Fig 2: KPT-330-mediated XPO1 inhibition drives stem cell-specific and pan-epithelial responses to induce differentiation.a, Violin plots of scRNA-seq log-normalized (transcripts per 10,000 – tp10k) expression of Xpo1 in all untreated control cells split by non-stem and stem I/II/III annotations. Wilcoxon rank sum test, Bonferroni correction stem I/II/III vs non-stem: ****P < 0.0001. b, Violin plots of module scores over all cells derived from genes with known NES in all untreated control cells split by non-stem and stem I/II/III annotations, with each score scaled on a range from 0 to 1. One-way ANOVA post-hoc Dunnett’s multiple comparisons test, ordered left to right: **P < 0.0063, ****P < 0.0001. c, Time course of KPT-330 treatment of ENR+CD differentiating organoids, with treatments over every continuous 2, 4 and 6 d interval. d, Flow cytometry analyses of 3D-cultured intestinal organoids treated with KPT-330 for the indicated time frame during 6 d culture in ENR+CD media. Paneth cells were identified as lysozyme-positive and CD24-positive cells. Means and individual values are shown (N = 4), and the dotted line represents the average of the Paneth cell fractions in control samples. One-way ANOVA post-hoc Dunnett’s multiple comparisons test, ordered left to right: ****P < 0.0001, *P = 0.0323, ***P = 0.001. e, Volcano plot of differentially expressed scRNA-seq log-normalized genes between KPT-330-treated and control cells within stem II/III populations in early timepoints (day 0.25–2). Red points, enriched in KPT-330 treatment; grey, enriched in control. Differential expression based on Wilcoxon rank sum test with significant log2 fold changes based on ±2s (dotted lines) of all genes; FDR (Bonferroni correction) cutoff P < 0.05. f, GSEA normalized enrichment score over all differentially expressed genes between KPT-330-treated and control cells within stem II/III populations in early timepoints (day 0.25–2). Gene sets shown from MSigDB Hallmark v7 with FDR < 0.05; red, enriched in KPT-330 treatment; grey, enriched in control. g, Violin plots split by KPT-330-treated and control for module scores derived from significantly enriched (stress response) and depleted (mitogen signalling) genes in KPT-330-treated and control cells within stem II/III populations in early timepoints (day 0.25–2); each score was scaled on a range from 0 to 1. Effect size (Cohen’s d) for each module between KPT-330-treated and control modules within each cell type is represented in bar chart below violin plots. h, Violin plots of module scores derived from genes expressed in active and quiescent ISCs between KPT-330-treated and control cells within stem II/III populations in early timepoints (day 0.25–2); each score was scaled on a range from 0 to 1. Two-sided t-test: ****P < 0.0001. i, LYZ secretion assay for organoids differentiated in ENR+CD, treated with 10 µM SR11302 (AP-1 inhibitor) or 20 nM Cobimetinib (MEK inhibitor) for 6 d. Organoids were incubated in fresh basal media with or without 10 µM Cch for 3 h on day 6. All data were normalized to ATP abundance and standardized to the control in each experiment. Means and individual values are shown (N = 5), dotted line represents the control value (1). One-way ANOVA post-hoc Tukey’s multiple comparisons test, ordered left to right, top to bottom: ****P < 0.0001, **P = 0.0014, **P = 0.0055, **P = 0.0035, **P = 0.0085, ****P < 0.0001. j, Proposed mechanism for XPO1 inhibition driving transcriptional changes manifesting as increased stress responses and reduced mitogen signalling, resulting in re-balanced cycling of stem cell fate decisions towards secretory Paneth cells and absorptive enterocytes.Source data

Fig 3: Small-molecule inhibition of XPO1 enhances Paneth cell differentiation.a, LYZ secretion assay for organoids differentiated in ENR+CD with increasing concentrations of KPT-330 for 6 d. Organoids were incubated in fresh basal media with or without 10 µM Cch for 3 h on day 6. All data are normalized to ATP abundance and standardized to the control in each experiment. Means and individual values are shown (N = 6), dotted line represents the control value (1). b, Representative immunofluorescence images of 25 total organoids differentiated in ENR+CD media with 160 nM KPT-330 for 6 d, Paneth cells are lysozyme (LYZ) positive. c, Quantification of immunofluorescence images for LYZ+ cells per organoid area for organoids differentiated in ENR+CD with 160 nM KPT-330 for 6 d. Means and individual values are shown (N = 25 organoids). Unpaired two-tailed t-test: ****P < 0.0001. d,e, Flow cytometry for Paneth cell fraction (lysozyme-positive and CD24-mid cells) of all live cells in 3D-cultured intestinal organoids treated with 160 nM KPT-8602 (d) and 2 ng ml-1 Leptomycin B (e) over 6 d culture in ENR+CD media. Means and individual values are shown (N = 4). Unpaired two-tailed t-test: (d) **P = 0.0019, (e) **P = 0.0051. f, Representative immunofluorescence images of 25 total organoids differentiated in ENR+CD media with 160 nM KPT-8602 or 2 ng ml-1 Leptomycin B for 3 d, Paneth cells are LYZ positive. g, Quantification of immunofluorescence images for LYZ+ cells per organoid area for organoids differentiated in ENR+CD with 160 nM KPT-8602 or 2 ng ml-1 Leptomycin B for 3 d. Means and individual values are shown (N = 25 organoids). One-way ANOVA post-hoc Dunnett’s multiple comparisons test: ****P = 0.0001, **P = 0.0035. h, LYZ secretion assay for organoids differentiated in ENR+CD with 160 nM KPT-330, 160 nM KPT-8602 or 2 ng ml-1 Leptomycin B for 6 d. Organoids were incubated in fresh basal media with or without 10 µM Cch for 3 h on day 6. All data were normalized to ATP abundance and standardized to the control in each experiment. Means and individual values are shown (N = 5), dotted line represents the control value (1). One-sample t-test compared to 1, ordered left to right: *P = 0.0242, *P = 0.0431, ***P = 0.0009, ***P = 0.0023. i, Western blotting of intracellular LYZ (immunoblot - IB) in 3D-cultured intestinal organoids cultured in ENR+CD media in the presence of KPT-330, KPT-8602 or Leptomycin B for 6 d (N = 1).Source data

Fig 4: Genetic labeling reveals broad expression of c-Kit in salivary glands. (A) Schematic of the submandibular gland (SMG) structure in rodents. AC, acini; ID, intercalated duct; GD, granular duct; SD, striated duct; ED, excretory duct. (B) Strategy used for genetic labeling of c-Kit-expressing cells with tdTomato (TdT) in adult mice (8 wks of age, n = 5 including 3 female and 2 male mice). Tamoxifen (TAM) was administered for 4 consecutive days and glands were harvested 3 days later and analyzed by flow cytometry (FC) and immunofluorescence microscopy (IF). (C) TdT expression in total population of SMG cells in control (-TAM) and labeled mice (+TAM). (D) IF images of TdT-labeled SMGs immunostained for Integrin a6 (green). Scale bar = 100 µm. (E) Quantification of TdT labeled cells in total population of SMG cells using FC or IF. (F–H) c-Kit immunoreactivity of TdT-labeled cells in tissue sections (F) or single cell suspensions of SMGs (G). Single channels and merged images of various ductal compartments are shown in F. Nuclear blue staining is dapi. Scale bar = 50 µm. Graph in (H) shows the percentage of TdT-labeled cells immunoreactive to c-Kit antibody in tissue sections (IF) and in single cell suspensions (FC) (n = 3). (I,J) Expression of surface markers including CD24, CD49f or Sca1 by TdT+ cells. For all graphs, values are the mean ± SD with n = 5 unless indicated otherwise.