Extended Data Fig. 6: Mesenchymal pathway activity and role of AP-1.
From: Decoding myofibroblast origins in human kidney fibrosis
a, KEGG pathway enrichment analysis along pseudotime for lineage 1 (see Fig. 2c.) b, Top, gene expression dynamics along pseudotime for lineage 2 (fibroblasts to myofibroblasts, see Fig. 2c). Cells (in columns) were ordered along pseudotime and genes (in rows) that correlate with pseudotime were selected and plotted along pseudotime (Methods). Each 10 cells were averaged in one column. Genes were grouped signifying their pseudotime expression pattern. Selected example genes for each group are indicated. See Supplementary Data 3 for gene cluster assignments. Bottom, cell cycle stage along pseudotime as percentage of each 500 cells along pseudotime. c, As in b but for lineage 3 cells (see Fig. 2c). d, PID signalling pathway enrichment analysis along pseudotime for lineage 2 cells ordered along pseudotime as in b. e, KEGG pathway enrichment analysis along pseudotime for lineage 2. f, As in d. but for lineage 3 cells (see Fig. 2c). g, As in e but for lineage 3 cells. h–k, Violin plots across mesenchymal cells types of selected genes of the human PDGFRβ+ dataset in Fig. 2. l, Transcription factor scores for proximal promoter regions (l) and distal regions (m) obtained by transcription factors equence motif enrichment analysis for top marker genes for the mesenchymal cell clusters of the human PDGFRβ+ dataset (Methods). Note enrichment of Fos and Jun motifs in promoters of fibroblast marker genes. m, Schematic of human kidney PDGFRβ+ cell generation and immortalization. n, Cell proliferation (WST-1) and expression of FOS, COL1A1, POSTN and OGN in immortalized human PDGFRβ kidney cells by RNA qPCR after treatment with the AP-1 inhibitor T-5224 and/or TGFβ. n = 3 per group. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, one-way ANOVA followed by Bonferroni’ post hoc test. Data are mean ± s.d. o, Expression of OGN (Fib1+3) and POSTN (MF1) visualized on the same UMAP embedding from Extended Data Fig. 5b. p, AP-1 average transcription factor expression (left) and average expression of putative AP-1-regulated genes (right) against collagen scores stratified by fibroblast and myofibroblast cells. Notably, the expression of AP-1 anti-correlates with collagen score but the expression of its target genes positively correlates with collagen score, potentially pointing towards an inhibitory role for AP-1. q, The number of statistically significant receptor–ligand interactions between mesenchymal cells and all other cell types (CD10− fraction; Fig. 1) according to CellphoneDB Analysis. Dendritic cells, monocytes, myofibroblasts, podocytes, arteriolar endothelial cells and injured tubules as the main sources of signalling ligands to pericytes fibroblasts and myofibroblasts. r, Dot plot for significant ligand–receptor interactions from the selected signalling pathways EGFR, PDGF, WNT, TGFβ, Notch and Hedgehog for pericytes, fibroblast and myofibroblasts. Interacting ligand–receptor and cell types are shown by pairs. The first cell type of the interacting pair expresses the ligand and the second cell type expresses the receptor (that is, first and second proteins from the interaction, respectively). Ligand–receptor interactions are grouped by signalling pathways. Yellow: EGFR, pink: PDGF, green: WNT, red: TGFβ, black: Notch, blue (light or dark): mixed of TGFβ and EGFR. None of the hedgehog interactions were significant. For details on statistics and reproducibility, see Methods.
