Fig 2: Characterization of Tracheal Macrophages(A) FACS analysis of CD45+;F4/80+ myeloid cells after 1 dpP; (n = 4).(B) Immunofluorescence and quantifications of CD45+ cells (green) near basal cells (KRT5, red) following polidocanol-induced injury. Scale bar, 25 µm.(C) Unsupervised clustering of all cells combined from sham and from 1, 3, and 7 dpP based on 15-marker myeloid panel; (n = 1/time point, consistent of 2 animals).(D) Heatmap of marker expression data, scaled from 0% to 100% of normalized, arcsin value of fluorescent intensity.(E) Cells isolated on indicated days post-injury, visualized by cluster assignment.(F) Relative abundance of cells from 24 distinct clusters across injury and repair, scaled 0.01% to 25.9% of total cells in analysis. See also Figure S1 and Table S1.

Fig 3: Blocking PGE2 signaling via the EP2 receptor increases AM numbers in aged mice.a–c Aged (18–22 months) C57BL/6 mice were given 7 daily i.p. injections of an EP2 antagonist. Concurrently, BrdU (0.8 mg/mL) was given in the drinking water ad libitum. AMs (i.e., CD45+ CD11c+ SiglecF+) were then collected from the BALF and analyzed by flow cytometry for BrdU incorporation, indicating proliferation, and Annexin V staining, indicating apoptosis. a Total cell count of lavaged AMs. n = 6/group. b Percentage of AMs that are BrdU+. n = 5/group. c Percentage of AMs that are Annexin V+. n = 6/group. d–f Aged mice were given clodronate-loaded liposomes to deplete resident AMs prior to an adoptive transfer of CFSE+ WT or EP2 KO AMs as depicted in (d). e, f AMs were analyzed by flow cytometry 4 weeks following transfer for CFSE dilution, a marker of proliferation n = 6/group. f Representative histogram of CFSE MFI. For panels a–e, statistical significance analyzed by Mann–Whitney, error bars represent SEM and each point represents a biological replicate. Source data are provided as a Source Data file.

Fig 4: TET loss induces the dysregulation of Wnt-associated transcription and methylation in premalignant lung epithelium. (A) Genetic lineage-labeling strategy to capture premalignant cells using Rosa26 floxed-STOP-tdTomato reporter. Rosa26LSL-tdTomato/+ (hereafter R), K, and KT mice were crossed to generate the following cohorts: KrasLSL-G12D/+; Rosa26LSL-tdTomato/+ (KR) and KrasLSL-G12D/+; Tet1 flox/flox; Tet2flox/flox; Tet3 flox/flox; Rosa26LSL-tdTomato/+ (KTR). Mice were intranasally administered with Ad-CMV-Cre. After 3 wk, lungs were collected to sort DAPI-/tdTomato+/EPCAM+/CD45-/CD31- epithelial cells. (B) PCA of RNA-seq data from K and KT premalignant cells. Each dot represents an individual mouse. n = 3 for K mice; n = 4 for KT mice. (C) Scatter plot showing the DEGs in KT premalignant cells versus K cells. nCount represents the normalized counts (mean of the counts divided by size factors). The dashed lines indicate the fourfold change threshold for defining DEGs. Red and blue dots depict significantly changed genes [log2(fold change) = 2 or = -2 and adjusted P value = 0.05], and gray dots depict genes without significant changes. (D) GO analysis of DEGs up-regulated or down-regulated in KT premalignant cells. The number of genes in each category is denoted. Wnt signaling pathway is indicated in red. DEGs here were those with log2(fold change) = 2 or = -2 and adjusted P value = 0.01. (E) Heat map showing relative expression levels of Wnt-associated DEGs. DEGs here were those with log2(fold change) = 1 or = -1 and adjusted P value = 0.05. (F) Global methylome comparison between K and KT premalignant cells using DMRs. DMRs were filtered by length (=1,000 bp) and CpG number (at least five CpG sites). DMRs with at least 20% absolute methylation level difference between K and KT samples were defined as hypermethylated or hypomethylated. Each dot represents the methylation level of an individual DMR. Red and blue triangles contain the identified hypermethylated and hypomethylated DMRs, respectively. (G) Distribution of hypermethylated DMRs in KT premalignant cells among various genomic elements. GO analysis of all DEGs harboring hypermethylated DMRs in (H) promoter and enhancer or (I) gene body regions in KT premalignant cells. The number of genes in each category is indicated. Wnt-related categories are denoted in red.

Fig 5: Generation and characterization of mammary-gland-specific MED1-overexpression mice(A) Schematic representation of MED1-overexpression transgenic construct (MMTV-MED1).(B) MED1 expression in mammary epithelial cells of MMTV-MED1 transgenic mouse was assessed by immunoblotting.(C) Whole-mount staining of pubertal mammary gland at age of 5, 7, and 8 weeks. Scale bar: 1 cm.(D–F) Analyses of the number of terminal end buds (TEBs) (D), secondary branches (E), and tertiary branches (F) of mice in (C).(G) Mammary cells from 7-week-old wild-type (WT) and MMTV-MED1 mice were analyzed by flow cytometry using antibodies against cell surface markers Lin (CD31, CD45, and Ter119), CD24, and CD29.(H) Flow cytometry analyses of Lin-CD24+Sca1+ cells in mammary glands of 7-week-old WT and MMTV-MED1 mice. The values are obtained from three independent experiments and shown as mean ± SD. *p < 0.05.