Fig 2: Pharmacological activation of FXR induces Lgr5 expression. (A-C) QRT-PCR analysis of FXR, Shp and Lgr5 expression in primary hepatocytes, Hep 1-6 cells and Hep G2 cells treated with GW4064 (10 µM) or DMSO for 24 h. (D) Mice were orally treated with either vehicle or CDCA (20 mg/kg) once a day for 7 days. Then, hepatic expression levels of Lgr5, Shp and Bsep were determined by QRT-PCR analysis. n = 5 per group. (E) Mice were orally treated with either vehicle or GW4064 (50 mg/kg) 2 days of twice-daily routines in a row. Then, hepatic expression levels of Lgr5, Shp and Bsep were determined by QRT-PCR analysis. n = 6 per group. (F) Representative images from RNAscope® ISH assays for Lgr5 and Gs on mouse liver cryosections treated with vehicle or CDCA. Red signal represents Lgr5, white represents Gs, blue shows DAPI. Scale bars, 20 µm. (G) ISH for Lgr5 combined with IF for Gs was performed on mouse liver paraffin sections treated with vehicle or GW4064. Red signal represents Lgr5, white represents Gs, blue shows DAPI. Scale bars, 50 µm. (H-I) Quantification of the IOD for (F-G) was presented in the corresponding diagram. Gene expression level of the samples normalized to internal control and the average expression of interest genes in vehicle group was set as 1. Data were expressed as means ± SD, *p < 0.05, **p < 0.01 and ***p < 0.001 were determined by the two-tailed Student's t-test.

Fig 3: LGR5 is highly expressed in nasal organoids. The stem cell marker Lgr5 mRNA expression levels in organoids and monolayer cultures, GAPDH was used as a housekeeping gene and data were normalised to GAPDH. The values are shown as means±s.e.m., n=5, ** P=0.001 (A). Representative immunofluorescence of 10–12-day-old organoid showing expression of MUC2 (mucus secreting cell, red) and Lgr5 (stem cell marker, green). DAPI stains nuclei blue (B), Scale bar: 20 µm and 20x objective.

Fig 4: Two-color immunofluorescence staining of primary colon cancer tissue with anti-LGR5 and BerEP4, and anti-CEA and BerEP4. (A) Anti-LGR5, (E) Anti-CEA both red color. (B,F) BerEP4 mAb, green color. (C,G) Overlays giving yellow color of double-stained areas. (D) FITC-conjugated mouse IgG; negative control for BerEP4. (H) Rabbit IgG; negative control for anti-LGR5 and anti-CEA. Original magnification: ×200.

Fig 5: EpCAM, LGR5, and LGR4 mRNA expression levels in tissues and cell lines. (A) mRNA expression levels in primary colon cancer (CC) tissues, resected normal colon tissues, and in a panel of colon cancer cell lines; LS174T, HT29, T84, HCT8 and Caco2, primary foreskin fibroblast cells (FSU) and endothelial cells (HUVECs). Red horizontal lines indicate median values. (B–D) mRNA expression levels in lymph nodes from noncancerous disease patients (Control) and colon cancer patients in different TNM stages (Stage I–IV). Red horizontal lines indicate median values. Dashed horizontal lines indicate clinical cutoff values of 0.07, 0.06 and 2.558 mRNA copies/18S rRNA unit for EpCAM, LGR5 and LGR4, respectively. n = number of lymph nodes. p-values were calculated by two-tailed Mann-Whitney test in (A) and Kruskal-Wallis nonparametric ANOVA followed by post hoc Dunn’s test for multiple comparisons in (B–D).