Fig 2: LY6D+ Prostate Cells Are Enriched for Organoid-Forming Potential(A) Representative phase images of solid, acinar, and translucent organoids from FACS-sorted Lin-SCA1highLY6D+, Lin-SCA1intLY6D+, and Lin-SCA1low/-LY6D+ prostate cells.(B) Co-IF analysis of organoids derived from FACS-sorted Lin-SCA1highLY6D+, Lin-SCA1intLY6D+, and Lin-SCA1low/-LY6D+ prostate cells for Keratins and p63 expression. Arrow depicts K5+K8+p63+ triple-positive cells. See Figure S4A for individual channels. Scale bars: 50 µm.(C) AR expression in K8+ cells in representative multipotent organoids derived from FACS-sorted Lin-SCA1high LY6D+, Lin-SCA1intLY6D+, or Lin-SCA1low/-LY6D+ prostate cells. Arrows depict AR+K8+ double-positive cells. See Figure S4B for individual channels. Scale bars: 50 µm.(D) Quantification of organoids formed from FACS-sorted LY6D+ and LY6D- (Lin-SCA1high, Lin-SCA1int, and Lin-SCA1low/-) prostate cell subsets (1,000–3,000 cells/well) from HN mice. n = 5 for each sorted subpopulation. p values: *p < 0.05 and ***p < 0.001. Error bars represent mean ± SEM.(E) Schematic diagram showing organoid culture in the presence or absence of androgen (DHT, dihydrotestosterone).(F) Quantification of organoids derived from FACS-sorted LY6D+ and LY6D- (Lin-SCA1high, Lin-SCA1int, and Lin-SCA1low/-) prostate cell subsets (1,000–3,000 cells/well) from HN mice, in the presence (+) or absence (-) of DHT. n = 5 for each sorted subpopulation. p value: ***p < 0.001. n.s., nonsignificant. Error bars represent mean ± SEM.(G) Quantification of unipotent (K5+p63+) and multipotent (K8+ K5+p63+) organoids formed from FACS-sorted LY6D+ (Lin-SCA1high, Lin-SCA1int, and Lin-SCA1low/-) prostate cell subsets (1,000–3,000 cells/well) from HN mice, in the presence (+) or absence (-) of DHT. n = 3 for each sorted subpopulation.(H) Representative co-IF analysis of LY6D, p63, and keratin markers for organoids derived from FACS-sorted LY6D+ (Lin-SCA1high, Lin-SCA1int, and Lin-SCA1low/-) prostate cells. Arrows depict double-positive cells. See Figure S4C for individual channels. Scale bars: 50 µm.(I) Androgen-response analysis of CR organoid outgrowth from FACS-sorted Lin-SCA1highLY6D+ subpopulation, in the absence of DHT, only or followed by DHT stimulation, measured by nuclear AR and Ki67 staining. See also Figure S4F. Scale bars: 50 µm.(J) Quantification of nuclear AR expression on LY6D+ (Lin-SCA1high, Lin-SCA1int, and Lin-SCA1low/-) derived organoids from castrated mice. Organoids were cultured in the absence of DHT for 14 days, and then stimulated for 7 days with 100 nM DHT. n = 3 for each sorted subpopulation. p values: ***p < 0.001 and **p < 0.01. Error bars represent mean ± SEM.(K) Quantification of Ki67 expression on LY6D+ (Lin-SCA1high, Lin-SCA1int, and Lin-SCA1low/-) derived organoids from castrated mice. Organoids were cultured in the absence of DHT for 14 days, and then stimulated for 7 days with 100 nM DHT. n = 3 for each sorted subpopulation. p value: * p < 0.05. n.s., non-significant. Error bars represent mean ± SEM.See also Figures S3 and S4.

Fig 3: Hippocampal neurogenesis in mice with acute and chronic DSS-induced colitis. (A) Schematic representation of adult neurogenesis in the DG and relative stage-specific markers. Representative images of cell labeled by glial fibrillary acidic protein, Gfap, sex determining region Y (SRY) box 2, Sox2, Ki67 and doublecortin, Dcx. The white line in the DG delineates the area in which all the analyses were carried out. (B–E) Relative quantification for stage-specific neurogenesis markers after DSS-acute treatment. (F–I) Relative quantification for stage-specific neurogenesis markers after DSS-chronic treatment. UN: untreated group; DSS: sodium dextran sulfate-treated group. The number of mice used per test is indicated in the graphs (n). Data are reported as mean ± SEM * p < 0.05; ** p < 0.01 for comparison with untreated controls.

Fig 4: Nuclear CYLD expression in correlation to tumor grading in human HCC tissues.(A) Representative staining of positive and negative nuclear CYLD expression in HCC specimens. 10- and 40-fold magnification (nucCYLD+; upper left and nucCYLD-, upper right panel). Representative staining of corresponding Ki67 expression in HCC specimens. 10-fold magnification (lower panels). (B) Nuclear expression of CYLD (IHS score 0–12) in HCC (G1 = 13, G2 = 54, G4 = 25 G4 = 3) and normal liver tissues (N = 7) correlated to grading (Spearman correlation coefficient: -0.423, P<0.001). (C) Nuclear expression of CYLD related to the ratio of Ki67 positive nuclei in HCC (nucCYLD+; N = 47; nucCYLD-; N = 41) and normal liver tissues (N = 7) (Spearman correlation coefficient: -0.271, P = 0.006).

Fig 5: Signaling pathways in KC and qKC mice. (A) Representative immunohistochemical staining images for Ki67, PCNA, cyclinD1, p-ERK, and COX-2 in the pancreases of control, qKO, KC, and qKC mice at 3 months of age, and 9 month old KC and qKC mice. Strong nuclear staining of Ki67 detected in PanIN lesions of KC and qKC mice, and acinar cells of control, qKO, KC, and qKC animals (black arrowheads). No Ki67 expression in islets of control mice (red arrow). Positive staining of PCNA in acinar cells, islets, and ducts in all experimental animals (black arrowheads). Absence of cyclin D1 immunoreactivity in control and qKO mice (red arrows). Strong nuclear labeling of cyclin D1 in all KC and qKC pancreases (black arrowheads). P-ERK expression visible in islets of control, qKO, KC, and qKC animals (yellow arrows). In addition, strong positive staining of p-ERK noted in PanINs (black arrowheads) and stroma of KC and qKC mice (black asterisks). No expression of p-ERK in acinar cells of all experimental animals (red arrows). Cytoplasmic labeling of COX-2 detected only in PanIN lesions occurring in KC and qKC mice (black arrow). Absence of COX-2 immunoreactivity in control and qKO pancreases. Scale bars represent 100 µm. (B) Histological score of Ki67-, PCNA-, cyclin D1-, p-ERK-, and COX-2-positive cells in pancreas of experimental mice groups (control mice, n = 10; qKO mice, n = 10; KC mice of age 3 months, n = 14, 9 months, n = 5; qKC mice of age 3 months, n = 9, 9 months, n = 8). Error bars represent mean ± SD; Error bars represent mean ± SD; * p < 0.05, ** p < 0.01, *** p = 0.0001, **** p < 0.0001; ns = not significant (Student’s t-test).