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R&D Systems human recombinant wisp 1
Human Recombinant Wisp 1, supplied by R&D Systems, used in various techniques. Bioz Stars score: 94/100, based on 14 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/human+wisp/Recombinant+Human+WISP-1%2FCCN4+Protein%2C+CF/pmc12781172-38-5-23
Average 94 stars, based on 14 article reviews

human recombinant wisp 1 - by Bioz Stars, 2026-09

94/100 stars

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Enzyme-linked Immunosorbent Assay:

Article Title: WNT1-inducible signaling pathway protein 1 (WISP1/CCN4) stimulates melanoma invasion and metastasis by promoting the epithelial–mesenchymal transition
Article Snippet: .. To measure WISP1 secretion from each line, cells were grown for 48 h to reach about 90% confluence, and the medium was filtered for ELISA analysis using the human WISP-1/CCN4 DuoSet ELISA Development Kit (R&D Systems, Minneapolis, MN). ..

Article Title: Circulating Wnt1-inducible signaling pathway protein-1 (WISP-1/CCN4) is a novel biomarker of adiposity in subjects with type 2 diabetes
Article Snippet: .. We used human WISP-1/CCN4 DuoSet® ELISA kits ( R&D Systems, USA), that were validated to ensure reliable results in the analysis of circulating CCN4 in clinical studies (Tacke et al. 2018 ). .. The high-sensitivity C-reactive protein (hsCRP), α1-acid glycoprotein (α1-AGP) and macrophage inflammatory protein-1α (MIP-1α) were measured by ELISA with commercially available kits ( Biomerica, USA, for hsCRP; AssayPro, USA, for α1-AGP).

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

Article Title: Cell Communication Network Factor 4 (CCN4/WISP1) Shifts Melanoma Cells from a Fragile Proliferative State to a Resilient Metastatic State
Article Snippet: .. CCN4 concentration in conditioned medium was determined by ELISA using Human WISP-1/CCN4 DuoSet ELISA Development Kit (R&D Systems, Minneapolis, MN). .. Creation of Ccn4 -Knockout Cell Pools Mouse Ccn4 CRISPR/Cas9 KO Plasmid (sc-423705) and Homology-Directed Repair (HDR) plasmids (sc-423705-HDR) were from Santa Cruz Biotechnology (Dallas, Texas).

Article Title: Cell Communication Network factor 4 (CCN4/WISP1) shifts melanoma cells from a fragile proliferative state to a resilient metastatic state
Article Snippet: .. CCN4 concentration in conditioned medium was determined by ELISA using Human WISP-1/CCN4 DuoSet ELISA Development Kit (R&D Systems, Minneapolis, MN). .. Mouse Ccn4 CRISPR/Cas9 KO Plasmid (sc-423705) and Homology-Directed Repair (HDR) plasmids (sc-423705-HDR) were from Santa Cruz Biotechnology (Dallas, Texas).

Article Title: Data-driven learning how oncogenic gene expression locally alters heterocellular networks
Article Snippet: .. The concentration of CCN4 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. ..

Concentration Assay:

Cell Culture:

Knock-Out:

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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

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

Journal: Cells

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

doi: 10.3390/cells13232005

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

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

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

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

Journal: Cells

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

doi: 10.3390/cells13232005

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

Techniques: Expressing, Control, Quantitative RT-PCR

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

Journal: Cells

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

doi: 10.3390/cells13232005

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

Techniques: Control, Staining, Fluorescence, Positive Control

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

Journal: Cells

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

doi: 10.3390/cells13232005

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

Techniques: Gene Expression, Control, Positive Control

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

Journal: Cells

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

doi: 10.3390/cells13232005

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

Techniques: Recombinant, Expressing, Control

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

Journal: Cells

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

doi: 10.3390/cells13232005

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

Techniques: Conjugation Assay, Control

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

Journal: Cells

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

doi: 10.3390/cells13232005

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

Techniques: Conjugation Assay, Control

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

Journal: Cells

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

doi: 10.3390/cells13232005

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

Techniques: Conjugation Assay, Control

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

Journal: Cells

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

doi: 10.3390/cells13232005

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

Techniques:

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: Gene Expression, 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

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