Extended Data Fig. 5: Purification and analysis of aorta-associated cells from adult animals (Related to Fig. 6).
From: Defining the lineage of thermogenic perivascular adipose tissue
a, Expression dot plot of indicated genes in cell clusters from adult aorta. b, mRNA in situ hybridization of Ly6a (green) in adult aorta. Arrowhead in inset shows progenitor cell. DAPI (blue) stains nuclei. (scale bar, 362.3 μm). Lu: lumen. Representative of n=2 experiments. c, mRNA in situ hybridization of Pi16 (green) and Bace2 (red). White arrowhead shows progenitor cell. Yellow arrowhead shows intermediate cell (scale bar, 145 μm). Representative of n=2 experiments. d, UMAPs showing Cd81 and Prdm16 expression. e, Violin plots showing expression of indicated genes used for sorting. f, FACS isolation of fibroblasts and smooth muscle cells (SMCs). To exclude debris and isolate single live cells, we gated on: (1) SSC-A and FSC-A ; (2) FSC-H vs FSC-W; and (3) SSC-H vs SSC-W. We then gated on Live (DAPI-); Lin- (CD45-,CD31-,TER119-) cells. Further selection was used to isolate: Intermediate Cells [PDGFRa+, MCAM(-), CD200+], Progenitors [PDGFRa+, MCAM(-), CD200(-)], SMC1 [PDGFRa(-), MCAM+, CD200+], SMC2 [PDGFRa(-), MCAM+, CD200(-)]. (Representative image of 6 separate experiments). Related to experiments shown in Figs. 6d and 7a,c. g, Expression of Seurat cluster-defining genes in the sorted cell bulk RNAseq datasets. Gene levels in sorted cell populations were compared to the row mean and annotated as “enriched” or “de-enriched” (Log2 FC>0.25). h,i, Expression heatmap of Seurat cluster-defining genes mapped on to sorted-cell RNASeq results for fibroblast (H) and SMC populations (I) (n=3).
