Fig 2: Single-cell transcriptome profiling of OHBBN-induced mouse bladder tumors.a tSNE plots showing a total of 22 clusters identified from CD45-negative and CD45-positive fractions isolated by flow cytometry from OHBBN-induced primary bladder tumors (pooled tumors, n = 3 tumors). b tSNE plots showing the proximity of cells with high expression of genes belonging to basal, luminal, and EMT-claudin lineage subtypes within Epcam-positive defined populations. c Single-cell gene plots gated on cells with high Krt5 and Krt8 gene expression followed by the assessment of EMT-claudin genes (Cldn4, Cldn7). Cells with Krt5 > 0 and Krt8 > 0 expression were gated as Krt5-Krt8 high, whereas cells with Krt5 = 0 and Krt8 = 0 were gated as Krt5-Krt8 low. Axis units are log (UMI) or transformed transcripts per cell. d tSNE plots showing the presence of bi-lineage-positive cells using gene expression overlays from (left) basal + luminal, (middle) luminal + EMT-claudin, and (right) basal + EMT-claudin subpopulations. Tumor identification and cell numbers sequenced were as follows: tumor 4950 (CD45-neg = 2939 cells, CD45-pos = 1307 cells), tumor 8524 (CD45-neg = 6119 cells, CD45-pos = 2736 cells), and tumor 8525 (CD45-neg = 5068 cells, CD45-pos = 7564 cells). Genes assessed in tSNE plots are shown in Table 1. Top up and down genes are shown in Supplementary Data 1.

Fig 3: Enhanced Notch signaling and decreased goblet cell frequencies in mouse small intestine organoids treated with IL-10.a Schematics for mouse SI organoid cultures. Organoids were grown in ENR media (containing EGF, Noggin, and R-Spondin) supplemented with recombinant murine IL-10 or BSA (Vehicle control). b RNA expression analysis by qRT-PCR from organoids treated with IL-10. Each dot represents SI organoids generated from an individual mouse (N = 5 independent mice). c Alcian blue staining of SI organoids in vehicle- and IL-10-treated organoids. Scale bar = 50 µm. d Percentage of the ab-stained area from the total organoid area. Dots represent single organoids imaged from 2 independent experiments/mice. e Flow cytometry analysis of organoids generated from SI crypts of Lgr5-GFP mice. Representative contour plots showing the percentage of intestinal stem cells (Epcam+ Lgr5-GFP+ cells) are shown. f Frequency of Lgr5+ intestinal stem cells (ISCs) after IL-10 treatments. Dots represent pools of organoids collected from 5 different mice. g Representative contour plots of WGA/CD24 stainings on disaggregated organoids treated with IL-10. h Frequencies of goblet cells (Epcam+ Lgr5-GFP- WGAhi CD24-) and paneth cells (Epcam+ Lgr5- WGAhi CD24+) from vehicle and IL-10-treated SI organoids. N = 5 independent mice. Independent experiments are represented by different symbol shapes in b, d, f and h. Conventional two-tailed t-test was performed in d, whereas paired two-tailed t tests were performed in b, f and h (*p < 0.05, **p < 0.01).

Fig 4: Characteristics of thymic epithelial cells (TECs) in postnatal ß-cat loss-of-function (LOF) mice.(A) Intracellular staining of ß-catenin in UEA1-Ly51+ cortical thymic epithelial cells (cTECs; left) and UEA1+Ly51- medullary thymic epithelial cells (mTECs; right) from control mice and ß-cat LOF mice at 2 wk. Histograms show ß-catenin expression in cTECs and mTECs from control mice (blue line) and ß-cat LOF mice (red line). Shaded area and black line represent the fluorescence in the absence of anti-ß-catenin antibody in control TECs and ß-cat LOF TECs, respectively. Plots show net MFI values for ß-catenin in cTECs and mTECs (means and standard error of the means [SEMs], n = 3). The numbers in parentheses indicate percentage of control value. (B) Quantitative RT-PCR analysis of mRNA expression levels (means and SEMs, n = 5) of indicated genes relative to Gapdh levels in UEA1-Ly51+ cTECs (top) and UEA1+Ly51- mTECs (bottom) in the thymus of control mice and ß-cat LOF mice at 2 wk. (C) Flow cytometric analysis of enzyme-digested thymic cells from control mice and ß-cat LOF mice at 2 wk. Shown are representative profiles of EpCAM and CD45 expression in PI- viable cells (left) and UEA1 reactivity and Ly51 expression in CD45-EpCAM+ viable cells (right). The numbers in dot plots indicate the frequency of cells within indicated area. (D) Plots show the number (means and SEMs, n = 6) of cTECs and mTECs in the thymus from control mice and ß-cat LOF mice at 2 wk. (E) Immunofluorescence analysis of ß5t (green), CCL21 (red), and Aire (cyan) on transverse sections of thymus from control mice and ß-cat LOF mice at 2 wk. Representative data from three independent experiments are shown. Bar: 100 µm. Ctrl: Control, LOF: ß-cat LOF. *p < 0.05; **p < 0.01; ***p < 0.001; N.S., not significant.

Fig 5: Deletion of Dhx9 in TECs compromised the development and maturation of TECs. (A–C) The representative pictures (A), weight (B) and cell numbers (C) of the thymus isolated from 4-week-old WT and Dhx9 cKO mice (n = 10 for each group). (D) Immunofluorescence staining of frozen thymic sections of WT and Dhx9 cKO mice. Red: KRT5-marked medullary regions; Green: KRT8-marked cortical regions; Scale bars: 500 µm. (E) The representative thymic H&E staining in 4-week-old WT and Dhx9 cKO mice; Scale bars: 1000 µm. (F) Flow cytometric profiles, frequencies, and cell numbers of CD45-EpCAM+ TECs in 4-week-old WT and Dhx9 cKO mice (n = 7 for each group). (G) Representative flow cytometry plots, frequencies, and cell numbers of UEA1+Ly51- mTECs and UEA1-Ly51+ cTECs in 4-week-old WT (n = 7) and Dhx9 cKO mice (n = 6). (H) Representative flow cytometry plots and frequencies showed the maturation of mTECs, as measured by the expression of CD40, CD80, and MHCII in 4-week-old WT (n = 8) and Dhx9 cKO mice (n = 6). (I) Representative flow cytometry plots and frequencies exhibited the maturation stage by the expression of CD80 and Aire in 4-week-old WT (n = 8) and Dhx9 cKO mice (n = 6). One representative histogram represented the mean ± SD. The unpaired, two-tailed student’s t-test was used. *P < 0.05, **P < 0.01, ***P < 0.001 compared with WT control mice.