate. Data shown as mean ± SEM (n = 8). Statistical analysis was performed using Mann–Whitney U test. * indicates p < 0.05. ( B ) Quantification of α-SMA protein expression and ( C ) quantification of PCNA protein expression using Western blotting analysis. Data were normalised to stain-free gel bands and expressed as the relative fold change to the control. Data shown as mean ± SEM (n = 9). Statistical analysis was performed using Mann–Whitney U test. * indicates p < 0.05. ( D ) Quantification of accumulated migration distance per cell over the duration of consecutive images (21 h 30 min). Data shown as mean ± SEM (n = 4). Statistical analysis was performed using Student’s t test. * indicates p < 0.05.

Article Snippet: Forty-eight hours later, SFM was replaced with fresh SFM in the presence or absence of recombinant human WISP-1 protein (500 ng/mL, Biotechne, Minneapolis, MN, USA, 1627-WS-050).

Techniques: Activation Assay, Cell Culture, Recombinant, Staining, MANN-WHITNEY, Expressing, Western Blot, Migration

WISP-1 deficiency attenuated angiotensin II (AngII)-induced coronary artery perivascular fibrosis. Cardiac fibrosis was induced by subcutaneous AngII infusion (1000 ng/kg/min) for 28 days via osmotic pumps in WISP-1 +/+ and WISP-1 −/− mice. Representative images showing type I collagen (dark brown) staining using anti-C-telo antibody in left ventricular tissues with and without AngII infusion. Nuclei are stained blue with haematoxylin. Non-immune IgG was used as the negative control. Quantification of positive type I collagen staining was expressed as the percentage of positive collagen I staining area to total tissue area. Data shown as mean ± SEM (n = 5–8). Red arrows indicate some positive staining (dark brown). Scale bar represents 100 μm. Statistical analysis was performed using Kruskal–Wallis H test. * indicates p < 0.05.

Journal: Cells

Article Title: WISP-1 Regulates Cardiac Fibrosis by Promoting Cardiac Fibroblasts’ Activation and Collagen Processing

doi: 10.3390/cells13110989

Figure Lengend Snippet: WISP-1 deficiency attenuated angiotensin II (AngII)-induced coronary artery perivascular fibrosis. Cardiac fibrosis was induced by subcutaneous AngII infusion (1000 ng/kg/min) for 28 days via osmotic pumps in WISP-1 +/+ and WISP-1 −/− mice. Representative images showing type I collagen (dark brown) staining using anti-C-telo antibody in left ventricular tissues with and without AngII infusion. Nuclei are stained blue with haematoxylin. Non-immune IgG was used as the negative control. Quantification of positive type I collagen staining was expressed as the percentage of positive collagen I staining area to total tissue area. Data shown as mean ± SEM (n = 5–8). Red arrows indicate some positive staining (dark brown). Scale bar represents 100 μm. Statistical analysis was performed using Kruskal–Wallis H test. * indicates p < 0.05.

Article Snippet: Forty-eight hours later, SFM was replaced with fresh SFM in the presence or absence of recombinant human WISP-1 protein (500 ng/mL, Biotechne, Minneapolis, MN, USA, 1627-WS-050).

Techniques: Staining, Negative Control

A schematic summary of the findings of this study. WISP-1 promotes cardiac fibroblasts’ phenotypic switch from quiescent fibroblasts to myofibroblasts (activated fibroblasts), promoting collagen processing and accumulation. WISP-1 activates Akt signalling via integrin β1/FAK/ILK in cardiac fibroblasts. Deletion of WISP-1 attenuates angiotensin II (AngII)-induced cardiac fibrotic remodelling in vivo. Figure key is illustrated on the top left-hand side of the figure. Purple ↓ denotes promotion; black ↑ denotes increase; ┤ denotes inhibition.

Journal: Cells

Article Title: WISP-1 Regulates Cardiac Fibrosis by Promoting Cardiac Fibroblasts’ Activation and Collagen Processing

doi: 10.3390/cells13110989

Figure Lengend Snippet: A schematic summary of the findings of this study. WISP-1 promotes cardiac fibroblasts’ phenotypic switch from quiescent fibroblasts to myofibroblasts (activated fibroblasts), promoting collagen processing and accumulation. WISP-1 activates Akt signalling via integrin β1/FAK/ILK in cardiac fibroblasts. Deletion of WISP-1 attenuates angiotensin II (AngII)-induced cardiac fibrotic remodelling in vivo. Figure key is illustrated on the top left-hand side of the figure. Purple ↓ denotes promotion; black ↑ denotes increase; ┤ denotes inhibition.

Article Snippet: Forty-eight hours later, SFM was replaced with fresh SFM in the presence or absence of recombinant human WISP-1 protein (500 ng/mL, Biotechne, Minneapolis, MN, USA, 1627-WS-050).

Techniques: In Vivo, Inhibition

Journal: Nature Communications

Article Title: Data-driven learning how oncogenic gene expression locally alters heterocellular networks

doi: 10.1038/s41467-022-29636-3

Figure Lengend Snippet: Digital cytometry deconvolutes a bulk transcriptomic profile using gene signatures that correspond to different stromal, malignant, and immune cell types. The results estimate the prevalence of the different cell types within the tissue sample, that is the digital cytometry features. By using bulk transcriptomic profiles of defined patient populations, underlying variation in the inferred cellular composition coupled with features associated with a patient sample, such as over-expression of a secreted gene product by malignant cells, can be used to estimate how the heterocellular network is impacted by a genetic alteration intrinsic to the malignant cell using Bayesian Network inference. To illustrate the approach, we focused on malignant cell expression of Cell Communication Network factor 4 (CCN4), a secreted matricellular protein. The resulting directed acyclic graph represents the collective conditional independence among the modeled features, or nodes, of the network.

Article Snippet: The concentration of CCN4 protein in the cell culture media from those wells was assayed using the Human WISP-1/CCN4 DuoSet ELISA Kit (R&D Systems, Minneapolis, MN) to confirm CCN4 knockout.

Techniques: Cytometry, Over Expression, Expressing

The nodes of the graph represent features, such as CCN4 gene expression (rectangle), sample attribute (hexagon), or the prevalence of a particular cell type/state (oval). The edges represent inferred causal relationships among the nodes. The black lines with arrow heads represent a positive causal relation while red lines with horizontal bars represent a negative or inhibitory causal relation, where the extent of influence of the parental node is annotated by the number beside the edge. The number included within the node symbol represents the average normalized value of the digital cytometry feature within the dataset with values of all of the parental nodes set to zero. The width of the edge is proportional to the posterior probability of inclusion into the DAG.

Journal: Nature Communications

Article Title: Data-driven learning how oncogenic gene expression locally alters heterocellular networks

doi: 10.1038/s41467-022-29636-3

Figure Lengend Snippet: The nodes of the graph represent features, such as CCN4 gene expression (rectangle), sample attribute (hexagon), or the prevalence of a particular cell type/state (oval). The edges represent inferred causal relationships among the nodes. The black lines with arrow heads represent a positive causal relation while red lines with horizontal bars represent a negative or inhibitory causal relation, where the extent of influence of the parental node is annotated by the number beside the edge. The number included within the node symbol represents the average normalized value of the digital cytometry feature within the dataset with values of all of the parental nodes set to zero. The width of the edge is proportional to the posterior probability of inclusion into the DAG.

Techniques: Expressing, Cytometry

A The percentage of live CD45+ cells isolated from tumors generated by inoculating s.c. with WT (red) and CCN4 KO (blue) variants of B16F0 (o and x’s) and YUMM1.7 (□ and +’s) cells, where the log-linear trends are highlighted by dotted lines. CD45+ values were obtained from three different antibody panels that quantified T cells, B/NK cells, and myeloid cells in TIL isolates from each mouse. B A comparison of the ratio of NK cells (black), CD8+ T cells (red), CD4+ T cells (blue), and B cells (green) to live CD45+ TILs in s.c. tumors generated using WT B16F0 and YUMM1.7 cells (mean ± s.d.). C The difference in the mean prevalence of the infiltrating immune cell types was compared when CCN4 is present (WT) versus absent (CCN4 KO) as predicted by digital cytometry from the BRCA (dark gray) and SKCM (light gray) datasets and as observed experimentally using the B16F0 (red) and YUMM1.7 (black) mouse models. D TIL comparison upon CCN4 KO in B16F0 and YUMM1.7 mouse models stratified by NK cells, CD8+ T cells, CD4+ T cells, and B cells (top to bottom) ( n = 7 biologically independent animals for YUMM1.7 and n = 4 biologically independent animals for B16F0 variants and mean ± s.d.). p -values calculated between WT and CCN4 KO pairs using two-sided Student’s t test.

Journal: Nature Communications

Article Title: Data-driven learning how oncogenic gene expression locally alters heterocellular networks

doi: 10.1038/s41467-022-29636-3

Figure Lengend Snippet: A The percentage of live CD45+ cells isolated from tumors generated by inoculating s.c. with WT (red) and CCN4 KO (blue) variants of B16F0 (o and x’s) and YUMM1.7 (□ and +’s) cells, where the log-linear trends are highlighted by dotted lines. CD45+ values were obtained from three different antibody panels that quantified T cells, B/NK cells, and myeloid cells in TIL isolates from each mouse. B A comparison of the ratio of NK cells (black), CD8+ T cells (red), CD4+ T cells (blue), and B cells (green) to live CD45+ TILs in s.c. tumors generated using WT B16F0 and YUMM1.7 cells (mean ± s.d.). C The difference in the mean prevalence of the infiltrating immune cell types was compared when CCN4 is present (WT) versus absent (CCN4 KO) as predicted by digital cytometry from the BRCA (dark gray) and SKCM (light gray) datasets and as observed experimentally using the B16F0 (red) and YUMM1.7 (black) mouse models. D TIL comparison upon CCN4 KO in B16F0 and YUMM1.7 mouse models stratified by NK cells, CD8+ T cells, CD4+ T cells, and B cells (top to bottom) ( n = 7 biologically independent animals for YUMM1.7 and n = 4 biologically independent animals for B16F0 variants and mean ± s.d.). p -values calculated between WT and CCN4 KO pairs using two-sided Student’s t test.

Techniques: Isolation, Generated, Comparison, Cytometry

A A comparison of the ratio of CD11c- (black) and CD11c+ (gray) macrophages, Dendritic cells (yellow), CD11c+ MDSC (green), MDSC (blue), and Neutrophils (red) to live CD45+ TILs in s.c. tumors generated using WT B16F0 and YUMM1.7 cells (mean ± s.d.). B The difference in prevalence of the myeloid cell types was compared when CCN4 is present (WT) versus absent (CCN4 KO) as predicted by digital cytometry of the BRCA (dark gray) and SKCM (light gray) data sets and as observed experimentally using the B16F0 (red) and YUMM1.7 (black) mouse models. Macrophages are the only myeloid cell subset inferred from the BRCA and SKCM datasets and are assumed to be related to CD11c+ macrophages in mouse models. C A representative scatter plot of GR1 versus CD11c expression in gated live CD45+ CD11b+ TILs obtained from WT (top) and CCN4 KO (bottom) YUMM1.7 tumors. D – F TIL comparison upon CCN4 KO in B16F0 and YUMM1.7 mouse models stratified by myeloid-derived suppressor cell subsets ( D : MDSC (top) and CD11c+ MDSC (bottom)) and other myeloid cell subsets ( E : CD11c- (top) and CD11c+ (bottom) macrophages, F : neutrophils (top) and dendritic cells (bottom)) ( n = 7 biologically independent animals for YUMM1.7 and n = 4 biologically independent animals for B16F0 variants and mean ± s.d.). p -values calculated between WT and CCN4 KO variants using two-sided Student’s t test.

Journal: Nature Communications

Article Title: Data-driven learning how oncogenic gene expression locally alters heterocellular networks

doi: 10.1038/s41467-022-29636-3

Figure Lengend Snippet: A A comparison of the ratio of CD11c- (black) and CD11c+ (gray) macrophages, Dendritic cells (yellow), CD11c+ MDSC (green), MDSC (blue), and Neutrophils (red) to live CD45+ TILs in s.c. tumors generated using WT B16F0 and YUMM1.7 cells (mean ± s.d.). B The difference in prevalence of the myeloid cell types was compared when CCN4 is present (WT) versus absent (CCN4 KO) as predicted by digital cytometry of the BRCA (dark gray) and SKCM (light gray) data sets and as observed experimentally using the B16F0 (red) and YUMM1.7 (black) mouse models. Macrophages are the only myeloid cell subset inferred from the BRCA and SKCM datasets and are assumed to be related to CD11c+ macrophages in mouse models. C A representative scatter plot of GR1 versus CD11c expression in gated live CD45+ CD11b+ TILs obtained from WT (top) and CCN4 KO (bottom) YUMM1.7 tumors. D – F TIL comparison upon CCN4 KO in B16F0 and YUMM1.7 mouse models stratified by myeloid-derived suppressor cell subsets ( D : MDSC (top) and CD11c+ MDSC (bottom)) and other myeloid cell subsets ( E : CD11c- (top) and CD11c+ (bottom) macrophages, F : neutrophils (top) and dendritic cells (bottom)) ( n = 7 biologically independent animals for YUMM1.7 and n = 4 biologically independent animals for B16F0 variants and mean ± s.d.). p -values calculated between WT and CCN4 KO variants using two-sided Student’s t test.

Techniques: Comparison, Generated, Cytometry, Expressing, Derivative Assay

A Expression of genes for transcription factors (left panel - Snai1: red triangle, Snai2: blue diamond, Zeb1: black circle, and Zeb2: gray square) and adhesion proteins (right panel - Cdh1: blue triangle, Cdh2: black circle, Fn1: red square) associated with the epithelial-mesenchymal transition were assayed as a function of time following addition of rmCCN4 to CCN4 KO YUMM1.7 (top row) and CCN4 KO B16F0 cells (bottom row). Colored asterisks indicate whether gene at a particular time point was significantly different than untreated cells, where n = 3 biological independent samples. B The distribution in cell trace staining among live CD4 + (left panel) and CD8 + (right panel) T cells stimulated with α CD3/ α CD28 (AP beads) alone or in the presence of media conditioned by WT B16F0 cells (AP beads + WT TCM), media conditioned by CCN4 KO B16F0 cells (AP beads + CCN4 KO TCM), or with 10 ng/ml of recombinant mouse CCN4 (AP beads + rCCN4). The distribution in the corresponding unstimulated cells (gray) are shown at the bottom. The colored vertical lines indicate the predicted dilution of cell trace staining in each generation based on the unstimulated controls. C Bivariate projection of the weights of genes within the resting (y-axis) and activated (x-axis) NK cell signatures. D Using spleens from C57BL/6 mice that were challenged with YUMM1.7 cells, isolated CD8+ T cells were assayed by in vitro ELISpot for IFN γ expression using variants of the YUMM1.7 cell line as targets (CCN4 KO YUMM1.7 with a blank inducible expression vector and CCN4 KO YUMM1.7 with a CCN4 inducible expression vector). To induce CCN4 expression, these YUMM1.7 variants were also cultured in the absence (−) or presence of doxycycline (+) and quantified following 24 h co-culture. Statistical significance between WT and CCN4 KO variants was assessed using two-way ANOVA followed by Tukey’s multiple comparison ad hoc post-test, where n = 6 biologically independent samples. Results summarized as mean ± s.d.

Journal: Nature Communications

Article Title: Data-driven learning how oncogenic gene expression locally alters heterocellular networks

doi: 10.1038/s41467-022-29636-3

Figure Lengend Snippet: A Expression of genes for transcription factors (left panel - Snai1: red triangle, Snai2: blue diamond, Zeb1: black circle, and Zeb2: gray square) and adhesion proteins (right panel - Cdh1: blue triangle, Cdh2: black circle, Fn1: red square) associated with the epithelial-mesenchymal transition were assayed as a function of time following addition of rmCCN4 to CCN4 KO YUMM1.7 (top row) and CCN4 KO B16F0 cells (bottom row). Colored asterisks indicate whether gene at a particular time point was significantly different than untreated cells, where n = 3 biological independent samples. B The distribution in cell trace staining among live CD4 + (left panel) and CD8 + (right panel) T cells stimulated with α CD3/ α CD28 (AP beads) alone or in the presence of media conditioned by WT B16F0 cells (AP beads + WT TCM), media conditioned by CCN4 KO B16F0 cells (AP beads + CCN4 KO TCM), or with 10 ng/ml of recombinant mouse CCN4 (AP beads + rCCN4). The distribution in the corresponding unstimulated cells (gray) are shown at the bottom. The colored vertical lines indicate the predicted dilution of cell trace staining in each generation based on the unstimulated controls. C Bivariate projection of the weights of genes within the resting (y-axis) and activated (x-axis) NK cell signatures. D Using spleens from C57BL/6 mice that were challenged with YUMM1.7 cells, isolated CD8+ T cells were assayed by in vitro ELISpot for IFN γ expression using variants of the YUMM1.7 cell line as targets (CCN4 KO YUMM1.7 with a blank inducible expression vector and CCN4 KO YUMM1.7 with a CCN4 inducible expression vector). To induce CCN4 expression, these YUMM1.7 variants were also cultured in the absence (−) or presence of doxycycline (+) and quantified following 24 h co-culture. Statistical significance between WT and CCN4 KO variants was assessed using two-way ANOVA followed by Tukey’s multiple comparison ad hoc post-test, where n = 6 biologically independent samples. Results summarized as mean ± s.d.

Techniques: Expressing, Staining, Recombinant, Isolation, In Vitro, Enzyme-linked Immunospot, Plasmid Preparation, Cell Culture, Co-Culture Assay, Comparison

Journal: Nature Communications

Article Title: Data-driven learning how oncogenic gene expression locally alters heterocellular networks

doi: 10.1038/s41467-022-29636-3

Article Snippet: In groups treated with recombinant mouse CCN4 protein (rmCCN4, 1680-WS-050,R&D systems), rmCCN4 was added at a final concentration of 5.0 μg/mL.

Techniques: Cytometry, Over Expression, Expressing

Journal: Nature Communications

Article Title: Data-driven learning how oncogenic gene expression locally alters heterocellular networks

doi: 10.1038/s41467-022-29636-3

Article Snippet: In groups treated with recombinant mouse CCN4 protein (rmCCN4, 1680-WS-050,R&D systems), rmCCN4 was added at a final concentration of 5.0 μg/mL.

Techniques: Expressing, Cytometry

Journal: Nature Communications

Article Title: Data-driven learning how oncogenic gene expression locally alters heterocellular networks

doi: 10.1038/s41467-022-29636-3

Article Snippet: In groups treated with recombinant mouse CCN4 protein (rmCCN4, 1680-WS-050,R&D systems), rmCCN4 was added at a final concentration of 5.0 μg/mL.

Techniques: Isolation, Generated, Comparison, Cytometry

Journal: Nature Communications

Article Title: Data-driven learning how oncogenic gene expression locally alters heterocellular networks

doi: 10.1038/s41467-022-29636-3

Article Snippet: In groups treated with recombinant mouse CCN4 protein (rmCCN4, 1680-WS-050,R&D systems), rmCCN4 was added at a final concentration of 5.0 μg/mL.

Techniques: Comparison, Generated, Cytometry, Expressing, Derivative Assay

Journal: Nature Communications

Article Title: Data-driven learning how oncogenic gene expression locally alters heterocellular networks

doi: 10.1038/s41467-022-29636-3

Article Snippet: In groups treated with recombinant mouse CCN4 protein (rmCCN4, 1680-WS-050,R&D systems), rmCCN4 was added at a final concentration of 5.0 μg/mL.

Techniques: Expressing, Staining, Recombinant, Isolation, In Vitro, Enzyme-linked Immunospot, Plasmid Preparation, Cell Culture, Co-Culture Assay, Comparison

( A ) F4/80 positive cells were accumulated in mice kidney (kidney cortex) with WISP1 administration and reduced by PTL, which was verified by counting the percentage of F4/80 positive cells. ( B ) The expression of inflammation markers was up-regulated by WISP1 and alleviated by PTL treatment. ( C ) M1 and M2 marker expression. Results are expressed as the mean ± SEM relative gene expression, *** P <0.001,** P <0.01, * P <0.05. n =3–5.

Journal: Clinical Science (London, England : 1979)

Article Title: WNT1-inducible signaling pathway protein 1 regulates kidney inflammation through the NF-κB pathway

doi: 10.1042/CS20210663

Figure Lengend Snippet: ( A ) F4/80 positive cells were accumulated in mice kidney (kidney cortex) with WISP1 administration and reduced by PTL, which was verified by counting the percentage of F4/80 positive cells. ( B ) The expression of inflammation markers was up-regulated by WISP1 and alleviated by PTL treatment. ( C ) M1 and M2 marker expression. Results are expressed as the mean ± SEM relative gene expression, *** P <0.001,** P <0.01, * P <0.05. n =3–5.

Article Snippet: Recombinant human WISP1 protein, IgG control and mouse WISP1 antibody were purchased commercially (R&D Systems, Minneapolis, MN) and used at specified concentrations.

Techniques: Expressing, Marker, Gene Expression

At 48 h after administration of WISP1 and/or PTL to healthy mice, there were: ( A ) no morphology changes (Masson staining) observed based on in the kidney at 48 h after administration of WISP1 and/or PTL in healthy mice. ( B ) No collagen (PSR staining) alteration was found in the WISP1-treated mice. ( C ) Q-PCR data showed no significant changes in collagen1a1 and fibronectin. ( D ) α-SMA expression were up-regulated with WISP1 treatment, reduced by PTL. Results are expressed as the mean ± SEM relative gene expression, ** P <0.01, * P <0.05. n =3–5.

Journal: Clinical Science (London, England : 1979)

Article Title: WNT1-inducible signaling pathway protein 1 regulates kidney inflammation through the NF-κB pathway

doi: 10.1042/CS20210663

Figure Lengend Snippet: At 48 h after administration of WISP1 and/or PTL to healthy mice, there were: ( A ) no morphology changes (Masson staining) observed based on in the kidney at 48 h after administration of WISP1 and/or PTL in healthy mice. ( B ) No collagen (PSR staining) alteration was found in the WISP1-treated mice. ( C ) Q-PCR data showed no significant changes in collagen1a1 and fibronectin. ( D ) α-SMA expression were up-regulated with WISP1 treatment, reduced by PTL. Results are expressed as the mean ± SEM relative gene expression, ** P <0.01, * P <0.05. n =3–5.

Article Snippet: Recombinant human WISP1 protein, IgG control and mouse WISP1 antibody were purchased commercially (R&D Systems, Minneapolis, MN) and used at specified concentrations.

Techniques: Staining, Expressing, Gene Expression

( A ) The translocation of P-p65 into nucleus induced by WISP1 was inhibited by treatment of NFκB inhibitor PTL, with the inhibition on the up-regulated expression of TNF-α. ( B , C ) PTL inhibited the LPS-induced nucleus translocation of P-p65 and the up-regulated expression of inflammation markers. ( D ) Results are expressed as the mean ± SEM relative gene expression, *** P <0.001,** P <0.01. n =3–5.

Journal: Clinical Science (London, England : 1979)

Article Title: WNT1-inducible signaling pathway protein 1 regulates kidney inflammation through the NF-κB pathway

doi: 10.1042/CS20210663

Figure Lengend Snippet: ( A ) The translocation of P-p65 into nucleus induced by WISP1 was inhibited by treatment of NFκB inhibitor PTL, with the inhibition on the up-regulated expression of TNF-α. ( B , C ) PTL inhibited the LPS-induced nucleus translocation of P-p65 and the up-regulated expression of inflammation markers. ( D ) Results are expressed as the mean ± SEM relative gene expression, *** P <0.001,** P <0.01. n =3–5.

Article Snippet: Recombinant human WISP1 protein, IgG control and mouse WISP1 antibody were purchased commercially (R&D Systems, Minneapolis, MN) and used at specified concentrations.

Techniques: Translocation Assay, Inhibition, Expressing, Gene Expression

( A ) Knockdown (KD) of WISP1 by shRNA. ( B ) Knockdown of WISP1 in LPS-stimulated RAW macrophages inhibited ( B ) the translocation of P-p65 into nucleus, and up-regulated ( C ) expression of inflammation markers. *** P <0.001,** P <0.01. n =3–5.

Journal: Clinical Science (London, England : 1979)

Article Title: WNT1-inducible signaling pathway protein 1 regulates kidney inflammation through the NF-κB pathway

doi: 10.1042/CS20210663

Figure Lengend Snippet: ( A ) Knockdown (KD) of WISP1 by shRNA. ( B ) Knockdown of WISP1 in LPS-stimulated RAW macrophages inhibited ( B ) the translocation of P-p65 into nucleus, and up-regulated ( C ) expression of inflammation markers. *** P <0.001,** P <0.01. n =3–5.

Article Snippet: Recombinant human WISP1 protein, IgG control and mouse WISP1 antibody were purchased commercially (R&D Systems, Minneapolis, MN) and used at specified concentrations.

Techniques: Knockdown, shRNA, Translocation Assay, Expressing

( A ) The addition of WISP1 antibody (Ab) inhibited the translocation of P-p65 induced by LPS and the expression of inflammation markers ( B,C ). ELISA showed that the up-regulation TNF-α was reduced by WISP1 knockdown and the addition of antibody. Results are expressed as the mean ± SEM relative gene expression, *** P <0.001,** P <0.01, * P <0.05. n =3–5.

Journal: Clinical Science (London, England : 1979)

Article Title: WNT1-inducible signaling pathway protein 1 regulates kidney inflammation through the NF-κB pathway

doi: 10.1042/CS20210663

Figure Lengend Snippet: ( A ) The addition of WISP1 antibody (Ab) inhibited the translocation of P-p65 induced by LPS and the expression of inflammation markers ( B,C ). ELISA showed that the up-regulation TNF-α was reduced by WISP1 knockdown and the addition of antibody. Results are expressed as the mean ± SEM relative gene expression, *** P <0.001,** P <0.01, * P <0.05. n =3–5.

Article Snippet: Recombinant human WISP1 protein, IgG control and mouse WISP1 antibody were purchased commercially (R&D Systems, Minneapolis, MN) and used at specified concentrations.

Techniques: Translocation Assay, Expressing, Enzyme-linked Immunosorbent Assay, Knockdown, Gene Expression

The representative cell images were taken using 4× light microscope. ( A ) PTL inhibited kidney fibroblast cells proliferation induced by recombinant WISP1 protein (1 μg/ml), validated by counting cell numbers. ( B ) Wisp1 knocked-down using shRNA reduced the proliferation of kidney fibroblast cells by TNF-α, confirmed by counting cell number. ( C ) The addition of WISP1 antibody reduced the fibroblast cell growth in the presence of TNF-α, as confirmed by counting cell number. n =3.

Journal: Clinical Science (London, England : 1979)

Article Title: WNT1-inducible signaling pathway protein 1 regulates kidney inflammation through the NF-κB pathway

doi: 10.1042/CS20210663

Figure Lengend Snippet: The representative cell images were taken using 4× light microscope. ( A ) PTL inhibited kidney fibroblast cells proliferation induced by recombinant WISP1 protein (1 μg/ml), validated by counting cell numbers. ( B ) Wisp1 knocked-down using shRNA reduced the proliferation of kidney fibroblast cells by TNF-α, confirmed by counting cell number. ( C ) The addition of WISP1 antibody reduced the fibroblast cell growth in the presence of TNF-α, as confirmed by counting cell number. n =3.

Article Snippet: Recombinant human WISP1 protein, IgG control and mouse WISP1 antibody were purchased commercially (R&D Systems, Minneapolis, MN) and used at specified concentrations.

Techniques: Light Microscopy, Recombinant, shRNA

( A ) STZ-induced DN mice at 18 weeks demonstrated the increased expression of TNF-α, MCP-1, CD68, α-SMA and WISP1 gene by qPCR, compared with control mice ( n =12–15 mice/group). Immunofluorescence staining indicated the elevated expression of WISP1 in DN model. Macrophage co-localized with WISP1 staining (white arrow). ( B ) High glucose-treated RAW cells expressed more WISP1, TNF-α and MCP-1 than low glucose-treated cells. ( C ) The kidneys of mice with 7 days post-UUO surgery showed an increased expression of inflammation genes, TNF-α, MCP-1 and IL6 and an up-regulation of WISP1, compared with control kidneys ( n =5 mice/group). Immunofluorescence staining indicated the elevated expression of WISP1 in UUO model. Macrophage co-localized with WISP1 staining (white arrow). *** P <0.001,** P <0.01.

Journal: Clinical Science (London, England : 1979)

Article Title: WNT1-inducible signaling pathway protein 1 regulates kidney inflammation through the NF-κB pathway

doi: 10.1042/CS20210663

Figure Lengend Snippet: ( A ) STZ-induced DN mice at 18 weeks demonstrated the increased expression of TNF-α, MCP-1, CD68, α-SMA and WISP1 gene by qPCR, compared with control mice ( n =12–15 mice/group). Immunofluorescence staining indicated the elevated expression of WISP1 in DN model. Macrophage co-localized with WISP1 staining (white arrow). ( B ) High glucose-treated RAW cells expressed more WISP1, TNF-α and MCP-1 than low glucose-treated cells. ( C ) The kidneys of mice with 7 days post-UUO surgery showed an increased expression of inflammation genes, TNF-α, MCP-1 and IL6 and an up-regulation of WISP1, compared with control kidneys ( n =5 mice/group). Immunofluorescence staining indicated the elevated expression of WISP1 in UUO model. Macrophage co-localized with WISP1 staining (white arrow). *** P <0.001,** P <0.01.

Article Snippet: Recombinant human WISP1 protein, IgG control and mouse WISP1 antibody were purchased commercially (R&D Systems, Minneapolis, MN) and used at specified concentrations.

Techniques: Expressing, Control, Immunofluorescence, Staining

( A ) Q-PCR analysis demonstrated that WISP1 antibody reduced inflammation gene expression in kidneys from UUO mice. ( B ) UUO kidneys without treatment or receiving IgG show an up-regulated F4/80 positive cells accumulation at 7 days. Administration of WISP1 antibody in UUO mice resulted in a reduction, verified by the percentage of F4/80 positive cells. ( C ) WISP1 antibody treatment rescued the degradation of IκBα expression in UUO kidneys, by Western blotting. Results are expressed as the mean ± SEM relative gene expression, *** P <0.001, ** P <0.01, * P <0.05. n =3–7.

Journal: Clinical Science (London, England : 1979)

Article Title: WNT1-inducible signaling pathway protein 1 regulates kidney inflammation through the NF-κB pathway

doi: 10.1042/CS20210663

Figure Lengend Snippet: ( A ) Q-PCR analysis demonstrated that WISP1 antibody reduced inflammation gene expression in kidneys from UUO mice. ( B ) UUO kidneys without treatment or receiving IgG show an up-regulated F4/80 positive cells accumulation at 7 days. Administration of WISP1 antibody in UUO mice resulted in a reduction, verified by the percentage of F4/80 positive cells. ( C ) WISP1 antibody treatment rescued the degradation of IκBα expression in UUO kidneys, by Western blotting. Results are expressed as the mean ± SEM relative gene expression, *** P <0.001, ** P <0.01, * P <0.05. n =3–7.

Article Snippet: Recombinant human WISP1 protein, IgG control and mouse WISP1 antibody were purchased commercially (R&D Systems, Minneapolis, MN) and used at specified concentrations.

Techniques: Gene Expression, Expressing, Western Blot

Figure 1. WISP2 inhibits WISP1-induced collagen linearization. A, Scanning electron microscopy of Col I lattices formed in the presence of PBS (Col I), 50 mg/mL recombinant WISP1, 100 mg/mL recombinant WISP2, or 50 mg/mL WISP1 þ 100 mg/mL WISP2 (1:3 WISP1:WISP2 molar ratio). Magenta arrows, examples of “knot-like” structures; yellow arrows, examples of “hairpin- or end-like structures.” Scale bars, 2 mm. B, Curvature ratios of Col I ﬁbrils in lattices from A (n ¼ 15; three independent experiments; ﬁve images/lattice). C, Binding of WISP1 to ﬁbrillar Col I (or BSA as negative control) as determined by solid-phase binding assay and detected with an anti-WISP1 antibody (n ¼ 6, from three independent experiments). D, Binding of WISP2 to ﬁbrillar Col I (or BSA as negative control) as determined by solid-phase binding assay and detected with an anti-WISP2 antibody (n ¼ 4, from two independent experiments). E, Inhibition of WISP1 (1 mmol/L) binding to ﬁbrillar Col I in the presence of increasing concentrations of WISP2 (n ¼ 6, from three independent experiments). F, Binding of prebound WISP1 (1 mmol/L) to ﬁbrillar Col I in the presence of increasing concentrations of WISP2 (n ¼ 6, from three independent experiments). G, Binding of prebound WISP2 (2 mmol/L) to ﬁbrillar Col I in the presence of increasing concentrations of WISP1 (n ¼ 4, from two independent experiments). H, Binding of WISP1 to WISP2 (or BSA as negative control) as determined by solid-phase binding assay (n ¼ 4, from two independent experiments). Mean SEM (B–H); one-way ANOVA followed by Tukey posttest (B). ns, nonsigniﬁcant, P > 0.05; , P < 0.001.

Journal: Cancer Research

Article Title: Matricellular Protein WISP2 Is an Endogenous Inhibitor of Collagen Linearization and Cancer Metastasis

doi: 10.1158/0008-5472.can-20-3982

Figure Lengend Snippet: Figure 1. WISP2 inhibits WISP1-induced collagen linearization. A, Scanning electron microscopy of Col I lattices formed in the presence of PBS (Col I), 50 mg/mL recombinant WISP1, 100 mg/mL recombinant WISP2, or 50 mg/mL WISP1 þ 100 mg/mL WISP2 (1:3 WISP1:WISP2 molar ratio). Magenta arrows, examples of “knot-like” structures; yellow arrows, examples of “hairpin- or end-like structures.” Scale bars, 2 mm. B, Curvature ratios of Col I ﬁbrils in lattices from A (n ¼ 15; three independent experiments; ﬁve images/lattice). C, Binding of WISP1 to ﬁbrillar Col I (or BSA as negative control) as determined by solid-phase binding assay and detected with an anti-WISP1 antibody (n ¼ 6, from three independent experiments). D, Binding of WISP2 to ﬁbrillar Col I (or BSA as negative control) as determined by solid-phase binding assay and detected with an anti-WISP2 antibody (n ¼ 4, from two independent experiments). E, Inhibition of WISP1 (1 mmol/L) binding to ﬁbrillar Col I in the presence of increasing concentrations of WISP2 (n ¼ 6, from three independent experiments). F, Binding of prebound WISP1 (1 mmol/L) to ﬁbrillar Col I in the presence of increasing concentrations of WISP2 (n ¼ 6, from three independent experiments). G, Binding of prebound WISP2 (2 mmol/L) to ﬁbrillar Col I in the presence of increasing concentrations of WISP1 (n ¼ 4, from two independent experiments). H, Binding of WISP1 to WISP2 (or BSA as negative control) as determined by solid-phase binding assay (n ¼ 4, from two independent experiments). Mean SEM (B–H); one-way ANOVA followed by Tukey posttest (B). ns, nonsigniﬁcant, P > 0.05; , P < 0.001.

Article Snippet: Recombinant murine WISP1 (R&D Systems, catalog no. 1680-WS) in PBS or recombinant human WISP2 (PeproTech, catalog no. 120-16) were added at a final concentration of 50 and 100 mg/mL, respectively, unless otherwise indicated.

Techniques: Electron Microscopy, Recombinant, Binding Assay, Negative Control, Inhibition

Figure 2. WISP2 inhibits WISP1-induced cell invasion through Col I. A–C, WISP2 (A) or WISP1 (B) gene expression levels, or WISP2/WISP1 gene expression ratio (C) in tumors and adjacent normal tissues from patients with different cancer types (BRCA, breast invasive carcinoma, n ¼ 113 normal and n ¼ 1,119 tumors; COAD, colon adenocarcinoma, n ¼ 41 normal and n ¼ 483 tumors; LUAD, lung adenocarcinoma, n ¼ 59 normal and n ¼ 541 tumors; LUSC, lung squamous cell carcinoma, n ¼ 51 normal and n ¼ 502 tumors; PRAD, prostate adenocarcinoma, n ¼ 52 normal and n ¼ 502 tumors; READ, rectum adenocarcinoma, n ¼ 10 normal and n ¼ 167 tumors). D, Migratory tracks of 4T1 cells plated on Col I (n ¼ 72 cells), Col I þ WISP1 (n ¼ 77 cells), Col I þ WISP2 (n ¼ 72 cells), or Col I þ WISP1 þ WISP2 (n ¼ 62 cells) lattices. E, Average cell speed from cells tracked in D. F, Migratory tracks of 4T1 cells plated on plastic in presence of PBS vehicle control (n ¼ 69 cells), WISP1 (n ¼ 85 cells), WISP2 (n ¼ 81 cells), or WISP1 þ WISP2 (n ¼ 65 cells). G, Average cell speed from cells tracked in F. H, Invasion of 4T1 cells through Col I layered on Transwell inserts in the presence of WISP1, WISP2, or WISP1 þ WISP2 (1:3 WISP1:WISP2 molar ratio; n ¼ 6 biological replicates). I, Migration of 4T1 cells through the microporous membrane of Transwell inserts (in the absence of Col I) in the presence of PBS (vehicle), WISP1, WISP2, or WISP1 þ WISP2 (n ¼ 6 biological replicates). J, Proliferation rate of 4T1 cells plated on Col I, Col I þ WISP1, or Col I þ WISP2 lattices (n ¼ 4 biological replicates). K, Invasion of MDA-MB-231 cells through Col I layered on Transwell inserts in the presence of WISP1, WISP2, or WISP1 þ WISP2 (n ¼ 6 biological replicates). L, Migration of MDA-MB-231 cells through the microporous membrane of Transwell inserts (in the absence of Col I) in the presence of PBS (vehicle), WISP1, WISP2, or WISP1 þ WISP2 (n ¼ 6 biological replicates). M, Proliferation rate of MDA-MB-231 cells plated on Col I, Col I þ WISP1, or Col I þ WISP2 lattices (n ¼ 4 biological replicates). Mean SEM (A–C, E, and G–M); Mann–Whitney test (A–C) and one-way ANOVA followed by Tukey posttest (E, G–I, K, and L). ns, nonsigniﬁcant, P > 0.05; , P < 0.001.

Journal: Cancer Research

Article Title: Matricellular Protein WISP2 Is an Endogenous Inhibitor of Collagen Linearization and Cancer Metastasis

doi: 10.1158/0008-5472.can-20-3982

Figure Lengend Snippet: Figure 2. WISP2 inhibits WISP1-induced cell invasion through Col I. A–C, WISP2 (A) or WISP1 (B) gene expression levels, or WISP2/WISP1 gene expression ratio (C) in tumors and adjacent normal tissues from patients with different cancer types (BRCA, breast invasive carcinoma, n ¼ 113 normal and n ¼ 1,119 tumors; COAD, colon adenocarcinoma, n ¼ 41 normal and n ¼ 483 tumors; LUAD, lung adenocarcinoma, n ¼ 59 normal and n ¼ 541 tumors; LUSC, lung squamous cell carcinoma, n ¼ 51 normal and n ¼ 502 tumors; PRAD, prostate adenocarcinoma, n ¼ 52 normal and n ¼ 502 tumors; READ, rectum adenocarcinoma, n ¼ 10 normal and n ¼ 167 tumors). D, Migratory tracks of 4T1 cells plated on Col I (n ¼ 72 cells), Col I þ WISP1 (n ¼ 77 cells), Col I þ WISP2 (n ¼ 72 cells), or Col I þ WISP1 þ WISP2 (n ¼ 62 cells) lattices. E, Average cell speed from cells tracked in D. F, Migratory tracks of 4T1 cells plated on plastic in presence of PBS vehicle control (n ¼ 69 cells), WISP1 (n ¼ 85 cells), WISP2 (n ¼ 81 cells), or WISP1 þ WISP2 (n ¼ 65 cells). G, Average cell speed from cells tracked in F. H, Invasion of 4T1 cells through Col I layered on Transwell inserts in the presence of WISP1, WISP2, or WISP1 þ WISP2 (1:3 WISP1:WISP2 molar ratio; n ¼ 6 biological replicates). I, Migration of 4T1 cells through the microporous membrane of Transwell inserts (in the absence of Col I) in the presence of PBS (vehicle), WISP1, WISP2, or WISP1 þ WISP2 (n ¼ 6 biological replicates). J, Proliferation rate of 4T1 cells plated on Col I, Col I þ WISP1, or Col I þ WISP2 lattices (n ¼ 4 biological replicates). K, Invasion of MDA-MB-231 cells through Col I layered on Transwell inserts in the presence of WISP1, WISP2, or WISP1 þ WISP2 (n ¼ 6 biological replicates). L, Migration of MDA-MB-231 cells through the microporous membrane of Transwell inserts (in the absence of Col I) in the presence of PBS (vehicle), WISP1, WISP2, or WISP1 þ WISP2 (n ¼ 6 biological replicates). M, Proliferation rate of MDA-MB-231 cells plated on Col I, Col I þ WISP1, or Col I þ WISP2 lattices (n ¼ 4 biological replicates). Mean SEM (A–C, E, and G–M); Mann–Whitney test (A–C) and one-way ANOVA followed by Tukey posttest (E, G–I, K, and L). ns, nonsigniﬁcant, P > 0.05; , P < 0.001.

Techniques: Gene Expression, Control, Migration, Membrane, MANN-WHITNEY

Figure 3. The CT domain of WISP1 drives WISP1-induced Col I linearization and cell invasion but is dispensable for WISP1-Col I binding. A, Scheme of WISP constructs stably transduced in 4T1 cells. IGFBP, insulin-like growth factor domain; VWC, von Willebrand factor type C repeat domain; TSP-1, thrombospondin type-1 repeat domain; CT, cysteine-rich knot-like C-terminal domain. B, Detection of WISP1, WISP1DCT, WISP2, and WISP2þCT binding to ﬁbrillar Col I, using conditioned media from 4T1 cells stably overexpressing these constructs or stably transduced with an empty vector (EV) control. Col I-bound proteins were detected with anti-WISP1 (left) or anti-WISP2 (right) antibodies. Binding was normalized to the molarity of each construct in the conditioned medium (left, n ¼ 9, from three independent experiments; right, n ¼ 8, from four independent experiments). C, Binding of WISP1-myc protein to ﬁbrillar Col I in the presence of WISP1, WISP2, WISP1DCT, or WISP2þCT using 1:1 (v:v) ratios of conditioned medium from 4T1 cells overexpressing these proteins. Conditionedmedium from 4T1-EV (4T1 cells stably transduced with anempty vector) was used as negative control. Col I-bound proteins were detected with an anti-Myc antibody (n ¼ 4, from two independent experiments). D, Scanning electron microscopy of Col I lattices formed in the presence of concentrated conditioned medium from 4T1 stable cell lines. Scale bars, 2 mm. E, Curvature ratios of Col I ﬁbrils in lattices from D (n ¼ 15, three independent experiments, ﬁve images/lattice). Statistical signiﬁcance versus Col I þ EV is shown. F, Average cell speed of 4T1-EV (n ¼ 123), 4T1-Wisp1 (n ¼ 75), 4T1-Wisp2 (n ¼ 69), 4T1-Wisp1DCT (n ¼ 106), and 4T1-Wisp2þCT (n ¼ 66) cells plated on Col I lattices. G, Invasion of 4T1-EV, 4T1-Wisp1, 4T1-Wisp2,4T1-Wisp1DCT, or 4T1-WISP2þCT cells through ColI lattices layeredon Transwell inserts (n ¼ 9, except 4T1-EV and 4T1-Wisp1, n ¼ 15 biological replicates). H, Average cell speed of 4T1-EV (n ¼ 115), 4T1-Wisp1 (n ¼ 89), 4T1-Wisp2 (n ¼ 54), 4T1-Wisp1DCT (n ¼ 53), and 4T1-Wisp2þCT (n ¼ 59) cells plated on plastic. I, Migration of 4T1-EV, 4T1-Wisp1, 4T1-Wisp2, 4T1-Wisp1DCT, or 4T1-WISP2þCT cells through uncoated Transwell inserts (n ¼ 9, except 4T1-EV, n ¼ 18 biological replicates). B, C, and E–I, Mean SEM; one-way ANOVA followed by Tukey posttest. ns, nonsigniﬁcant, P > 0.05; , P < 0.01; , P < 0.001.

Journal: Cancer Research

Article Title: Matricellular Protein WISP2 Is an Endogenous Inhibitor of Collagen Linearization and Cancer Metastasis

doi: 10.1158/0008-5472.can-20-3982

Figure Lengend Snippet: Figure 3. The CT domain of WISP1 drives WISP1-induced Col I linearization and cell invasion but is dispensable for WISP1-Col I binding. A, Scheme of WISP constructs stably transduced in 4T1 cells. IGFBP, insulin-like growth factor domain; VWC, von Willebrand factor type C repeat domain; TSP-1, thrombospondin type-1 repeat domain; CT, cysteine-rich knot-like C-terminal domain. B, Detection of WISP1, WISP1DCT, WISP2, and WISP2þCT binding to ﬁbrillar Col I, using conditioned media from 4T1 cells stably overexpressing these constructs or stably transduced with an empty vector (EV) control. Col I-bound proteins were detected with anti-WISP1 (left) or anti-WISP2 (right) antibodies. Binding was normalized to the molarity of each construct in the conditioned medium (left, n ¼ 9, from three independent experiments; right, n ¼ 8, from four independent experiments). C, Binding of WISP1-myc protein to ﬁbrillar Col I in the presence of WISP1, WISP2, WISP1DCT, or WISP2þCT using 1:1 (v:v) ratios of conditioned medium from 4T1 cells overexpressing these proteins. Conditionedmedium from 4T1-EV (4T1 cells stably transduced with anempty vector) was used as negative control. Col I-bound proteins were detected with an anti-Myc antibody (n ¼ 4, from two independent experiments). D, Scanning electron microscopy of Col I lattices formed in the presence of concentrated conditioned medium from 4T1 stable cell lines. Scale bars, 2 mm. E, Curvature ratios of Col I ﬁbrils in lattices from D (n ¼ 15, three independent experiments, ﬁve images/lattice). Statistical signiﬁcance versus Col I þ EV is shown. F, Average cell speed of 4T1-EV (n ¼ 123), 4T1-Wisp1 (n ¼ 75), 4T1-Wisp2 (n ¼ 69), 4T1-Wisp1DCT (n ¼ 106), and 4T1-Wisp2þCT (n ¼ 66) cells plated on Col I lattices. G, Invasion of 4T1-EV, 4T1-Wisp1, 4T1-Wisp2,4T1-Wisp1DCT, or 4T1-WISP2þCT cells through ColI lattices layeredon Transwell inserts (n ¼ 9, except 4T1-EV and 4T1-Wisp1, n ¼ 15 biological replicates). H, Average cell speed of 4T1-EV (n ¼ 115), 4T1-Wisp1 (n ¼ 89), 4T1-Wisp2 (n ¼ 54), 4T1-Wisp1DCT (n ¼ 53), and 4T1-Wisp2þCT (n ¼ 59) cells plated on plastic. I, Migration of 4T1-EV, 4T1-Wisp1, 4T1-Wisp2, 4T1-Wisp1DCT, or 4T1-WISP2þCT cells through uncoated Transwell inserts (n ¼ 9, except 4T1-EV, n ¼ 18 biological replicates). B, C, and E–I, Mean SEM; one-way ANOVA followed by Tukey posttest. ns, nonsigniﬁcant, P > 0.05; , P < 0.01; , P < 0.001.

Techniques: Binding Assay, Construct, Stable Transfection, Transduction, Plasmid Preparation, Control, Negative Control, Electron Microscopy, Migration

Figure 4. WISP2 and WISP1DCT block TGFb1-induced cell invasion through Col I by acting as WISP1 antagonists. A, Relative Wisp1 mRNA expression levels in 4T1-EV (4T1 cells stably transduced with an empty vector), 4T1-Wisp1DCT, and 4T1-Wisp2 cells cultured in the presence of vehicle or TGFb1 (2 ng/mL). Primers targeting sequences coding in the CT domain of Wisp1 were used to detect endoge- nous Wisp1 expression levels but not overexpressed Wisp1DCT (n ¼ 3 biological replicates). B, Average cell speed of 4T1-EV þ vehicle (n ¼ 44), 4T1-EV þ 2 ng/mL TGFb1 (n ¼ 37), 4T1-Wisp2 þ vehicle (n ¼ 36), 4T1- Wisp2 cells þ TGFb1 (n ¼ 44), 4T1-Wisp1DCT þ vehicle (n ¼ 36), and 4T1-Wisp1DCT þ TGFb1 (n ¼ 35) cells plated on Col I lattices. C, Invasion of 4T1-EV, 4T1-Wisp2, and 4T1-Wisp1DCT cells treated with TGFb1 (2 ng/mL) or vehicle control through Col I lattices layered on Trans- well inserts (n ¼ 6 biological replicates). D, WISP1 protein concentration in conditioned medium from 4T1-gCont (4T1-indCas9 cells stably transduced with a pool of control nontargeting gRNAs) and 4T1-Wisp1KO (4T1 cells with CRISPR/Cas9 knockout of Wisp1) treated with TGFb1 (2 ng/mL) or vehicle control (n ¼ 4, from two independent experiments). E, Average cell speed of 4T1-gCont þ vehicle (n ¼ 72), 4T1-gCont þ TGFb1 (n ¼ 64), 4T1-Wisp1KO (n ¼ 68), 4T1-Wisp1KO þ TGFb1 (n ¼ 48) cells plated on Col I lattices. F, Invasion of 4T1-gCont and 4T1-Wisp1KO cells, treated with TGFb1 (2 ng/mL) or vehicle control, through Col I lattices layered on Transwell inserts (n ¼ 6 biological replicates). Mean SEM; two-way ANOVA followed by Tukey posttest. ns, nonsigniﬁcant, P > 0.05; , P < 0.05; , P < 0.01; , P < 0.001.

Journal: Cancer Research

Article Title: Matricellular Protein WISP2 Is an Endogenous Inhibitor of Collagen Linearization and Cancer Metastasis

doi: 10.1158/0008-5472.can-20-3982

Figure Lengend Snippet: Figure 4. WISP2 and WISP1DCT block TGFb1-induced cell invasion through Col I by acting as WISP1 antagonists. A, Relative Wisp1 mRNA expression levels in 4T1-EV (4T1 cells stably transduced with an empty vector), 4T1-Wisp1DCT, and 4T1-Wisp2 cells cultured in the presence of vehicle or TGFb1 (2 ng/mL). Primers targeting sequences coding in the CT domain of Wisp1 were used to detect endoge- nous Wisp1 expression levels but not overexpressed Wisp1DCT (n ¼ 3 biological replicates). B, Average cell speed of 4T1-EV þ vehicle (n ¼ 44), 4T1-EV þ 2 ng/mL TGFb1 (n ¼ 37), 4T1-Wisp2 þ vehicle (n ¼ 36), 4T1- Wisp2 cells þ TGFb1 (n ¼ 44), 4T1-Wisp1DCT þ vehicle (n ¼ 36), and 4T1-Wisp1DCT þ TGFb1 (n ¼ 35) cells plated on Col I lattices. C, Invasion of 4T1-EV, 4T1-Wisp2, and 4T1-Wisp1DCT cells treated with TGFb1 (2 ng/mL) or vehicle control through Col I lattices layered on Trans- well inserts (n ¼ 6 biological replicates). D, WISP1 protein concentration in conditioned medium from 4T1-gCont (4T1-indCas9 cells stably transduced with a pool of control nontargeting gRNAs) and 4T1-Wisp1KO (4T1 cells with CRISPR/Cas9 knockout of Wisp1) treated with TGFb1 (2 ng/mL) or vehicle control (n ¼ 4, from two independent experiments). E, Average cell speed of 4T1-gCont þ vehicle (n ¼ 72), 4T1-gCont þ TGFb1 (n ¼ 64), 4T1-Wisp1KO (n ¼ 68), 4T1-Wisp1KO þ TGFb1 (n ¼ 48) cells plated on Col I lattices. F, Invasion of 4T1-gCont and 4T1-Wisp1KO cells, treated with TGFb1 (2 ng/mL) or vehicle control, through Col I lattices layered on Transwell inserts (n ¼ 6 biological replicates). Mean SEM; two-way ANOVA followed by Tukey posttest. ns, nonsigniﬁcant, P > 0.05; , P < 0.05; , P < 0.01; , P < 0.001.

Techniques: Blocking Assay, Expressing, Stable Transfection, Transduction, Plasmid Preparation, Cell Culture, Control, Protein Concentration, CRISPR, Knock-Out

Figure 5. WISP2 limits collagen linearization in tumors and inhibits breast cancer metastasis. A, Primary tumor mass, 28 days after orthotopic inoculation of 4T1-EV, 4T1-Wisp1, 4T1-Wisp2, or 4T1-Wisp1DCT cells into the 4th mammary fat pad of BALB/c female mice (n ¼ 10 mice/group, except 4T1-Wisp2, n ¼ 9 mice). B, Number of lung metastases in mice from A. C, Representative images of ﬁbrillar collagen in primary tumors from A visualized by Picrosirius red staining, followed by polarized light microscopy. Corresponding bright-ﬁeld images show tissue integ- rity. Scale bars, 200 mm. D, Average polarized light intensity in Picrosirius red staining images of tumors from A. E, Average stiffness (Young’s modulus) of tumors from A, measured by atomic force microsco- py. F, Scheme of experimental design. Recombinant WISP2 (rWISP2; 100 mg in 200 mL PBS) was admin- istered via intraperitoneal injection starting on day 8 post-injection of 4T1 cells. Tissues were collected 2 hours after the last dose of rWISP2. G, Primary tumor mass, 28 days after orthotopic inoculation of 4T1 cells into the 4th mammary fat pad (PBS, n ¼ 10; rWISP2, n ¼ 8 mice/group). H, Number of lung metastases in mice from G. I, Representative images of ﬁbrillar collagen in primary tumors from G visual- ized by Picrosirius red staining, followed by polarized light microscopy. Corresponding bright-ﬁeld images show tissue integrity. Scale bars, 200 mm. J, Aver- age polarized light intensity in Picrosirius red staining images of tumors from G. Mean SEM (A, B, D, E, G, H, and J); one-way ANOVA followed by Tukey posttest (A, B, D, and E). G, H, and J, Unpaired two-sided t test. ns, nonsigniﬁcant, P > 0.05; , P < 0.05; , P < 0.01, , P < 0.001.

Journal: Cancer Research

Article Title: Matricellular Protein WISP2 Is an Endogenous Inhibitor of Collagen Linearization and Cancer Metastasis

doi: 10.1158/0008-5472.can-20-3982

Figure Lengend Snippet: Figure 5. WISP2 limits collagen linearization in tumors and inhibits breast cancer metastasis. A, Primary tumor mass, 28 days after orthotopic inoculation of 4T1-EV, 4T1-Wisp1, 4T1-Wisp2, or 4T1-Wisp1DCT cells into the 4th mammary fat pad of BALB/c female mice (n ¼ 10 mice/group, except 4T1-Wisp2, n ¼ 9 mice). B, Number of lung metastases in mice from A. C, Representative images of ﬁbrillar collagen in primary tumors from A visualized by Picrosirius red staining, followed by polarized light microscopy. Corresponding bright-ﬁeld images show tissue integ- rity. Scale bars, 200 mm. D, Average polarized light intensity in Picrosirius red staining images of tumors from A. E, Average stiffness (Young’s modulus) of tumors from A, measured by atomic force microsco- py. F, Scheme of experimental design. Recombinant WISP2 (rWISP2; 100 mg in 200 mL PBS) was admin- istered via intraperitoneal injection starting on day 8 post-injection of 4T1 cells. Tissues were collected 2 hours after the last dose of rWISP2. G, Primary tumor mass, 28 days after orthotopic inoculation of 4T1 cells into the 4th mammary fat pad (PBS, n ¼ 10; rWISP2, n ¼ 8 mice/group). H, Number of lung metastases in mice from G. I, Representative images of ﬁbrillar collagen in primary tumors from G visual- ized by Picrosirius red staining, followed by polarized light microscopy. Corresponding bright-ﬁeld images show tissue integrity. Scale bars, 200 mm. J, Aver- age polarized light intensity in Picrosirius red staining images of tumors from G. Mean SEM (A, B, D, E, G, H, and J); one-way ANOVA followed by Tukey posttest (A, B, D, and E). G, H, and J, Unpaired two-sided t test. ns, nonsigniﬁcant, P > 0.05; , P < 0.05; , P < 0.01, , P < 0.001.

Techniques: Staining, Light Microscopy, Recombinant, Injection

Figure 6. WISP1 promotes whereas WISP2 inhi- bits human breast cancer metastasis. A, Invasion of MDA-MB-231 cells over- expressing WISP1 (MDA-WISP1) or WISP2 (MDA-WISP2), or stably trans- duced with an empty vector control (MDA-EV) through Col I lattices layered on Transwell inserts (n ¼ 9 biological replicates). B, Migration of MDA-EV, MDA-WISP1, or MDA-WISP2 cells through uncoated Transwell inserts (n ¼ 6 biological replicates). C, Prolifer- ation rate of MDA-EV, MDA-WISP1, or MDA-WISP2 cells plated on Col I lattices. (n ¼ 12 biological replicates). D, Primary tumor mass, 70 days after orthotopic inoculation of MDA-EV, MDA-WISP1, or MDA-WISP2 cells into the 4th mammary fat pad of NSG female mice (n ¼ 7 mice/ group). E, Number of lung metastases in mice from D. F, Representative images of ﬁbrillar collagen in primary tumors from D visualized by Picrosirius red staining, followed by polarized light microscopy. Corresponding bright- ﬁeld images show tissue integrity. Scale bars, 200 mm. G, Average polarized light intensity in Picrosirius red staining images of tumors from D. Mean SEM (A–E and G); one-way ANOVA followed by Tukey posttest (A, B, D, E, and G). ns, nonsigniﬁcant, P > 0.05; , P < 0.05; , P < 0.01; , P < 0.001.

Journal: Cancer Research

Article Title: Matricellular Protein WISP2 Is an Endogenous Inhibitor of Collagen Linearization and Cancer Metastasis

doi: 10.1158/0008-5472.can-20-3982

Figure Lengend Snippet: Figure 6. WISP1 promotes whereas WISP2 inhi- bits human breast cancer metastasis. A, Invasion of MDA-MB-231 cells over- expressing WISP1 (MDA-WISP1) or WISP2 (MDA-WISP2), or stably trans- duced with an empty vector control (MDA-EV) through Col I lattices layered on Transwell inserts (n ¼ 9 biological replicates). B, Migration of MDA-EV, MDA-WISP1, or MDA-WISP2 cells through uncoated Transwell inserts (n ¼ 6 biological replicates). C, Prolifer- ation rate of MDA-EV, MDA-WISP1, or MDA-WISP2 cells plated on Col I lattices. (n ¼ 12 biological replicates). D, Primary tumor mass, 70 days after orthotopic inoculation of MDA-EV, MDA-WISP1, or MDA-WISP2 cells into the 4th mammary fat pad of NSG female mice (n ¼ 7 mice/ group). E, Number of lung metastases in mice from D. F, Representative images of ﬁbrillar collagen in primary tumors from D visualized by Picrosirius red staining, followed by polarized light microscopy. Corresponding bright- ﬁeld images show tissue integrity. Scale bars, 200 mm. G, Average polarized light intensity in Picrosirius red staining images of tumors from D. Mean SEM (A–E and G); one-way ANOVA followed by Tukey posttest (A, B, D, E, and G). ns, nonsigniﬁcant, P > 0.05; , P < 0.05; , P < 0.01; , P < 0.001.

Techniques: Expressing, Stable Transfection, Plasmid Preparation, Control, Migration, Staining, Light Microscopy

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