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tgfβ3 dy243 plates  (R&D Systems)


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    R&D Systems tgfβ3 dy243 plates
    a , Visualization of kernel density estimates of POSTN (top) and COMP (bottom) expression within a representative COL1A1 -high region. b , Heat map showing mean pixel correlations across synovial samples ( n = 4) between kernel densities for selected fibrogenic genes within COL1A1 -high regions. c , Representative examples ( n = 4 RA tissues) of COMP transcript expression in perivascular and pauci-cellular regions. Scale bar, 50 µm. d , Correlation between the percentage of fibroblasts that express COMP (count > 0) per sample and abundance of endothelial and mural cells as a percentage of total cells in the same sample. Statistics calculated by two-sided Pearson’s correlation coefficient test. Top: 4 samples analyzed with a 50-gene custom panel. Bottom: 16 samples analyzed with a 5,101-gene panel. e , Mean pixel correlations across samples ( n = 4) for TGFB isoform expression compared to selected fibroblast and endothelial markers. f , Heat map representing relative expression of TGFB isoforms across different cell types in synovial samples ( n = 17) analyzed with a 5,101-gene panel. g , Example of data in f showing TGFB transcripts overlaid on cell types. Scale bar, 50 µm. h , Immunofluorescence staining of RA synovial tissue showing protein expression of TGFβ isoforms relative to EC marker vWF. <t>TGFβ3</t> and vWF staining were performed on serial sections. i , Immunofluorescence data showing Notch3, Collagen 1 and pSMAD3 staining in perivascular and pauci-cellular regions of the RA synovium. Scale bars, 200 µm. Immunofluorescence is representative of n > 5 RA synovial tissue.
    Tgfβ3 Dy243 Plates, supplied by R&D Systems, used in various techniques. Bioz Stars score: 94/100, based on 42 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/product/tgf%CE%B23/Human+TGF-beta+3+DuoSet+ELISA/pmc12956583-329-28-24
    Average 94 stars, based on 42 article reviews
    tgfβ3 dy243 plates - by Bioz Stars, 2026-09
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    Images

    1) Product Images from "Spatial patterning of fibroblast TGFβ signaling underlies treatment resistance in rheumatoid arthritis"

    Article Title: Spatial patterning of fibroblast TGFβ signaling underlies treatment resistance in rheumatoid arthritis

    Journal: Nature Immunology

    doi: 10.1038/s41590-025-02386-2

    a , Visualization of kernel density estimates of POSTN (top) and COMP (bottom) expression within a representative COL1A1 -high region. b , Heat map showing mean pixel correlations across synovial samples ( n = 4) between kernel densities for selected fibrogenic genes within COL1A1 -high regions. c , Representative examples ( n = 4 RA tissues) of COMP transcript expression in perivascular and pauci-cellular regions. Scale bar, 50 µm. d , Correlation between the percentage of fibroblasts that express COMP (count > 0) per sample and abundance of endothelial and mural cells as a percentage of total cells in the same sample. Statistics calculated by two-sided Pearson’s correlation coefficient test. Top: 4 samples analyzed with a 50-gene custom panel. Bottom: 16 samples analyzed with a 5,101-gene panel. e , Mean pixel correlations across samples ( n = 4) for TGFB isoform expression compared to selected fibroblast and endothelial markers. f , Heat map representing relative expression of TGFB isoforms across different cell types in synovial samples ( n = 17) analyzed with a 5,101-gene panel. g , Example of data in f showing TGFB transcripts overlaid on cell types. Scale bar, 50 µm. h , Immunofluorescence staining of RA synovial tissue showing protein expression of TGFβ isoforms relative to EC marker vWF. TGFβ3 and vWF staining were performed on serial sections. i , Immunofluorescence data showing Notch3, Collagen 1 and pSMAD3 staining in perivascular and pauci-cellular regions of the RA synovium. Scale bars, 200 µm. Immunofluorescence is representative of n > 5 RA synovial tissue.
    Figure Legend Snippet: a , Visualization of kernel density estimates of POSTN (top) and COMP (bottom) expression within a representative COL1A1 -high region. b , Heat map showing mean pixel correlations across synovial samples ( n = 4) between kernel densities for selected fibrogenic genes within COL1A1 -high regions. c , Representative examples ( n = 4 RA tissues) of COMP transcript expression in perivascular and pauci-cellular regions. Scale bar, 50 µm. d , Correlation between the percentage of fibroblasts that express COMP (count > 0) per sample and abundance of endothelial and mural cells as a percentage of total cells in the same sample. Statistics calculated by two-sided Pearson’s correlation coefficient test. Top: 4 samples analyzed with a 50-gene custom panel. Bottom: 16 samples analyzed with a 5,101-gene panel. e , Mean pixel correlations across samples ( n = 4) for TGFB isoform expression compared to selected fibroblast and endothelial markers. f , Heat map representing relative expression of TGFB isoforms across different cell types in synovial samples ( n = 17) analyzed with a 5,101-gene panel. g , Example of data in f showing TGFB transcripts overlaid on cell types. Scale bar, 50 µm. h , Immunofluorescence staining of RA synovial tissue showing protein expression of TGFβ isoforms relative to EC marker vWF. TGFβ3 and vWF staining were performed on serial sections. i , Immunofluorescence data showing Notch3, Collagen 1 and pSMAD3 staining in perivascular and pauci-cellular regions of the RA synovium. Scale bars, 200 µm. Immunofluorescence is representative of n > 5 RA synovial tissue.

    Techniques Used: Expressing, Immunofluorescence, Staining, Marker

    a , UMAP projection and dotplots of single-cell 3D co-culture data displaying COMP and POSTN expression . b , Chip-seq analysis of RBPJ binding to TGFBR2 (-350 bp to -138 bp upstream of transcriptional start site (TSS)) and TGFBR3 (-185 bp to +84 bp relative to TSS) promoter regions in HepG2 cells (data from GSE127388 ). Significant peaks are annotated with blue bars and were called using the narrowPeak function in MACS2. c , ELISA quantification of supernatant TGFβ1, TGFβ2, and TGFβ3 from fibroblasts stimulated with DLL4 for 3 days. d , RT-qPCR analysis of TGFβ isoform and fibrogenic gene expression in unstimulated or DLL4-stimulated fibroblasts (72 h) and fibroblasts that were exposed to conditioned media from unstimulated and DLL4-stimulated fibroblasts for 72 h. e , RT-qPCR analysis of TGFβ isoform and fibrogenic gene expression in unstimulated or DLL4-stimulated cells plated in the bottom chamber and fibroblasts that were cultured in the above transwell insert for 72 h. f , Top: ELISA quantification of supernatant COMP, PRO-COL1 and POSTN from fibroblasts stimulated with or without recombinant TGFβ1 (10 ng ml−1) and with or without a pan-TGFβ blocking antibody (5 ug mL-1) for 72 h. Below: ELISA quantification of COMP, PRO-COL1 and POSTN on unstimulated or DLL4-stimulated fibroblasts that were treated with antibodies against individual or pan-TGFβ isoforms (5 ug mL-1) for 72 h. g , RT-qPCR analysis of fibroblasts with or without DLL4 stimulation that were treated with siRNA (20 nM) for 96 h. h-i , ELISA quantification of fibroblast production of PRO-COL1, POSTN and COMP over 24 h after 72 h treatment with indicated siRNA combinations (10 nM each) with or without DLL4 stimulation. For c-i Data are shown as mean +/- s.d with n = 3 biological replicates, and representative of at least two independent experiments. Statistical analysis was performed by unpaired two-sided t-test.
    Figure Legend Snippet: a , UMAP projection and dotplots of single-cell 3D co-culture data displaying COMP and POSTN expression . b , Chip-seq analysis of RBPJ binding to TGFBR2 (-350 bp to -138 bp upstream of transcriptional start site (TSS)) and TGFBR3 (-185 bp to +84 bp relative to TSS) promoter regions in HepG2 cells (data from GSE127388 ). Significant peaks are annotated with blue bars and were called using the narrowPeak function in MACS2. c , ELISA quantification of supernatant TGFβ1, TGFβ2, and TGFβ3 from fibroblasts stimulated with DLL4 for 3 days. d , RT-qPCR analysis of TGFβ isoform and fibrogenic gene expression in unstimulated or DLL4-stimulated fibroblasts (72 h) and fibroblasts that were exposed to conditioned media from unstimulated and DLL4-stimulated fibroblasts for 72 h. e , RT-qPCR analysis of TGFβ isoform and fibrogenic gene expression in unstimulated or DLL4-stimulated cells plated in the bottom chamber and fibroblasts that were cultured in the above transwell insert for 72 h. f , Top: ELISA quantification of supernatant COMP, PRO-COL1 and POSTN from fibroblasts stimulated with or without recombinant TGFβ1 (10 ng ml−1) and with or without a pan-TGFβ blocking antibody (5 ug mL-1) for 72 h. Below: ELISA quantification of COMP, PRO-COL1 and POSTN on unstimulated or DLL4-stimulated fibroblasts that were treated with antibodies against individual or pan-TGFβ isoforms (5 ug mL-1) for 72 h. g , RT-qPCR analysis of fibroblasts with or without DLL4 stimulation that were treated with siRNA (20 nM) for 96 h. h-i , ELISA quantification of fibroblast production of PRO-COL1, POSTN and COMP over 24 h after 72 h treatment with indicated siRNA combinations (10 nM each) with or without DLL4 stimulation. For c-i Data are shown as mean +/- s.d with n = 3 biological replicates, and representative of at least two independent experiments. Statistical analysis was performed by unpaired two-sided t-test.

    Techniques Used: Single Cell, Co-Culture Assay, Expressing, ChIP-sequencing, Binding Assay, Enzyme-linked Immunosorbent Assay, Quantitative RT-PCR, Gene Expression, Cell Culture, Recombinant, Blocking Assay

    Related Articles

    Transfection:

    Article Title: TGFβ links EBV to multisystem inflammatory syndrome in children.
    Article Snippet: Six hours after transfection, cells were incubated with 10 μg ml−1 blasticitidin (ThermoFisher) for 18–48 h for positive selection of transfected cells. .. T cells and transfected HEK293T cells were serum-starved overnight prior to incubation with 10% v/v serum from patients with MIS-C for 30 min. For T cells as a control, patients’ sera were pre-incubated with antibodies directed against TGFβ1, TGFβ2 and TGFβ3 (50 μg ml−1, R&D Systems, MAB1835-SP) for 10 min. .. Cells were collected after 30 min and lysed using a buffer containing 0.5% v/v NP40 (Sigma), 150 mM NaCl, 50 mM TrisHCl, cOmplete protease inhibitors (Roche) and phosSTOP phosphatase inhibitors (Roche).

    Article Title: TGFβ links EBV to multisystem inflammatory syndrome in children
    Article Snippet: Six hours after transfection, cells were incubated with 10 μg ml −1 blasticitidin (ThermoFisher) for 18–48 h for positive selection of transfected cells. .. T cells and transfected HEK293T cells were serum-starved overnight prior to incubation with 10% v/v serum from patients with MIS-C for 30 min. For T cells as a control, patients’ sera were pre-incubated with antibodies directed against TGFβ1, TGFβ2 and TGFβ3 (50 μg ml −1 , R&D Systems, MAB1835-SP) for 10 min. .. Cells were collected after 30 min and lysed using a buffer containing 0.5% v/v NP40 (Sigma), 150 mM NaCl, 50 mM TrisHCl, cOmplete protease inhibitors (Roche) and phosSTOP phosphatase inhibitors (Roche).

    Incubation:

    Article Title: TGFβ links EBV to multisystem inflammatory syndrome in children.
    Article Snippet: Six hours after transfection, cells were incubated with 10 μg ml−1 blasticitidin (ThermoFisher) for 18–48 h for positive selection of transfected cells. .. T cells and transfected HEK293T cells were serum-starved overnight prior to incubation with 10% v/v serum from patients with MIS-C for 30 min. For T cells as a control, patients’ sera were pre-incubated with antibodies directed against TGFβ1, TGFβ2 and TGFβ3 (50 μg ml−1, R&D Systems, MAB1835-SP) for 10 min. .. Cells were collected after 30 min and lysed using a buffer containing 0.5% v/v NP40 (Sigma), 150 mM NaCl, 50 mM TrisHCl, cOmplete protease inhibitors (Roche) and phosSTOP phosphatase inhibitors (Roche).

    Article Title: TGFβ links EBV to multisystem inflammatory syndrome in children
    Article Snippet: Six hours after transfection, cells were incubated with 10 μg ml −1 blasticitidin (ThermoFisher) for 18–48 h for positive selection of transfected cells. .. T cells and transfected HEK293T cells were serum-starved overnight prior to incubation with 10% v/v serum from patients with MIS-C for 30 min. For T cells as a control, patients’ sera were pre-incubated with antibodies directed against TGFβ1, TGFβ2 and TGFβ3 (50 μg ml −1 , R&D Systems, MAB1835-SP) for 10 min. .. Cells were collected after 30 min and lysed using a buffer containing 0.5% v/v NP40 (Sigma), 150 mM NaCl, 50 mM TrisHCl, cOmplete protease inhibitors (Roche) and phosSTOP phosphatase inhibitors (Roche).

    Control:

    Article Title: TGFβ links EBV to multisystem inflammatory syndrome in children.
    Article Snippet: Six hours after transfection, cells were incubated with 10 μg ml−1 blasticitidin (ThermoFisher) for 18–48 h for positive selection of transfected cells. .. T cells and transfected HEK293T cells were serum-starved overnight prior to incubation with 10% v/v serum from patients with MIS-C for 30 min. For T cells as a control, patients’ sera were pre-incubated with antibodies directed against TGFβ1, TGFβ2 and TGFβ3 (50 μg ml−1, R&D Systems, MAB1835-SP) for 10 min. .. Cells were collected after 30 min and lysed using a buffer containing 0.5% v/v NP40 (Sigma), 150 mM NaCl, 50 mM TrisHCl, cOmplete protease inhibitors (Roche) and phosSTOP phosphatase inhibitors (Roche).

    Article Title: TGFβ links EBV to multisystem inflammatory syndrome in children
    Article Snippet: Six hours after transfection, cells were incubated with 10 μg ml −1 blasticitidin (ThermoFisher) for 18–48 h for positive selection of transfected cells. .. T cells and transfected HEK293T cells were serum-starved overnight prior to incubation with 10% v/v serum from patients with MIS-C for 30 min. For T cells as a control, patients’ sera were pre-incubated with antibodies directed against TGFβ1, TGFβ2 and TGFβ3 (50 μg ml −1 , R&D Systems, MAB1835-SP) for 10 min. .. Cells were collected after 30 min and lysed using a buffer containing 0.5% v/v NP40 (Sigma), 150 mM NaCl, 50 mM TrisHCl, cOmplete protease inhibitors (Roche) and phosSTOP phosphatase inhibitors (Roche).

    Recombinant:

    Article Title: Transforming growth factor-β isoforms drive common and distinct signaling pathways in dermal fibroblasts: insights into the signaling diversity in keloid pathogenesis
    Article Snippet: .. The medium was then replaced with serum- and antibiotic-free DMEM, and cells were stimulated with 5 ng/mL of recombinant human TGF-β1, TGF-β2, or TGFβ3 (R&D Systems, USA) in separate wells, with three biological replicates per condition. .. After 24 hours of stimulation, the total RNA was extracted from the cells using RNAiso Plus reagent (Takara, Japan) according to manufacturer’s instructions.

    other:

    Article Title: Hepatic stellate cells control liver zonation, size and functions via R-spondin 3.
    Article Snippet: The mouse hepatocyte line AML12, obtained from the American Type Culture Collection, was cultured in DMEM (Thermo Fisher Scientific, 11965118) with 10% (v/v) antibiotic–antimycotic (Gibco, 150062) and 10% (v/v) fetal bovine serum (FBS; GeminBio, 900-108) at 37 °C under 5% CO2.



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    a , Visualization of kernel density estimates of POSTN (top) and COMP (bottom) expression within a representative COL1A1 -high region. b , Heat map showing mean pixel correlations across synovial samples ( n = 4) between kernel densities for selected fibrogenic genes within COL1A1 -high regions. c , Representative examples ( n = 4 RA tissues) of COMP transcript expression in perivascular and pauci-cellular regions. Scale bar, 50 µm. d , Correlation between the percentage of fibroblasts that express COMP (count > 0) per sample and abundance of endothelial and mural cells as a percentage of total cells in the same sample. Statistics calculated by two-sided Pearson’s correlation coefficient test. Top: 4 samples analyzed with a 50-gene custom panel. Bottom: 16 samples analyzed with a 5,101-gene panel. e , Mean pixel correlations across samples ( n = 4) for TGFB isoform expression compared to selected fibroblast and endothelial markers. f , Heat map representing relative expression of TGFB isoforms across different cell types in synovial samples ( n = 17) analyzed with a 5,101-gene panel. g , Example of data in f showing TGFB transcripts overlaid on cell types. Scale bar, 50 µm. h , Immunofluorescence staining of RA synovial tissue showing protein expression of TGFβ isoforms relative to EC marker vWF. <t>TGFβ3</t> and vWF staining were performed on serial sections. i , Immunofluorescence data showing Notch3, Collagen 1 and pSMAD3 staining in perivascular and pauci-cellular regions of the RA synovium. Scale bars, 200 µm. Immunofluorescence is representative of n > 5 RA synovial tissue.
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    Image Search Results


    a , Visualization of kernel density estimates of POSTN (top) and COMP (bottom) expression within a representative COL1A1 -high region. b , Heat map showing mean pixel correlations across synovial samples ( n = 4) between kernel densities for selected fibrogenic genes within COL1A1 -high regions. c , Representative examples ( n = 4 RA tissues) of COMP transcript expression in perivascular and pauci-cellular regions. Scale bar, 50 µm. d , Correlation between the percentage of fibroblasts that express COMP (count > 0) per sample and abundance of endothelial and mural cells as a percentage of total cells in the same sample. Statistics calculated by two-sided Pearson’s correlation coefficient test. Top: 4 samples analyzed with a 50-gene custom panel. Bottom: 16 samples analyzed with a 5,101-gene panel. e , Mean pixel correlations across samples ( n = 4) for TGFB isoform expression compared to selected fibroblast and endothelial markers. f , Heat map representing relative expression of TGFB isoforms across different cell types in synovial samples ( n = 17) analyzed with a 5,101-gene panel. g , Example of data in f showing TGFB transcripts overlaid on cell types. Scale bar, 50 µm. h , Immunofluorescence staining of RA synovial tissue showing protein expression of TGFβ isoforms relative to EC marker vWF. TGFβ3 and vWF staining were performed on serial sections. i , Immunofluorescence data showing Notch3, Collagen 1 and pSMAD3 staining in perivascular and pauci-cellular regions of the RA synovium. Scale bars, 200 µm. Immunofluorescence is representative of n > 5 RA synovial tissue.

    Journal: Nature Immunology

    Article Title: Spatial patterning of fibroblast TGFβ signaling underlies treatment resistance in rheumatoid arthritis

    doi: 10.1038/s41590-025-02386-2

    Figure Lengend Snippet: a , Visualization of kernel density estimates of POSTN (top) and COMP (bottom) expression within a representative COL1A1 -high region. b , Heat map showing mean pixel correlations across synovial samples ( n = 4) between kernel densities for selected fibrogenic genes within COL1A1 -high regions. c , Representative examples ( n = 4 RA tissues) of COMP transcript expression in perivascular and pauci-cellular regions. Scale bar, 50 µm. d , Correlation between the percentage of fibroblasts that express COMP (count > 0) per sample and abundance of endothelial and mural cells as a percentage of total cells in the same sample. Statistics calculated by two-sided Pearson’s correlation coefficient test. Top: 4 samples analyzed with a 50-gene custom panel. Bottom: 16 samples analyzed with a 5,101-gene panel. e , Mean pixel correlations across samples ( n = 4) for TGFB isoform expression compared to selected fibroblast and endothelial markers. f , Heat map representing relative expression of TGFB isoforms across different cell types in synovial samples ( n = 17) analyzed with a 5,101-gene panel. g , Example of data in f showing TGFB transcripts overlaid on cell types. Scale bar, 50 µm. h , Immunofluorescence staining of RA synovial tissue showing protein expression of TGFβ isoforms relative to EC marker vWF. TGFβ3 and vWF staining were performed on serial sections. i , Immunofluorescence data showing Notch3, Collagen 1 and pSMAD3 staining in perivascular and pauci-cellular regions of the RA synovium. Scale bars, 200 µm. Immunofluorescence is representative of n > 5 RA synovial tissue.

    Article Snippet: For measurement of pro-collagen 1 alpha 1 (R&D Systems, DY6220-05), COMP (R&D Systems, DY3134) or POSTN (R&D Systems, DY3548B), TGFβ1 (R&D Systems, DY240-05), TGFβ2 (R&D Systems, DY302) and TGFβ3 (DY243) plates were incubated overnight at 4 °C with diluted capture antibody solution, washed with PBS-T and blocked for 1 h at 20 °C in blocking buffer (1% BSA in PBS).

    Techniques: Expressing, Immunofluorescence, Staining, Marker

    a , UMAP projection and dotplots of single-cell 3D co-culture data displaying COMP and POSTN expression . b , Chip-seq analysis of RBPJ binding to TGFBR2 (-350 bp to -138 bp upstream of transcriptional start site (TSS)) and TGFBR3 (-185 bp to +84 bp relative to TSS) promoter regions in HepG2 cells (data from GSE127388 ). Significant peaks are annotated with blue bars and were called using the narrowPeak function in MACS2. c , ELISA quantification of supernatant TGFβ1, TGFβ2, and TGFβ3 from fibroblasts stimulated with DLL4 for 3 days. d , RT-qPCR analysis of TGFβ isoform and fibrogenic gene expression in unstimulated or DLL4-stimulated fibroblasts (72 h) and fibroblasts that were exposed to conditioned media from unstimulated and DLL4-stimulated fibroblasts for 72 h. e , RT-qPCR analysis of TGFβ isoform and fibrogenic gene expression in unstimulated or DLL4-stimulated cells plated in the bottom chamber and fibroblasts that were cultured in the above transwell insert for 72 h. f , Top: ELISA quantification of supernatant COMP, PRO-COL1 and POSTN from fibroblasts stimulated with or without recombinant TGFβ1 (10 ng ml−1) and with or without a pan-TGFβ blocking antibody (5 ug mL-1) for 72 h. Below: ELISA quantification of COMP, PRO-COL1 and POSTN on unstimulated or DLL4-stimulated fibroblasts that were treated with antibodies against individual or pan-TGFβ isoforms (5 ug mL-1) for 72 h. g , RT-qPCR analysis of fibroblasts with or without DLL4 stimulation that were treated with siRNA (20 nM) for 96 h. h-i , ELISA quantification of fibroblast production of PRO-COL1, POSTN and COMP over 24 h after 72 h treatment with indicated siRNA combinations (10 nM each) with or without DLL4 stimulation. For c-i Data are shown as mean +/- s.d with n = 3 biological replicates, and representative of at least two independent experiments. Statistical analysis was performed by unpaired two-sided t-test.

    Journal: Nature Immunology

    Article Title: Spatial patterning of fibroblast TGFβ signaling underlies treatment resistance in rheumatoid arthritis

    doi: 10.1038/s41590-025-02386-2

    Figure Lengend Snippet: a , UMAP projection and dotplots of single-cell 3D co-culture data displaying COMP and POSTN expression . b , Chip-seq analysis of RBPJ binding to TGFBR2 (-350 bp to -138 bp upstream of transcriptional start site (TSS)) and TGFBR3 (-185 bp to +84 bp relative to TSS) promoter regions in HepG2 cells (data from GSE127388 ). Significant peaks are annotated with blue bars and were called using the narrowPeak function in MACS2. c , ELISA quantification of supernatant TGFβ1, TGFβ2, and TGFβ3 from fibroblasts stimulated with DLL4 for 3 days. d , RT-qPCR analysis of TGFβ isoform and fibrogenic gene expression in unstimulated or DLL4-stimulated fibroblasts (72 h) and fibroblasts that were exposed to conditioned media from unstimulated and DLL4-stimulated fibroblasts for 72 h. e , RT-qPCR analysis of TGFβ isoform and fibrogenic gene expression in unstimulated or DLL4-stimulated cells plated in the bottom chamber and fibroblasts that were cultured in the above transwell insert for 72 h. f , Top: ELISA quantification of supernatant COMP, PRO-COL1 and POSTN from fibroblasts stimulated with or without recombinant TGFβ1 (10 ng ml−1) and with or without a pan-TGFβ blocking antibody (5 ug mL-1) for 72 h. Below: ELISA quantification of COMP, PRO-COL1 and POSTN on unstimulated or DLL4-stimulated fibroblasts that were treated with antibodies against individual or pan-TGFβ isoforms (5 ug mL-1) for 72 h. g , RT-qPCR analysis of fibroblasts with or without DLL4 stimulation that were treated with siRNA (20 nM) for 96 h. h-i , ELISA quantification of fibroblast production of PRO-COL1, POSTN and COMP over 24 h after 72 h treatment with indicated siRNA combinations (10 nM each) with or without DLL4 stimulation. For c-i Data are shown as mean +/- s.d with n = 3 biological replicates, and representative of at least two independent experiments. Statistical analysis was performed by unpaired two-sided t-test.

    Article Snippet: For measurement of pro-collagen 1 alpha 1 (R&D Systems, DY6220-05), COMP (R&D Systems, DY3134) or POSTN (R&D Systems, DY3548B), TGFβ1 (R&D Systems, DY240-05), TGFβ2 (R&D Systems, DY302) and TGFβ3 (DY243) plates were incubated overnight at 4 °C with diluted capture antibody solution, washed with PBS-T and blocked for 1 h at 20 °C in blocking buffer (1% BSA in PBS).

    Techniques: Single Cell, Co-Culture Assay, Expressing, ChIP-sequencing, Binding Assay, Enzyme-linked Immunosorbent Assay, Quantitative RT-PCR, Gene Expression, Cell Culture, Recombinant, Blocking Assay

    a , Visualization of kernel density estimates of POSTN (top) and COMP (bottom) expression within a representative COL1A1 -high region. b , Heat map showing mean pixel correlations across synovial samples ( n = 4) between kernel densities for selected fibrogenic genes within COL1A1 -high regions. c , Representative examples ( n = 4 RA tissues) of COMP transcript expression in perivascular and pauci-cellular regions. Scale bar, 50 µm. d , Correlation between the percentage of fibroblasts that express COMP (count > 0) per sample and abundance of endothelial and mural cells as a percentage of total cells in the same sample. Statistics calculated by two-sided Pearson’s correlation coefficient test. Top: 4 samples analyzed with a 50-gene custom panel. Bottom: 16 samples analyzed with a 5,101-gene panel. e , Mean pixel correlations across samples ( n = 4) for TGFB isoform expression compared to selected fibroblast and endothelial markers. f , Heat map representing relative expression of TGFB isoforms across different cell types in synovial samples ( n = 17) analyzed with a 5,101-gene panel. g , Example of data in f showing TGFB transcripts overlaid on cell types. Scale bar, 50 µm. h , Immunofluorescence staining of RA synovial tissue showing protein expression of TGFβ isoforms relative to EC marker vWF. TGFβ3 and vWF staining were performed on serial sections. i , Immunofluorescence data showing Notch3, Collagen 1 and pSMAD3 staining in perivascular and pauci-cellular regions of the RA synovium. Scale bars, 200 µm. Immunofluorescence is representative of n > 5 RA synovial tissue.

    Journal: Nature Immunology

    Article Title: Spatial patterning of fibroblast TGFβ signaling underlies treatment resistance in rheumatoid arthritis

    doi: 10.1038/s41590-025-02386-2

    Figure Lengend Snippet: a , Visualization of kernel density estimates of POSTN (top) and COMP (bottom) expression within a representative COL1A1 -high region. b , Heat map showing mean pixel correlations across synovial samples ( n = 4) between kernel densities for selected fibrogenic genes within COL1A1 -high regions. c , Representative examples ( n = 4 RA tissues) of COMP transcript expression in perivascular and pauci-cellular regions. Scale bar, 50 µm. d , Correlation between the percentage of fibroblasts that express COMP (count > 0) per sample and abundance of endothelial and mural cells as a percentage of total cells in the same sample. Statistics calculated by two-sided Pearson’s correlation coefficient test. Top: 4 samples analyzed with a 50-gene custom panel. Bottom: 16 samples analyzed with a 5,101-gene panel. e , Mean pixel correlations across samples ( n = 4) for TGFB isoform expression compared to selected fibroblast and endothelial markers. f , Heat map representing relative expression of TGFB isoforms across different cell types in synovial samples ( n = 17) analyzed with a 5,101-gene panel. g , Example of data in f showing TGFB transcripts overlaid on cell types. Scale bar, 50 µm. h , Immunofluorescence staining of RA synovial tissue showing protein expression of TGFβ isoforms relative to EC marker vWF. TGFβ3 and vWF staining were performed on serial sections. i , Immunofluorescence data showing Notch3, Collagen 1 and pSMAD3 staining in perivascular and pauci-cellular regions of the RA synovium. Scale bars, 200 µm. Immunofluorescence is representative of n > 5 RA synovial tissue.

    Article Snippet: Membranes were blocked for 15 min in Everyblot blocking buffer (Bio-Rad, 12010020), then incubated overnight at 4 °C with primary antibodies against TGFβ1 (Proteintech, 21898-1-AP; 1:500 dilution), TGFβ2 (Proteintech, 19999-1-AP; 1:500 dilution), TGFβ3 (Proteintech, 18942-1-AP; 1:500 dilution), TGFβR1 (RD Biosciences, AF3025; 1:300 dilution), TGFβR2 (Bioss, bs-0117R; 1:500 dilution), TGFβR3 (Cell Signaling Technology, 2519S; 1:500 dilution), GAPDH (Thermo Fisher Scientific, MA5-15738) or beta-actin (Cell Signaling Technology, 3700).

    Techniques: Expressing, Immunofluorescence, Staining, Marker

    a , UMAP projection and dotplots of single-cell 3D co-culture data displaying COMP and POSTN expression . b , Chip-seq analysis of RBPJ binding to TGFBR2 (-350 bp to -138 bp upstream of transcriptional start site (TSS)) and TGFBR3 (-185 bp to +84 bp relative to TSS) promoter regions in HepG2 cells (data from GSE127388 ). Significant peaks are annotated with blue bars and were called using the narrowPeak function in MACS2. c , ELISA quantification of supernatant TGFβ1, TGFβ2, and TGFβ3 from fibroblasts stimulated with DLL4 for 3 days. d , RT-qPCR analysis of TGFβ isoform and fibrogenic gene expression in unstimulated or DLL4-stimulated fibroblasts (72 h) and fibroblasts that were exposed to conditioned media from unstimulated and DLL4-stimulated fibroblasts for 72 h. e , RT-qPCR analysis of TGFβ isoform and fibrogenic gene expression in unstimulated or DLL4-stimulated cells plated in the bottom chamber and fibroblasts that were cultured in the above transwell insert for 72 h. f , Top: ELISA quantification of supernatant COMP, PRO-COL1 and POSTN from fibroblasts stimulated with or without recombinant TGFβ1 (10 ng ml−1) and with or without a pan-TGFβ blocking antibody (5 ug mL-1) for 72 h. Below: ELISA quantification of COMP, PRO-COL1 and POSTN on unstimulated or DLL4-stimulated fibroblasts that were treated with antibodies against individual or pan-TGFβ isoforms (5 ug mL-1) for 72 h. g , RT-qPCR analysis of fibroblasts with or without DLL4 stimulation that were treated with siRNA (20 nM) for 96 h. h-i , ELISA quantification of fibroblast production of PRO-COL1, POSTN and COMP over 24 h after 72 h treatment with indicated siRNA combinations (10 nM each) with or without DLL4 stimulation. For c-i Data are shown as mean +/- s.d with n = 3 biological replicates, and representative of at least two independent experiments. Statistical analysis was performed by unpaired two-sided t-test.

    Journal: Nature Immunology

    Article Title: Spatial patterning of fibroblast TGFβ signaling underlies treatment resistance in rheumatoid arthritis

    doi: 10.1038/s41590-025-02386-2

    Figure Lengend Snippet: a , UMAP projection and dotplots of single-cell 3D co-culture data displaying COMP and POSTN expression . b , Chip-seq analysis of RBPJ binding to TGFBR2 (-350 bp to -138 bp upstream of transcriptional start site (TSS)) and TGFBR3 (-185 bp to +84 bp relative to TSS) promoter regions in HepG2 cells (data from GSE127388 ). Significant peaks are annotated with blue bars and were called using the narrowPeak function in MACS2. c , ELISA quantification of supernatant TGFβ1, TGFβ2, and TGFβ3 from fibroblasts stimulated with DLL4 for 3 days. d , RT-qPCR analysis of TGFβ isoform and fibrogenic gene expression in unstimulated or DLL4-stimulated fibroblasts (72 h) and fibroblasts that were exposed to conditioned media from unstimulated and DLL4-stimulated fibroblasts for 72 h. e , RT-qPCR analysis of TGFβ isoform and fibrogenic gene expression in unstimulated or DLL4-stimulated cells plated in the bottom chamber and fibroblasts that were cultured in the above transwell insert for 72 h. f , Top: ELISA quantification of supernatant COMP, PRO-COL1 and POSTN from fibroblasts stimulated with or without recombinant TGFβ1 (10 ng ml−1) and with or without a pan-TGFβ blocking antibody (5 ug mL-1) for 72 h. Below: ELISA quantification of COMP, PRO-COL1 and POSTN on unstimulated or DLL4-stimulated fibroblasts that were treated with antibodies against individual or pan-TGFβ isoforms (5 ug mL-1) for 72 h. g , RT-qPCR analysis of fibroblasts with or without DLL4 stimulation that were treated with siRNA (20 nM) for 96 h. h-i , ELISA quantification of fibroblast production of PRO-COL1, POSTN and COMP over 24 h after 72 h treatment with indicated siRNA combinations (10 nM each) with or without DLL4 stimulation. For c-i Data are shown as mean +/- s.d with n = 3 biological replicates, and representative of at least two independent experiments. Statistical analysis was performed by unpaired two-sided t-test.

    Article Snippet: Membranes were blocked for 15 min in Everyblot blocking buffer (Bio-Rad, 12010020), then incubated overnight at 4 °C with primary antibodies against TGFβ1 (Proteintech, 21898-1-AP; 1:500 dilution), TGFβ2 (Proteintech, 19999-1-AP; 1:500 dilution), TGFβ3 (Proteintech, 18942-1-AP; 1:500 dilution), TGFβR1 (RD Biosciences, AF3025; 1:300 dilution), TGFβR2 (Bioss, bs-0117R; 1:500 dilution), TGFβR3 (Cell Signaling Technology, 2519S; 1:500 dilution), GAPDH (Thermo Fisher Scientific, MA5-15738) or beta-actin (Cell Signaling Technology, 3700).

    Techniques: Single Cell, Co-Culture Assay, Expressing, ChIP-sequencing, Binding Assay, Enzyme-linked Immunosorbent Assay, Quantitative RT-PCR, Gene Expression, Cell Culture, Recombinant, Blocking Assay

    Effects of hypoxic environment on ER stress response, autophagy pathway, and AMFR-FAM134B axis regulation in OS cells. Note: ( A ) Experimental flowchart showing the design and treatment process under hypoxic and normoxic conditions. ( B ) mRFP-GFP-LC3 fluorescence reporter system detection of changes in autophagosome and autolysosome numbers in OS cells (bar = 15 μm). ( C-E ) Western blot analysis of autophagy-related proteins, LC3-II/I ratio, and P62 expression levels in OS cells. ( F-J ) Western blot analysis of ER stress markers, GRP78, CHOP, FAM134B, and AMFR protein expression and quantitative analysis. ( K ) Ubiquitination pull-down assay detecting FAM134B ubiquitination levels. ( L ) Co-IP assay detecting the binding ability of FAM134B with LC3B-II. All data are expressed as mean ± SD. Comparisons between two groups were performed using an independent-samples t-test, while comparisons among multiple groups were conducted using ANOVA. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001

    Journal: Journal of Nanobiotechnology

    Article Title: AMFR-mediated ER-phagy regulation and therapeutic targeting in osteosarcoma: a multifunctional nanoplatform strategy

    doi: 10.1186/s12951-025-03754-8

    Figure Lengend Snippet: Effects of hypoxic environment on ER stress response, autophagy pathway, and AMFR-FAM134B axis regulation in OS cells. Note: ( A ) Experimental flowchart showing the design and treatment process under hypoxic and normoxic conditions. ( B ) mRFP-GFP-LC3 fluorescence reporter system detection of changes in autophagosome and autolysosome numbers in OS cells (bar = 15 μm). ( C-E ) Western blot analysis of autophagy-related proteins, LC3-II/I ratio, and P62 expression levels in OS cells. ( F-J ) Western blot analysis of ER stress markers, GRP78, CHOP, FAM134B, and AMFR protein expression and quantitative analysis. ( K ) Ubiquitination pull-down assay detecting FAM134B ubiquitination levels. ( L ) Co-IP assay detecting the binding ability of FAM134B with LC3B-II. All data are expressed as mean ± SD. Comparisons between two groups were performed using an independent-samples t-test, while comparisons among multiple groups were conducted using ANOVA. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001

    Article Snippet: Subsequently, cells were incubated overnight at 4 °C with primary antibodies targeting AMFR (#sc-166861, 1:100, Santa Cruz, USA), LC3B (#ab192890, 1:100, Abcam, UK), and P62 (#ab91526, 1:500, Abcam, UK).

    Techniques: Fluorescence, Western Blot, Expressing, Ubiquitin Proteomics, Pull Down Assay, Co-Immunoprecipitation Assay, Binding Assay

    AMFR knockdown regulates ER-phagy flux and cellular stress response by inhibiting FAM134B ubiquitination. Note: ( A ) Experimental design flowchart. ( B ) Ubiquitination pull-down assay detecting FAM134B ubiquitination levels. ( C ) Co-IP assay detecting the binding ability of FAM134B with LC3B. ( D-E ) Western blot analysis detecting ER-phagy flux (LC3-II/I, P62) and expression levels of ER stress markers (CHOP, GRP78). ( F ) TEM observation of changes in ER structure following AMFR knockdown (bar = 500 nm). ( G ) ER-Tracker staining detecting changes in ER area and fluorescence intensity (bar = 15 μm). ( H ) mRFP-GFP-LC3 fluorescence reporter system detecting dynamic changes in ER-phagy flux (bar = 15 μm). ( I ) DCF-DA staining detecting intracellular ROS levels (bar = 15 μm). ( J ) Intracellular ROS levels measured using EPR. ( K ) Antioxidant enzyme activity detection shows changes in GSH/GSSG ratio, SOD, and CAT enzyme activities. All data are presented as mean ± SD, with experiments repeated three times. Comparisons between two groups were performed using an independent-samples t-test. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001

    Journal: Journal of Nanobiotechnology

    Article Title: AMFR-mediated ER-phagy regulation and therapeutic targeting in osteosarcoma: a multifunctional nanoplatform strategy

    doi: 10.1186/s12951-025-03754-8

    Figure Lengend Snippet: AMFR knockdown regulates ER-phagy flux and cellular stress response by inhibiting FAM134B ubiquitination. Note: ( A ) Experimental design flowchart. ( B ) Ubiquitination pull-down assay detecting FAM134B ubiquitination levels. ( C ) Co-IP assay detecting the binding ability of FAM134B with LC3B. ( D-E ) Western blot analysis detecting ER-phagy flux (LC3-II/I, P62) and expression levels of ER stress markers (CHOP, GRP78). ( F ) TEM observation of changes in ER structure following AMFR knockdown (bar = 500 nm). ( G ) ER-Tracker staining detecting changes in ER area and fluorescence intensity (bar = 15 μm). ( H ) mRFP-GFP-LC3 fluorescence reporter system detecting dynamic changes in ER-phagy flux (bar = 15 μm). ( I ) DCF-DA staining detecting intracellular ROS levels (bar = 15 μm). ( J ) Intracellular ROS levels measured using EPR. ( K ) Antioxidant enzyme activity detection shows changes in GSH/GSSG ratio, SOD, and CAT enzyme activities. All data are presented as mean ± SD, with experiments repeated three times. Comparisons between two groups were performed using an independent-samples t-test. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001

    Article Snippet: Subsequently, cells were incubated overnight at 4 °C with primary antibodies targeting AMFR (#sc-166861, 1:100, Santa Cruz, USA), LC3B (#ab192890, 1:100, Abcam, UK), and P62 (#ab91526, 1:500, Abcam, UK).

    Techniques: Knockdown, Ubiquitin Proteomics, Pull Down Assay, Co-Immunoprecipitation Assay, Binding Assay, Western Blot, Expressing, Staining, Fluorescence, Activity Assay