mouse rwisp 1 1680 ws  (R&D Systems)

Journal: Frontiers in Immunology

Article Title: WISP1 drives esophageal squamous cell carcinoma progression via modulation of cancer-associated fibroblasts and immune microenvironment

doi: 10.3389/fimmu.2025.1586790

Figure Lengend Snippet: Differential expression of WISP1 in normal and tumor tissue samples. (A) Expression of WISP1 in pan-cancer tissues and adjacent normal tissues via TIMER2.0. (B) Expression levels of WISP1 in ESCA from GEPIA2 (|Log2FC|> 1, P < 0.01, log scale: log2 (TPM + 1), Jitter Size: 0.4; T: Tumor, N: Normal). (C) Expression of WISP1 mRNA in the ESCC dataset. (D–G) Results of IHC and WB analyses using 12 paired ESCC tissues and adjacent control samples (T: Tumor, N: Normal; scale bars = 100μm). (H) ROC curves predicting the prognostic ability of high WISP1 expression for 1-year, 3-year, and 5-year patient survival. *p < 0.05; **p < 0.01; ***p < 0.001,;****P < 0.0001.

Article Snippet: Primary fibroblasts were infected with shWISP1 lentivirus or treated with recombinant human WISP1 (rhWISP1) protein( NP_003873.1 , Sino Biological, China; endotoxin level <0.1 EU/μg, as determined by Limulus Amebocyte Lysate assay), followed by three washes with PBS.

Techniques: Quantitative Proteomics, Expressing, Control

Journal: Frontiers in Immunology

Article Title: WISP1 drives esophageal squamous cell carcinoma progression via modulation of cancer-associated fibroblasts and immune microenvironment

doi: 10.3389/fimmu.2025.1586790

Figure Lengend Snippet: The impact of WISP1 expression on the survival of ESCC patients. (A–C) Kaplan-Meier analysis of overall survival for high-expression versus low-expression groups in the datasets GSE53624 , GSE53625 , and TCGA. (D–F) Time-dependent ROC analysis for patients in the datasets.

Article Snippet: Primary fibroblasts were infected with shWISP1 lentivirus or treated with recombinant human WISP1 (rhWISP1) protein( NP_003873.1 , Sino Biological, China; endotoxin level <0.1 EU/μg, as determined by Limulus Amebocyte Lysate assay), followed by three washes with PBS.

Techniques: Expressing

Journal: Frontiers in Immunology

Article Title: WISP1 drives esophageal squamous cell carcinoma progression via modulation of cancer-associated fibroblasts and immune microenvironment

doi: 10.3389/fimmu.2025.1586790

Figure Lengend Snippet: Identification of DEGs in ESCC, functional enrichment analysis, and functional annotation of WISP1. (A) Volcano plot shows the DEGs from four datasets. (B) A Venn diagram shows genes that are differentially expressed across all four datasets. (C) GO and KEGG analyses reveal the potential biological mechanisms of DEGs in the four datasets. (D, E) GeneMANIA and STRING databases identify target proteins and genes associated with WISP1, followed by enrichment analysis of the functions of these proteins and genes. (F) Visualization of enrichment analysis results.

Article Snippet: Primary fibroblasts were infected with shWISP1 lentivirus or treated with recombinant human WISP1 (rhWISP1) protein( NP_003873.1 , Sino Biological, China; endotoxin level <0.1 EU/μg, as determined by Limulus Amebocyte Lysate assay), followed by three washes with PBS.

Techniques: Functional Assay

Journal: Frontiers in Immunology

Article Title: WISP1 drives esophageal squamous cell carcinoma progression via modulation of cancer-associated fibroblasts and immune microenvironment

doi: 10.3389/fimmu.2025.1586790

Figure Lengend Snippet: Tumor microenvironment and immune cell infiltration analysis in the GSE53624 dataset. (A) Differences in the abundance of infiltrating immune cells between high-expression and low-expression groups. (B–D) Differences in stromal scores, immune scores, and estimate scores between high-expression and low-expression groups. (E) Evaluation of the expression of immune checkpoint molecules (CD274, PDCD1, TIGIT, CD276, CTLA4, LAG3) between high-risk and low-risk groups. (F) Correlation between WISP1 expression and immune checkpoint molecules (CD274, PDCD1, TIGIT, CD276, CTLA4, LAG3). Blue indicates positive correlation, red indicates negative correlation, and the numbers inside the boxes represent the magnitude of the correlation. *p < 0.05; **p < 0.01; ***p < 0.001; ****P < 0.0001.

Article Snippet: Primary fibroblasts were infected with shWISP1 lentivirus or treated with recombinant human WISP1 (rhWISP1) protein( NP_003873.1 , Sino Biological, China; endotoxin level <0.1 EU/μg, as determined by Limulus Amebocyte Lysate assay), followed by three washes with PBS.

Techniques: Expressing

Journal: Frontiers in Immunology

Article Title: WISP1 drives esophageal squamous cell carcinoma progression via modulation of cancer-associated fibroblasts and immune microenvironment

doi: 10.3389/fimmu.2025.1586790

Figure Lengend Snippet: Gene set enrichment analysis (GSEA) of hallmark pathways for esophageal squamous cell carcinoma (ESCC) patients stratified by WISP1 expression levels. (A–C) Enrichment patterns of associated pathways in the GSE53624 , GSE53625 , and TCGA datasets. The analyses were evaluated using a significance threshold corrected for the False Discovery Rate (FDR).

Article Snippet: Primary fibroblasts were infected with shWISP1 lentivirus or treated with recombinant human WISP1 (rhWISP1) protein( NP_003873.1 , Sino Biological, China; endotoxin level <0.1 EU/μg, as determined by Limulus Amebocyte Lysate assay), followed by three washes with PBS.

Techniques: Expressing

Journal: Frontiers in Immunology

Article Title: WISP1 drives esophageal squamous cell carcinoma progression via modulation of cancer-associated fibroblasts and immune microenvironment

doi: 10.3389/fimmu.2025.1586790

Figure Lengend Snippet: Computational prediction of drug sensitivity for ESCC patients stratified by WISP1 expression levels. Using WISP1 expression grouping derived from the GSE53624 dataset, the “oncoPredict” R package was employed to evaluate the drug response spectrum. **** indicates statistical significance at p < 0.0001.

Article Snippet: Primary fibroblasts were infected with shWISP1 lentivirus or treated with recombinant human WISP1 (rhWISP1) protein( NP_003873.1 , Sino Biological, China; endotoxin level <0.1 EU/μg, as determined by Limulus Amebocyte Lysate assay), followed by three washes with PBS.

Techniques: Expressing, Derivative Assay

Journal: Frontiers in Immunology

Article Title: WISP1 drives esophageal squamous cell carcinoma progression via modulation of cancer-associated fibroblasts and immune microenvironment

doi: 10.3389/fimmu.2025.1586790

Figure Lengend Snippet: Functional characterization of WISP1 in a subpopulation of fibroblasts. (A) Gene Set Enrichment Analysis (GSEA) shows enrichment results of differentially expressed genes between WISP1_Fib_Negative and WISP1_Fib_Positive clusters within the c5.all.v2024.1.Hs.symbols gene set; (B, C) display the enrichment results of DEGs in the h.all.v7.1.symbols gene set. (D) Proportional distribution of WISP1_Fib_Negative and WISP1_Fib_Positive clusters in normal esophageal tissues and ESCC samples. (E, F) Comparative analysis of intercellular communication signal intensity among fibroblast subpopulations. (G, H) Molecular stratification of fibroblasts using lineage-specific markers (DCN/Decorin, IGFBP6/Insulin Like Growth Factor Binding Protein 6, MFAP5/Microfibril Associated Protein 5, ACTA2/Actin Alpha 2 Smooth Muscle, TAGLN/Transgelin, CTHRC1/Collagen Triple Helix Repeat Containing 1)into NFs and CAFs subtypes. (I) Validation of CAF-specific markers (FAP, COL1A1, COL3A1, COL4A1, COL10A1, MMP1, MMP11, MMP14) and expression patterns of WISP1 in CAFs via single-cell RNA sequencing.

Article Snippet: Primary fibroblasts were infected with shWISP1 lentivirus or treated with recombinant human WISP1 (rhWISP1) protein( NP_003873.1 , Sino Biological, China; endotoxin level <0.1 EU/μg, as determined by Limulus Amebocyte Lysate assay), followed by three washes with PBS.

Techniques: Functional Assay, Binding Assay, Biomarker Discovery, Expressing, RNA Sequencing

Journal: Frontiers in Immunology

Article Title: WISP1 drives esophageal squamous cell carcinoma progression via modulation of cancer-associated fibroblasts and immune microenvironment

doi: 10.3389/fimmu.2025.1586790

Figure Lengend Snippet: WISP1 regulates cancer-associated fibroblast function and extracellular matrix remodeling. (A, B) Immunofluorescence multiplex staining of α-smooth muscle actin (αSMA) and fibroblast activation protein (FAP) in paired cancer-associated fibroblasts (CAFs, tumor-derived) and normal fibroblasts (NFs, adjacent non-tumor tissues) (scale bars = 50μm). (C) Western blot (WB) analysis of WISP1, αSMA, and FAP expression in CAFs versus NFs. (D, E) Lentiviral short hairpin RNA (shRNA)-mediated WISP1 knockdown in CAFs, validated by quantitative reverse transcription PCR (qRT-PCR) and WB. (F–K) Functional characterization of CAF proliferation (CCK-8/EdU), migration, and invasion (Transwell) post-WISP1 silencing (scale bars = 50μm). (M) ELISA quantification of secreted WISP1 in supernatants from untransfected CAFs, CAFs-shVector, and CAFs-shWISP1 at 0 h, 24 h, and 48 h (N) Schematic of indirect co-culture system for CAF-ESCC interaction analysis. (L, P) WB assessment of extracellular matrix (ECM)-remodeling markers (COL1A1, MMP14) in WISP1-depleted CAFs and rescue via recombinant human WISP1 (rhWISP1). (O, Q) Transwell assay comparing the migration and invasion capacities of KYSE150 (left) and Eca109 (right) cells co-cultured with untransfected CAFs, CAFs-shVector, or CAFs-shWISP1(scale bars = 50μm). (R, S) Transwell assay assessing the migration and invasion capacities of KYSE150 (left) and Eca109 (right) cells in the CAFs-shWISP1 co-culture system following rescue experiments with rhWISP1 supplementation (scale bar = 50μm).Data are presented as mean ± standard deviation from three independent experiments. Statistical significance was analyzed by two-tailed Student’s t-tests. ns = not significant, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Article Snippet: Primary fibroblasts were infected with shWISP1 lentivirus or treated with recombinant human WISP1 (rhWISP1) protein( NP_003873.1 , Sino Biological, China; endotoxin level <0.1 EU/μg, as determined by Limulus Amebocyte Lysate assay), followed by three washes with PBS.

Techniques: Immunofluorescence, Multiplex Assay, Staining, Activation Assay, Derivative Assay, Western Blot, Expressing, shRNA, Knockdown, Reverse Transcription, Quantitative RT-PCR, Functional Assay, CCK-8 Assay, Migration, Enzyme-linked Immunosorbent Assay, Co-Culture Assay, Recombinant, Transwell Assay, Cell Culture, Standard Deviation, Two Tailed Test

Journal: Frontiers in Immunology

Article Title: WISP1 drives esophageal squamous cell carcinoma progression via modulation of cancer-associated fibroblasts and immune microenvironment

doi: 10.3389/fimmu.2025.1586790

Figure Lengend Snippet: WISP1 regulates ECM remodeling in CAFs through STAT3 signaling. (A, B) Representative phospho-kinase antibody array membrane comparing CAFs-shVector and CAFs-shWISP1. Red boxes highlight phosphorylated STAT3 (Y705) signals. (C) Dose-response curve of STAT3 inhibitor Stattic in CAFs. (D) Western blot analysis of phosphorylated STAT3 (Y705), total STAT3, and GAPDH (loading control) in: CAFs-shVector, CAFs-shWISP1, CAFs-shWISP1 + rhWISP1 (0.8 μg/mL), and CAFs-shWISP1 + rhWISP1 (0.8 μg/mL) + Stattic (7 μM). (E) Western blot analysis of COL1A1, MMP14, and GAPDH in CAFs-shWISP1 + rhWISP1 (0.8 μg/mL) and CAFs-shWISP1 + rhWISP1 (0.8 μg/mL) + Stattic (7 μM).

Article Snippet: Primary fibroblasts were infected with shWISP1 lentivirus or treated with recombinant human WISP1 (rhWISP1) protein( NP_003873.1 , Sino Biological, China; endotoxin level <0.1 EU/μg, as determined by Limulus Amebocyte Lysate assay), followed by three washes with PBS.

Techniques: Ab Array, Membrane, Western Blot, Control

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

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

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

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

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

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

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.

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: 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

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.

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: 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

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.

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: 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

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.

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