R-spondin1 augments β-catenin signaling.

<p>(A) Co-transfection of increasing doses of wild-type (WT) RSPO1 (0–100 ng/well) with β-catenin (0–5 ng/well) showed dose-dependent augmentation of β-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 β-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).</p

R-spondin1 can show nuclear and nucleolar localization.

<p>(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<i>)</i> (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.</p

<i>RSPO1</i> expression increases at key stages of early human ovary development.

<p>(A) Analysis of <i>CTNNB1</i>, <i>WNT4</i>, and <i>RSPO1</i> in human fetal gonads between 6–9 weeks post-conception. Testis and ovary samples showed higher expression of <i>CTNNB1</i> (ANOVA, p<0.001), <i>WNT</i>4 (ANOVA, p<0.01) and <i>RSPO1</i> (ANOVA, p<0.001) compared to control. Significantly higher expression of <i>RSPO1</i> was detected in the ovary compared to testis (**p<0.01). (B) Analysis of <i>RSPO1</i> expression levels in the testis and ovary during this period of development. A significant increase in <i>RSPO1</i> was found in the ovary across this time course (ANOVA, P<0.0001; 8w >6–7w and 9w >6–7w, both ***p<0.001) (control, 8 wpc heart).</p

Effect of Ca²⁺-ICR frequency on NT2 cell metabolic activity and proliferation.

<p>(A-B) The Ca²⁺-ICR exposed cells increased their metabolic activity and the proliferation trend from week 1 to week 4 compared to the control ones followed by a statistical significant decrease at the 5^th week of treatment. The proliferative status of these cells at week 5 was also studied analyzing the expression of the Ki67. (C) Data are shown as mean ± standard deviation (SD). Asterisks identify statistical significance referring to the control sample (P<0.05).</p

Effect of DKK1 treatment on R-spondin1 augmentation of β-catenin signaling.

<p>(A) RSPO1/β-catenin co-transfected cells were treated 2 hours before transfection with different doses of DKK1 (0–400 ng/ml). After 24 h, cells were lysed and assayed for luciferase activity. No significant reduction in stimulation was seen (ANOVA: RSPO1 50 ng, p = 0.15). (B) Cells were treated with DKK1 (0–1000 ng/ml) and stimulated with Rspo1 peptide (0–2000 ng/ml). Luciferase activity was measured 24 h later. Luciferase data are reported as a mean ± SEM of at least three triplicate experiments, standardized for Renilla co-expression. (N.S., not significant; *p<0.05).</p

Schematic representation and quantitative description for the Ca²⁺-ICR exposure apparatus for electromagnetic field generation.

<p>(A) The Ca²⁺-ICR exposure system illustration. (B) Magnitude and flux lines of the magnetic flux density of the overall exposure system (zoom on the right).</p

Real Time-PCR analysis for TGF-α and FGF-4 expression on Ca²⁺-ICR exposed NT2 cells.

<p>(A-B) TGF-α and FGF-4 mRNAs expression analysis of cells in three different conditions treated for 1 to 5 weeks. Data are shown as mean ± standard deviation (SD). Asterisks identify statistical significance referring to the control sample (P<0.05).</p

Effect of Ca²⁺-ICR exposure on NT2 cell morphology by phase contrast microscope analysis.

<p>(A)The control (ctr), (B) exposed (exp) and (C) retinoic acid (RA) treated NT2 cells were grown as spheres for 5 weeks and seeded on Petri dishes in a matrigel substrate to study their cell morphology. Exp NT2 cells similarly to the RA treated cells show a differentiated cellular morphology in which the cells have developed some neuritic like-structures (×20 objective).</p

Effects of Ca²⁺-ICR exposure on NT2 cell colony formation in soft agar.

<p>The NT2 cells were grown as spheres for 5 weeks in three different conditions (ctr, exp, RA) and then seeded in soft agar substrate for another 7 days to study their capability of forming colonies which was measured using CyQuant GR Dye in a fluorescence plate reader. Data were reported as fluorescence intensity which is directly proportional to the cell number. Data are shown as mean ± standard deviation (SD). The asterisks identify statistical significance referring to the control sample (P<0.05).</p

R-spondin1 weakly activates a β-catenin-responsive promoter.

<p>(A) The effect of WT RSPO1 on TCF-dependent transcriptional activation (left panel) was compared with that of a naturally-occurring RSPO1 mutant (MT) vector generating a protein lacking the first furin domain (right panel), using embryonic kidney tsa201 cells and the TOPFLASH reporter construct. Transient gene expression assays of increasing concentrations of plasmids encoding wild-type (WT) or mutant (MT) RSPO1 (0–100 ng/well) showed a mild activation (maximum 1.8-fold) of the TOPFLASH reporter with WT <i>RSPO1</i> vector (ANOVA, p<0.001) and a complete lack of activity for the mutant. (B) Dose-dependent activation of the TOPFLASH reporter with increasing doses of beta-catenin (0–10 ng/well) (ANOVA, p<0.001). Luciferase data are reported as a mean ± SEM of at least three triplicate experiments, standardized for Renilla co-expression (compared to basal value, **p<0.01; ***p<0.001).</p