Fig 2: Lipid-stabilized Wnt3a protein supports serum-free derivation and self-renewal of multipotent human intestinal stem cells.(a) Expansion of single human duodenum organoid cells (passage 7) able to form new organoids (n=3, mean±s.e.m.). (b) Human duodenum organoids derived in the presence of Wnt3a liposomes (400 ng Wnt3a per ml) in serum-free medium. (c) Quantitative RT–PCR analysis for the intestinal stem cell markers LGR5 and TROY in passage 7 human duodenum organoids derived and maintained in Wnt3a-conditioned medium or liposomes (n=3, mean±s.e.m.). (d) Histochemical and immunocytochemical stainings as indicated of sections of passage 6 human duodenum organoids derived and maintained in Wnt3a-conditioned medium or liposomes, and of human small intestine. Staining for Ki67, EPHB2 and chromogranin A (CHRA) in brown, for alkaline phosphatase (ALPI) in blue and periodic acid-Schiff (PAS) in purple (see arrows). (e) Confocal images (z-stack projection) of human duodenum organoids derived and maintained in Wnt3a liposomes, then either differentiated for 5 days or maintained in expansion medium. Immunostaining for Villin (VIL) and lysozyme (LYSZ) in red, and for Mucin2 (MUC2) and CHRA in green. Nuclei were counterstained with DAPI (4,6-diamidino-2-phenylindole; blue). Scale bars, (b) 100 μm, (d) 20 μm (Ki67) and (d,e) 50 μm.

Fig 3: Wnt3a liposomes support serum-free establishment of human jejunum and liver organoids.(a) Human jejunum organoids derived in the presence of Wnt3a liposomes (800 ng Wnt3a per ml) in serum-free medium. (b) Human liver organoids derived in the presence of Wnt3a liposomes (800 ng Wnt3a per ml) in serum-free medium from patient biopsies with (i) hepatitis C combined with hepatocellular carcinoma, (ii) α-1 antitrypsin deficiency and (iii) Wilson's disease. Scale bars, (a) 100 μm and (b) 50 μm. P, passage.

Fig 4: After the treatment of MDA-MB-231 cells with 9a for 48 h, the western blotting results showed that the global levels of asymmetrical dimethylarginine (ADMA), dimethylaminohydrolases (DDAH) and H4R3me2a were significantly decreased in concentration-dependent manners, and the global level of symmetrical dimethylarginine (SDMA) was not significantly changed. On the Wnt pathway, the western blotting results showed that the global levels of Wnt3A and β-catenin were significantly decreased in concentration-dependent manners, while the level of Wnt5a/b was not changed. The experiments were repeated in triplicates.

Fig 5: Abnormal overexpression of cilia caused by gastric cancer activates the Wnt/β-catenin signaling pathway in MSCs. (A) Analysis of primary cilia in MSC, MSC+HGC27, MSC+ HGC27+DKK1 and MSC+HGC27+chloral hydrate by immunofluorescence. Primary cilia were stained with anti-acetylated α-tubulin (axoneme, green) and anti-γ-tubulin (basal body, red) antibodies. Nuclei were stained with DAPI (blue). White arrows indicate cilia. Scale bars, 20 μm. (B) Western blot analysis of acetylated α-tubulin and γ-tubulin expression in MSC, MSC+HGC27 and MSC+HGC27+DKK1. (C) Quantitative analysis of protein levels in (B). Protein levels were normalized to GAPDH. (D) Analysis of β-catenin (red) expression by immunofluorescence in MSC, MSC+HGC27, and MSC+HGC27+DKK1 groups. Nuclei were stained with DAPI (blue). Scale bars, 20 μm. (E) MSC, or MSC co-cultured with HGC27 with or without DKK1 treatment were analyzed for Wnt3a (red) expression. Nuclei were stained with DAPI (blue). Scale bar, 100 μm. (F) Quantitative analysis of the fluorescence intensity in (E). (G) Western blot analysis comparing Naked1, Axin1, or β-catenin expression in the MSC, MSC+HGC27, and MSC+HGC27+DKK1 groups. (H) Quantitative analysis of protein levels in (G). The protein levels were normalized to GAPDH. (I) Western blot analysis comparing Naked1, Axin1, or β-catenin expression in the MSC, MSC+HGC27 and MSC+HGC27+chloral hydrate groups. (J) Quantitative analysis of protein levels in (I). The protein levels were normalized to GAPDH. (K) qRT-PCR results showing Wnt3a, β-catenin, TCF-1, and GSK-3β transcription levels in MSC, MSC+HGC27, MSC+HGC27+DKK1 on day 3 following OS induction. The gene expression levels were normalized to GAPDH expression. Data are shown as mean ± SEM. Statistical differences were obtained using One-way ANOVA with post-hoc testing, *, p<0.05, **, p<0.01, ***, p<0.001. NS, not statistically significant, n=3, per-group. Ace-tubulin, acetylated α-tubulin.