Fig. 3: Single-cell RNA sequencing reveals fibroblast subsets with distinct ferroptosis sensitivity.

a tSNE plot displaying 4735 CD45− cells from 3 STIA mice separated into 7 major cell types, including fibroblasts (9 clusters), vasculature (2 clusters), pericytes (1 cluster), chondrocytes (1 cluster), muscle cells (1 cluster), osteoblasts (2 clusters), and erythrocytes (2 clusters). b Heatmap showing the expression of high-risk ferroptosis genes in fibroblast clusters in the inflamed STIA joints. c Predicted ferroptosis-sensitive and ferroptosis-resistant fibroblast subsets based on ferroptosis-related genes listed in (b). d Expression of identified conserved marker genes in the ferroptosis-sensitive and ferroptosis-resistant fibroblast subsets. e Left, representative fluorescent multiplex IHC staining and quantification of inflamed joints of CIA mice labeled with anti-Mfap4 (red), anti-Sparcl1 (purple), anti-FAPα (green), and DAPI (blue). Mfap4^low, n = 9905 cells from 10 inflamed joints. Mfap4^high, n = 3336 cells from 10 inflamed joints. Right, representative fluorescent multiplex IHC staining (top panel) and quantification (bottom panel) of hyperplastic synovium of RA patients labeled with anti-Mfap4 (green), anti-Sparcl1 (red), anti-FAPα (orange), and DAPI (blue). Mfap4^low, n = 8222 cells from 10 independent human synovium samples. Mfap4^high, n = 6730 cells from 10 independent human synovium samples. Scale bars, 200 μm. ****P < 0.0001; two-tailed t-test. f Quantification of the mean fluorescence intensity (MFI) of Mfap4 (top) and Sparcl1 (bottom) in hyperplastic tissue invading cartilage and in the remaining tissue. ****P < 0.0001; two-tailed t-test. Cartilage invaded area, n = 684 cells from 3 inflamed joints of CIA mice. Non-invaded area, n = 1257 cells from 3 inflamed joints of CIA mice. g Representative immunofluorescent staining of inflamed joints of CIA model mice with or without treatment with a low dose of IKE and labeled with anti-Mfap4, anti-Sparcl1, anti-FAPα, and DAPI. Similar results were observed from 15 joints tested for each group. Scale bars, 100 μm. h Enrichment plots of the GO_Proteinaceous extracellular matrix gene set (left) and KEGG_Glycosaminoglycan biosynthesis gene set (right) among the ferroptosis-sensitive subsets identified by GSEA. The p-value is calculated through permutation tests. i Enrichment plots of the GO_Cell division gene set (left) and KEGG_DNA replication gene set (right) among the ferroptosis-resistant subset identified by GSEA. The p-value is calculated through permutation tests. j Expression of selected mRNA transcripts in the TNF pathway gene set among ferroptosis-sensitive and ferroptosis-resistant subsets. Source data are provided as a Source data file.