Fig 2: R-spondin1 augments ß-catenin signaling.(A) Co-transfection of increasing doses of wild-type (WT) RSPO1 (0–100 ng/well) with ß-catenin (0–5 ng/well) showed dose-dependent augmentation of ß-catenin signaling (left panel) (ANOVA: CTNNB1 0 ng, p<0.05; CTNNB1 2 ng, p<0.01; CTNNB1 5 ng, p = 0.07). A statistically significant synergistic effect was seen when doses of 10 ng RSPO1 and 50 ng RSPO1 were transfected on the background of increasing doses of CTNNB1 (ANOVA: RSPO1 10 ng, p<0.05; RSPO1 50 ng, p<0.05). No activity was seen after co-transfection of mutant (MT) RSPO1 (0–100 ng/well) (right panel). (B) Relative luciferase activity after stimulation of ß-catenin transfected cells with Rspo1 peptide (0–3000 ng/ml) (ANOVA: CTNNB1 -, p<0.01; CTNNB1 +, p<0.001). Luciferase data are reported as a mean ± SEM of at least three triplicate experiments, standardized for Renilla co-expression (*p<0.05; **p<0.01; ***p<0.001 compared to basal value without RSPO1 vector or peptide for that study).

Fig 3: R-spondin1 can show nuclear and nucleolar localization.(A) Immunofluorescent microscopy was used to detect the cellular distribution of a WT pRSPO1-GFP vector in H295R cells (left panel). DAPI stained nuclei and merged images are shown (center and right panels, respectively) (magnification 20X). (B) A similar pattern of cellular distribution was obtained in a CHO cell line (left panel). Fluorescent labeling performed with an antibody against C23-nucleolar protein (red) revealed that the GFP-tagged WT RSPO1 protein shows strong nucleolar localization in some of these cells (center and right panels) (magnification 20X). (C) Immunofluorescent analysis of MT pRSPO1-GFP showed similar cellular distribution. (D) The strong nuclear localization was confirmed following Western blot analysis of nuclear and cytosolic extracts prepared from HEK293T cells that had been transfected with either WT or MT pRSPO1-HA constructs.

Fig 4: PECAM1 distribution pattern in cultured 12.5 dpc male UGRs.Confocal microscopy analysis of PECAM1 localization, observed by whole mount immunofluorescence in 12.5 dpc male gonads cultured for 48 hours in medium alone (A), in the presence of DKK1 at concentration of 1 µg/ml (C), 2 µg/ml (E) and 4 µg/ml (G) and in the presence of DKK1/RSPO1 at concentration of 2 and 3 µg/ml respectively (I). The corresponding TO-PRO3 staining (nuclei) are reported in B,D,F,H, and L. White arrowheads indicate the coelomic vessel. ms: mesonephros; ts: testis.

Fig 5: PECAM1 and ß-catenin distribution pattern in 3 and 6 hours cultured 12.5 dpc male UGRs.Confocal microscopy analysis of PECAM1 distribution, observed by whole mount immunofluorescence in 12.5 dpc male gonads cultured for 3 (A-D) and 6 (E-H) hours in medium alone (A and E), and in the presence of RSPO1 (C and G). The corresponding TO-PRO3 staining (nuclei) are reported in B,D,F, and H. Arrowheads indicate the coelomic vessel. Confocal microscopy analysis of ß-catenin distribution, observed by whole mount immunofluorescence in 12.5 dpc male gonads cultured for 3 (I-N) and 6 (O-R) hours in medium alone (I and L, O and P), and in the presence of RSPO1 (M and N, Q and R). All the images are merged with the respective TO-PRO3 staining. I,M,O, and Q show representative optical sections withdrawn at the organ surface while L, N, P, and R show representative optical sections withdrawn in the inner part of the same organs. ce: coelomic epithelium; tc: testicular cords. White arrowheads indicate ß-catenin nuclear staining.