Figure 6. SMCs with downregulated contractile and synthetic molecules are characterized by markers of degradative organelles and cellular activation.

Myh11-CreER^T2 mT/mG (Tsc1^+/+) and Tsc1^fl/fl Myh11-CreER^T2 mT/mG (Tsc1^−/−) mice were treated with tamoxifen at 1.5 weeks and the thoracic aortas and isolated GFP⁺ SMCs were analyzed at 24 weeks. (A) Multidimensional scaling (MDS) of bulk RNA-seq data (n = 4) shows clear delineation of Tsc1^+/+ (circular symbols) from Tsc1^−/− (triangular symbols) experimental conditions (Exp Cond). (B) T-distributed stochastic neighbor embedding (tSNE) of single-cell RNA-seq data identifying 13 cell clusters by a deep learning framework using a filtered data matrix of 2,788 cells by 8,645 genes shows clear delineation of Tsc1^+/+ (circular symbols) from Tsc1^−/− (triangular symbols) experimental conditions. (C) Heatmaps reflecting bulk and single-cell RNA expression changes for genes of interest representing contractile, synthetic, innate immunity, lysosome, other degradative organelles (autophagosomes, endosomes, and phagosomes), and cell activation (proteases, adhesion molecules, and cytokines) phenotypes. There was coordinated upregulation or downregulation of functionally related genes among Tsc1^+/+ (blue header bars) versus Tsc1^−/− (red header bars) aortas (numbered i–iv for each experimental condition) and SMC clusters (numbered 0–12).