Fig 2: Single-Cell Proteomic Analysis of Cellular Subpopulations in EME6/7t EECs and Primary Tumors(A and B) tSNE plots of mass cytometry (CyTOF) analysis visualizing 74,048 epithelial cells, which were divided into clusters (n = 27) based on their proteomic profiles (A). These cells included immortalized EEC line EME6/7t (exposed to ASC-CM ± Tip) and primary endometrial tumors of patients with known BMIs (B).(C) tSNE-based phenographs showing abundance and cellular distributions of JAC proteins.(D) tSNE-based phenographs showing EpCAM and CK8/18 as epithelial cell markers and TGFR-2, SMAD2, and SMAD4 as TGF-ß factors.See also Figure S6 and Tables S2 and S3.

Fig 3: Stratification of Endometrial Cell Subpopulations Based on SMAD4 and JAC Expression Levels and Patients’ BMIs(A–C) Expression profiles of SMAD4 and JAC based on CyTOF were classified into five categories: I, II, III, IV, and V. Violin plots show ordered SMAD4 expression level (low to high) and distribution in the individual subpopulations (A). Expression heatmaps of JAC were aligned according to SMAD4 expression levels (in descending order) in different cellular subpopulations (B). Area of circle denotes relative cell proportion of a subpopulation within each sample and ordered in the aforementioned five categories in cellular subpopulations of EME6/7t lines and primary tumors (C). Tumors were ordered from top to bottom based on increased patients’ BMIs and histology classes.(D) Balloon plots of subpopulation distributions of endometrial tumors in the five categories based on histology and BMI. Circle area indicates the size of each subpopulation. Categories IV and V were associated with relatively high expression levels of SMAD4 and JAC, whereas low levels of SMAD4 and JAC were noted in categories I, II, and III.(E) Violin plots showing the average expression levels of four JAC markers—ADAM15, ROCK1, Cx43, and Gas1—based on patients’ tumor histology: papillary serous and endometrioid.(F) Balloon plots of subpopulation distributions in the five categories stratified by BMI into three groups: normal weight (NW), obesity (OB), and morbid obesity (MOB).(G) Violin plots showing the average expression levels of ADAM15, ROCK1, Cx43, and Gas1, based on three BMI groups: NW, OB, and MOB. Lower JAC expressions corresponded with patients with higher BMIs. ****p < 0.0001.See also Figure S7.

Fig 4: TGF-ß/SMAD4-Mediated Downregulation of Candidate JAC Genes(A) Bar plots showing mRNA expression levels of selected PAI1-regulated JAC genes by qRT-PCR in the two shSMAD4 knockdown clones, compared to shCtrl in the presence or absence of ASC-CM.(B) ChIP-qPCR analysis of SMAD4 binding to promoter regions of the JAC genes in control (Ctrl) cells and ASC-CM-exposed cells.Data represent mean ± SD, n = 3 technical replicates; *p < 0.05, **p < 0.01, ***p < 0.001, Student’s t test. See also Figure S5.