Extended Data Fig. 5: ImP induces transcriptional changes in fibroblasts, macrophages and endothelial cells.
From: Imidazole propionate is a driver and therapeutic target in atherosclerosis
a-g, Chow-fed ApoE−/− mice were administered ImP and the aorta-derived cells from the scRNAseq were analysed. a-c, Dot plot of expression levels of cell type markers used to identify cell subclusters for macrophages (MFs) (a), fibroblasts (FBs) (b), and endothelial cells (ECs) (c). d-f, Table of representative gene ontology (GO) terms enriched in the subclusters of MFs (d), FBs (e) and ECs (f). All shown GO terms are significant (adjusted p-value ≤ 0.05). g, Relative abundance (log-ratio) of the subcluster cell proportions comparing ImP treatment vs CTR, regardless of the treatment duration. h, MCP1 concentration measured by ELISA in the supernatant of MEFs treated with ImP for 24 h. n = 10. i, Number of post-migration monocytes collected in the bottom chamber in response to supernatant from (h) and quantified by flow cytometry. n = 7. j, Heat map of gene set enrichment analysis (GSEA) from RNAseq analysis of bone marrow-derived macrophages (BMDMs), mouse embryonic fibroblasts (MEFs) and mouse aortic ECs (MAECs) comparing ImP stimulation after 1 h and 2 h vs unstimulated conditions within each cell type. k, GSEA enrichment plots of phosphopeptides related to the mTOR pathway from BMDMs (top) and MEFs (down) after ImP treatment for 180 min compared with unstimulated cells. Global abundances of all peptides related to the mTOR pathway were used for the analysis. l,m, BMDMs (left) and MEFs (right) after ImP stimulation for 24 h with or without rapamycin co-incubation. l, Flow cytometry staining for pS6 ribosomal protein. Left: BMDM n = 8; MEF CTR = 10; ImP=10; ImP + AGN = 9; Right: representative histograms for CTR (top), ImP (middle) and ImP + rapamycin (bottom). m,TNF production measured by ELISA. BMDM n = 7; MEF n = 9 n, Flow cytometry staining for pS6 in peritoneal macrophages harvested from chow-fed ApoE−/− mice administered ImP for 8 weeks. Left: n = 9. Right: representative histograms for CTR (top), ImP (bottom). o, ImP was administered (ImP) or not (CTR) to Ldlr−/− mice grafted with BM from control Raptorf/f (CTR = 17; ImP=18) or Lyz2ΔRaptor (CTR = 15; ImP=18) and fed chow diet for 12 weeks. Quantification of atherosclerotic lesions by Oil red O en face staining of the aorta, showing quantification in whole aorta. a-c, Wilcoxon rank sum test comparing cell clusters. Adjusted p-value < 0.05. g, Two-proportions Z-test. h,i, Data are pooled from n = 7 independent experiments. h, Two-tailed Unpaired Student’s t test. i, Two-tailed Mann–Whitney U test. j, Kolmogorov Smirnov test. k, Nominal p-values were calculated using a 1000-permutation test. l,m,o, Individual data (each one represent an independent experiment) and mean ± SEM. One-way ANOVA with Tukey post-hoc correction. n, Individual data and mean ± SEM of at least two pooled independent experiments. Two tailed unpaired Student’s t test between CTR and ImP. *p < 0.05; **p < 0.01; ***p < 0.005; ****p < 0.001.
