tnf receptor 1  (Santa Cruz Biotechnology)

Journal: JCI Insight

Article Title: TNF- α represses fibroblast to myofibroblast transition through the histone methyltransferase Setdb2

doi: 10.1172/jci.insight.190836

Figure Lengend Snippet: ( A ) Flow gating on wound fibroblasts that were isolated from wounds 7 days after injury in C57BL/6 mice utilizing magnetic separation for Tie2 – , Ter119 – , CD45 – , CD31 – , and EPCAM – cells. Cells were plated and passaged 3 times prior to flow cytometry ( n = 5 mice). ( B ) Acta2 , Tagln , Myl9 , and Cald1 gene expression in wound fibroblasts isolated 7 days after wounding following TNF-α stimulation for 6 hours (25 ng/μL) ( n = 4 mice/group, pooled and run in triplicate). ( C ) Unbiased epigenetic array comparing expression of chromatin modifying enzymes in dermal fibroblasts following 6 hours of TNF-α (25 ng/μL) stimulation with control. Chromatin modifying enzymes with less than 1.5- to 0.5-fold regulation were excluded. Fold regulation was normalized to arithmetic mean of 5 housekeeping genes ( n = 4 mice/group, run in singlicate). ( D ) Setdb2 expression in wound fibroblasts 7 days after wounding following TNF-α (25 ng/μL) stimulation for 6 hours ( n = 4 mice/group, run in triplicate). ( E ) Cluster analysis uniform manifold approximation and projection (UMAP) of single-cell RNA sequencing from human T2D and non-T2D wounds showed 10 unique cell clusters (representative). Dot plots detailing ACTA2 , TAGLN , CALD1 , and MYL9 gene expression between human wound fibroblasts expressing high amounts of Setdb2 compared with those expressing low levels of Setdb2 ( n = 10). ( F ) Cluster analysis UMAP of single-cell RNA sequencing from human T2D and non-T2D wounds showed 8 unique fibroblast cell clusters (representative) ( n = 10). ( G ) Dot plots detailing SETDB2 , ACTA2 , TAGLN , CALD1 , and MYL9 gene expression between human wound fibroblast subtypes ( n = 10). * P < 0.05, *** P < 0.001, **** P < 0.0001. Data are presented as the mean ± SEM. Data were first analyzed for normal distribution, and if data passed the normality test, 2-tailed Student’s t test was used. Representative figures are displayed for panel B and D , which were repeated 3 times independently.

Article Snippet: Addition of etanercept (4 μM, MedChemExpress), a TNF-α decoy receptor that decreases TNF-α receptor signaling to the activated BMDM supernatants for 4 hours prevented the increase in expression of Setdb2 , suggesting that Setdb2 is regulated by TNF-α in the inflammatory macrophage supernatants ( ).

Techniques: Isolation, Flow Cytometry, Gene Expression, Expressing, Control, RNA Sequencing

Journal: JCI Insight

Article Title: TNF- α represses fibroblast to myofibroblast transition through the histone methyltransferase Setdb2

doi: 10.1172/jci.insight.190836

Figure Lengend Snippet: ( A ) Setdb2 gene expression in wound fibroblasts isolated from unwounded dermis of wild-type mice following 72 hours of siRNA transfection with nontargeting, negative control (siNTC) or siRNA specific to Setdb2 (40 nM) ( n = 5 mice/group, run in triplicate). ( B ) Acta2 , Tagln , Myl9 , and Cald1 gene expression in wound fibroblasts isolated from day 7 of wild-type mice following 72 hours of siRNA transfection with siNTC or siRNA specific to Setdb2 (40 nM) ( n = 5 mice/group, run in triplicate). ( C ) Acta2 , Tagln , Myl9 , and Cald1 gene expression in dermal fibroblasts isolated from Setdb2 fl/fl ColCreERT – / – and Setdb2 fl/fl ColCreERT +/+ ( n = 5 mice/group, run in triplicate). ( D ) Real-time qPCR values for chromatin immunoprecipitation (ChIP) for H3K9me3 at the promoters of Acta2 , Tagln , Myl9 , and Cald1 genes following 6 hours of TNF-α stimulation of Setdb2 fl/fl ColCreERT – / – and Setdb2 fl/fl ColCreERT +/+ dermal fibroblasts ( n = 5 mice/group, run in triplicate). ( E ) Cluster analysis UMAP of single-cell RNA sequencing from Setdb2 fl/fl ColCreERT – / – and Setdb2 fl/fl ColCreERT +/+ day 7 wounds showed 5 unique fibroblast cell clusters (representative). Dot plots detailing ACTA2 , TAGLN , CALD1 , and MYL9 gene expression between Setdb2 fl/fl ColCreERT – / – and Setdb2 fl/fl ColCreERT +/+ day 7 murine wound fibroblasts ( n = 3 per group). ( F ) Histograms showing the proportion of fibroblasts by subtype in day 7 wounds isolated from Setdb2 fl/fl ColCreERT – / – and Setdb2 fl/fl ColCreERT +/+ ( n = 3 per group). * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. Data are presented as the mean ± SEM. Data were first analyzed for normal distribution, and if data passed the normality test, 2-tailed Student’s t test was used. Panel A utilized Tukey’s multiple-comparison test, with a single pooled variance. Representative figures are displayed for panel A – D , which were repeated 3 times independently.

Article Snippet: Addition of etanercept (4 μM, MedChemExpress), a TNF-α decoy receptor that decreases TNF-α receptor signaling to the activated BMDM supernatants for 4 hours prevented the increase in expression of Setdb2 , suggesting that Setdb2 is regulated by TNF-α in the inflammatory macrophage supernatants ( ).

Techniques: Gene Expression, Isolation, Transfection, Negative Control, Chromatin Immunoprecipitation, RNA Sequencing, Comparison

Journal: JCI Insight

Article Title: TNF- α represses fibroblast to myofibroblast transition through the histone methyltransferase Setdb2

doi: 10.1172/jci.insight.190836

Figure Lengend Snippet: ( A ) Stat3 expression in dermal fibroblasts after 6 hours of TNF-α (25 ng/μL) stimulation, compared with control ( n = 5 mice/group, run in triplicate). ( B ) Western blot for p-STAT3 and β-actin in isolated wound fibroblasts after 2 hours of stimulation of TNF-α ( n = 5 mice/group, run in triplicate). ( C ) Setdb2 gene expression in wound fibroblasts isolated from day 7 of wild-type mice following 72 hours of siRNA transfection with siNTC or siRNA specific to Stat3 (siStat3) (40 nM) ( n = 4 mice/group, run in triplicate). ( D ) Setdb2 expression in isolated wound fibroblasts isolated from day 7 following injury from Stat3 fl/fl ColCreERT +/+ compared with Stat3 fl/fl ColCreERT – / – ( n = 3 mice/group, run in triplicate). ( E ) Western blot for p-STAT3 and β-actin in isolated wound fibroblasts after 2 hours of stimulation of TNF-α or TNF-α+tofacitinib (50 nM) ( n = 4 mice/group, run in triplicate). ( F ) Setdb2 expression in isolated wound fibroblasts treated with TNF-α, or TNF-α+tofacitinib, compared with control ( n = 4 mice/group, run in triplicate). * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. Data are presented as the mean ± SEM. Data were first analyzed for normal distribution, and if data passed the normality test, 2-tailed Student’s t test was used. Panel F utilized Tukey’s multiple-comparison test, with a single pooled variance. Representative figures are displayed for panels A , C , D , and F , which were repeated 3 times independently.

Article Snippet: Addition of etanercept (4 μM, MedChemExpress), a TNF-α decoy receptor that decreases TNF-α receptor signaling to the activated BMDM supernatants for 4 hours prevented the increase in expression of Setdb2 , suggesting that Setdb2 is regulated by TNF-α in the inflammatory macrophage supernatants ( ).

Techniques: Expressing, Control, Western Blot, Isolation, Gene Expression, Transfection, Comparison

Journal: JCI Insight

Article Title: TNF- α represses fibroblast to myofibroblast transition through the histone methyltransferase Setdb2

doi: 10.1172/jci.insight.190836

Figure Lengend Snippet: ( A ) Cluster analysis UMAP of single-cell RNA sequencing from human T2D and non-T2D wounds showed 10 unique cell clusters (representative), with both fibroblasts and macrophages identified. River plots depicting receptor-ligand interactions affect outgoing (signal source) TNF-α interactions from macrophages and incoming (signal responder) patterns from fibroblast subtypes. The thickness of the flow indicates the contribution of the cell group or signaling pathway to each latent pattern ( n = 10). ( B ) Setdb2 , Acta2 , Tagln , Myl9 , and Cald1 expression in isolated dermal fibroblasts treated with supernatants from activated BMDMs or control BMDMs. Supernatants were mixed 1:1 with serum-free DMEM and applied to fibroblasts for 4 hours ( n = 4 mice/group for fibroblasts, n = 8 mice/group for BMDMs, run in triplicate). ( C ) Setdb2 expression in isolated dermal fibroblasts treated with supernatants from activated BMDMs or control BMDMs. Supernatants were mixed 1:1 with serum-free DMEM ± etanercept (4 μM) and applied to fibroblasts for 4 hours ( n = 4 mice/group for fibroblasts, n = 8 mice/group for BMDMs, run in triplicate). ** P < 0.01, *** P < 0.001. Data are presented as the mean ± SEM. Data were first analyzed for normal distribution, and if data passed the normality test, 2-tailed Student’s t test was used. Representative figures are displayed for panel B and C , which were repeated 3 times independently.

Article Snippet: Addition of etanercept (4 μM, MedChemExpress), a TNF-α decoy receptor that decreases TNF-α receptor signaling to the activated BMDM supernatants for 4 hours prevented the increase in expression of Setdb2 , suggesting that Setdb2 is regulated by TNF-α in the inflammatory macrophage supernatants ( ).

Techniques: RNA Sequencing, Expressing, Isolation, Control

Journal: JCI Insight

Article Title: TNF- α represses fibroblast to myofibroblast transition through the histone methyltransferase Setdb2

doi: 10.1172/jci.insight.190836

Figure Lengend Snippet: ( A ) Wound expression of TNF-α harvested on day 7 following tissue injury (DIO and littermate controls) ( n = 4 mice/group, run in triplicate). ( B ) Setdb2 expression in fibroblasts isolated day 7 following tissue injury (DIO and littermate controls) ( n = 4 mice/group, run in triplicate). ( C ) Cluster analysis UMAP of single-cell RNA-Seq from human T2D and non-T2D wounds. Dot plot comparing Setdb2 expression in human T2D with non-T2D wound fibroblasts ( n = 10). ( D ) Wound healing curve for DIO Setdb2 fl/fl ColCreERT fl/fl mice. Mice were wounded via 6 mm punch biopsy and images were taken daily. Scale bar is 4 mm ( n = 10/group for Setdb2 fl/fl ColCreERT – / – and 5 for Setdb2 fl/fl ColCreER +/+ ). ( E ) Wound healing curve for DIO mice injected daily with tofacitinib (1 mg/kg) or DMSO control. Tofacitinib injections were started on day 1. Scale bar is 4 mm ( n = 6/group). ( F ) Acta2 , Tagln , Myl9 , and Cald1 gene expression from wound fibroblasts isolated from mice injected daily with tofacitinib or DMSO control following wounding ( n = 5 mice/group, run in triplicate). ( G ) Setdb2 gene expression from wound fibroblasts isolated from mice injected daily with tofacitinib or DMSO control following wounding, following stimulation with TNF-α for 6 hours ( n = 5 mice/group, run in triplicate). ( H ) Receptor-ligand plots depicting TNF-α receptor-ligand interactions in human scRNA-Seq between wound macrophage and wound fibroblast subtypes ( n = 10). ( I ) Significantly downregulated pathways in human T2D wound fibroblasts compared with non-T2D wound fibroblasts. Red box indicates most strongly downregulated pathways ( n = 10). * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. Data are presented as the mean ± SEM. Data were first analyzed for normal distribution, and if data passed the normality test, 2-tailed Student’s t test was used. Representative figures are displayed for panel A , B , F , and G , which were repeated 3 times independently.

Article Snippet: Addition of etanercept (4 μM, MedChemExpress), a TNF-α decoy receptor that decreases TNF-α receptor signaling to the activated BMDM supernatants for 4 hours prevented the increase in expression of Setdb2 , suggesting that Setdb2 is regulated by TNF-α in the inflammatory macrophage supernatants ( ).

Techniques: Expressing, Isolation, RNA Sequencing, Injection, Control, Gene Expression