Fig 2: Generation of CSCC Organoids. (A) Schematic illustration of the derivation of CSCC organoids from patient samples. (B) Representative brightfield microscopy images of CSCC organoids post-seeding (exemplified by CSCC-2; see Fig. S4 for more examples). Scale bar, 25 μm. (C) Measurements of the growth diameters of CSCC organoids at days 1, 7, and 11. Horizontal lines and error bars represent the mean diameter ± SEM for five organoids (n = 5). (D) Immunohistological features of CSCC organoids derived from patients, compared to original tumors (exemplified by CSCC-2, see Fig. S5 for more examples). Representative immunofluorescence images display markers Ki67, p53, PanCK, PAX8, and CAR (all in red), with nuclei counterstained using DAPI (blue) and cell membranes with phalloidin (green). Inset images from the left panels are magnified three-fold in the right panels for enhanced detail. Scale bar, 50 μm. (E) Proportion of immunofluorescence staining-positive cells in primary tumors and organoids (n = 3). Statistical significance was assessed using two-way ANOVA. T, tumor; O, organoid; ns, not significant; *p < 0.05; ***p < 0.001; ****p < 0.0001; ns, not significant

Fig 3: Expression of kidney markers (SIX2, PAX8, Nephrin, and Podocin), endothelial marker (CD31), mesenchymal markers (PDGFR-β and Vimentin), and pluripotency-associated markers (TRA-1-81, NANOG, and SSEA4) in 3D-NPCs. Cell nuclei were stained with Hoechst. Zoom-in pictures show the subcellular localization of the respective proteins. Pictures were taken under 20x magnification. The scale bars represent a length of 50 μm.

Fig 4: PAX8, SOX17, and MECOM are candidate MTFs in HGSOC.(A) PAX8, SOX17, and MECOM coincide with SEs in primary ovarian tumors. H3K27ac ChIP-seq data were generated in 12 primary tumors, and data were normalized to counts per million (CPM) mapped reads. (B) SEs were identified in 12 primary HGSOCs; PAX8, SOX17, and MECOM are associated with SEs in most tumors. Stitched enhancers were ranked by H3K27ac ChIP-seq signal. (C) PAX8, SOX17, and MECOM occupy their own and each other’s SEs. Enhancers are indicated as black bars. (D) Proposed core regulatory circuit for HGSOC based on TF co-occupancy. (E) PAX8, SOX17, and MECOM ChIP-seq peaks were ranked by CPM-normalized signal. Strong binding sites for each factor are detected proximal to their own and each other’s gene loci. (F) PAX8, SOX17, and MECOM bind to enhancers of HGSOC clinical biomarkers. (G) Global cobinding of PAX8, SOX17, and MECOM. Each row is a PAX8 ChIP-seq peak. CPM-normalized ChIP-seq reads were plotted for a 4-kb window centered on each binding site. Rows are ordered by decreasing PAX8 signal. (H) PLA performed in Kuramochi cells. Each red dot represents a single interaction. Nuclei were counterstained blue with 4′,6-diamidino-2-phenylindole (blue). Scale bar, 100 μm. Image magnification, ×40. (I) Quantified PLA signal per cell. In (C) and (E) to (G), ChIP-seq data were generated in Kuramochi cells and normalized to CPM mapped reads. ***P < 0.001; Student’s paired t test. Error bars indicate SD of the mean values from three independent experiments (performed with technical triplicates).

Fig 5: Bioinformatics analysis of TTFs and thyroid iodine-metabolizing genes in the combined GEO cohort of thyroid cancers. (A−F) Robust RMA expression values of TTF-1 (A), TTF-2 (B), PAX8 (C), TPO (D), TSHR (E), and SLC5A5 (F) were compared among normal thyroid tissues, PTCs, FTCs, PDTCs, and ATCs in the combined GEO cohort. *P < 0.05, **P < 0.01, ***P < 0.001, one-way ANOVA.