CD45^–GFP⁺ MVPCs were sorted from WT or Tsc2KD mouse lungs and cultured to establish primary cell lines from matched WT (2 cell lines), mTOR regulated (1 cell line), and mTOR+ (1 cell line). Each independent cell line was analyzed in triplicate using bulk RNA-Seq. (A) Heatmap comparisons of DEGs with an average normalized expression above 2 and a log₂ fold change greater than 1 in either direction are represented. There are 224 significantly differentially expressed genes (KD vs. WT) in the mTOR+ comparison and 887 significantly differentially expressed genes (KD vs. WT) in the Tsc2-regulated comparison, log fold-change (lfc) ≥ 1. A Venn diagram of DEGs from mTOR-activated (mTOR+) MVPCs relative to mTOR-regulated (mTOR=) MVPCs is shown. (B) Functional interaction network of mTOR-regulated DEGs. The STRING database of protein-protein interactions was used to derive the interactions. The shapes of nodes in the network correspond to functions of pathways, while the colors are scaled to biological significance score (61) of the respective gene (log₂ fold change × –log₁₀ adjusted P value). Genes identified as unique regulators are gray. mTOR, gray; p53, red; Tsc2, yellow. All interactions shown here have STRING scores of 0.4 or above, representing medium confidence or higher in the evidence of interaction. (C) Dot plot of Reactome functional categories that were significantly enriched in the mTOR-activated DEGs list versus regulated MVPCs generated using the CompareCluster function from the Bioconductor package ClusterProfiler (102). The size of the dots corresponds to the gene ratio, the number of genes in the list annotated to the given Reactome category divided by the total number of DEGs with unique Entrez identifiers in the list. The color scale represents the adjusted P values obtained for enrichment of the category in each gene list.