Extended Data Fig. 4: NOTCH3 signalling in synovial fibroblasts and mural cells.

a, b, Expression of NOTCH1 and NOTCH3 in synovial stromal cells as assessed by scRNA-seq. a, Expression of NOTCH1 (top) and NOTCH3 (bottom) in lining fibroblasts (orange), mural cells (red), sublining fibroblasts (green) and endothelial cells (blue) from the human primary synovial tissue scRNA-seq dataset (n = 35,153 cells from the synovial tissues of 6 patients with rheumatoid arthritis and 6 patients with osteoarthritis). b, Mural cells were subdivided into two major mural subpopulations: vascular smooth muscle cells (orange) and pericytes (blue). No difference in the expression of NOTCH3 was observed (Wilcoxon rank-sum test P = 0.86) between these two subpopulations. c, Representative synovial tissue sections (n = 6) showing RNAscope staining for NOTCH3 in purple, immunohistochemistry staining (IHC) for CD90 in brown and nuclei in blue. The arteriole is marked with a triangle. d, The mean fluorescence intensity (MFI) of NOTCH3 in synovial mural cells (n = 18), sublining fibroblasts (n = 40), lining fibroblasts (n = 20), endothelial cells (n = 15) and leukocytes (n = 19). Box plots summarize the median, interquartile range, and 95% quantile range, and significance is determined by Spearman correlation. e, Representative immunohistochemistry image showing the staining of the NOTCH3 intracellular ___domain (NOTCH3-ICD) in the synovial tissue of a patient with rheumatoid arthritis (n = 3). A, arterial endothelium; V, venous endothelium. f, Immunoblot of synovial fibroblast lysates treated with antibodies against the NOTCH3 intracellular ___domain (NICD3, top) or GAPDH (bottom). The treatments were as follows: 10 mmol EDTA (Notch activation), plate-coated DLL4 (5 μg ml⁻¹), or plate-coated JAG1 (5 μg ml⁻¹), in the presence or absence of 10 μM DAPT (γ-secretase inhibitor). n = 3 independent experiments. For gel source data, see Supplementary Data 1.

Source data