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

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

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

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

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

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