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