Fig 2: WNT3A increases neurogenesis in AH-NSPC differentiation assays. (A) Percentage of Tubulin βIII (Tuj1) positive cells out of the total number of cells differentiated from AH-NSPCs in the presence of increasing concentrations of WNT3A. Data correspond to the average ± sem of n = 3 (*P < 0.05; ***P < 0.001 by ANOVA). (B) Immunofluorescence images showing the levels of Tuj1 positive cells (red) and DAPI stain (blue) at 0, 50 and 100 ng/ml of WNT3A. As illustrated, WNT3A induces neurogenesis in vitro in a dose-dependent manner. (C) Percentage of neurons (N, Tubulin βIII positive cells), astrocytes (A, Glial Fibrillary Acidic Protein, GFAP positive cells) and oligodendrocytes (O, Myelin Basic Protein, MBP positive cells) in the absence (N2) or presence of 100 ng/ml of WNT3A. The cell lineage analysis shows an increase in neurons and a decrease in oligodendrocytes in the presence of WNT3A (*P < 0.05; **P < 0.01 by ANOVA). (D) Western immunoblot of whole cell lysates from AH-NSPCs treated with WNT3A (100 ng/ml), separated by SDS-PAGE and blotted sequentially with antibodies against P-LRP6 and β-actin as loading control. Full-length blots are included in Supplementary Fig. S7. (E) Relative gene expression (RE) pattern for the WNT pathway target gene Axin2 upon WNT3A (100 ng/ml) stimulation at 6 h. Sdha was used as the housekeeping gene and expression levels were referred to untreated AH-NSPCs (0 h). Data correspond to average ± sem of n = 3 independent experiments analysed by the 2−ΔΔCt method (two-tailed T-test: P = 0.06). Scale bar in B, 100 µm.

Fig 3: Time-course of Rspo2- and Wnt3a-dependent LRP5/6 phosphorylation and β-catenin stabilization.C57MG cells were treated with BSA, Rspo2 (100 ng/ml), Wnt3a (100 ng/ml) alone or in combination for the indicated periods. Cell lysates (30 µg protein/lane) were immunoblotted for phosphorylated LRP5/6, total LRP5, total LRP6 and Hsp70, the latter serving as a loading control. Soluble β-catenin was measured by immunoblotting after pull-down with GST-E-cadherin.

Fig 4: VHHs targeting LRP6P3E3P4E4 block cellular responses to Wnt3a. a Schematic representation of LRP6. The P3E3P4E4 module of the extracellular domain was used to generate anti-LRP6 VHHs. Coloring scheme: LRP6P1E1; yellow/orange, LRP6P2E2; pink/orange, LRP6P3E3; blue/orange and LRP6P4E4; green/orange. LA domains are shown in brown. b Wnt luciferase reporter assay performed in LRP6-overexpressing HEK293T cells stimulated with Wnt3a-conditioned medium and treated with 10 μM of the indicated anti-LRP6P3E3P4E4 VHHs. c Wnt luciferase reporter assay performed in HEK293T cells stimulated with Wnt3a-conditioned medium and treated with 10 μM of the indicated anti-LRP6P3E3P4E4 VHHs. Graphs show average (bars) and range (dots) of luciferase activity in duplicate cell cultures transfected in parallel

Fig 5: Constitutively active β-catenin rescues the Ccnys depletion-induced basal stem/progenitor cell defect.(A) Lrp6 Ser1490 phosphorylation staining in a basal colony. Basal cells were isolated from two 8-week old CD1 mice and culture in Matrigel for 6 days. Colonies were stained with anti-phospho-Ser1490 Lrp6 antibody (green). DNA was stained by DAPI (blue). The arrow indicates a mitotic cell in the colony. (B) Ccnys-depletion attenuates Lrp6 Ser1490 phosphorylation and β-Catenin activation in primary mammary cells. All mammary cells were isolated from 10-week-old Ccny-/- mice and their Ccny+/- littermate, respectively, and infected with scramble or sh-Ccnyl1 lentivirus. After 72 h of culture, the cells were treated with 200 ng ml-1 Wnt3A for another 16 h. Total cell lysates were then prepared and analyzed by western blotting. GAPDH served as loading control. Shown data are representative of three independent experiments.(C) LiCl restored the size of basal colonies to the normal range. Primary basal cells (Lin-,CD24+,CD29hi) were sorted from 8-week-old Ccny-/- mammary glands, followed by infection of scramble or sh-Ccnyl1 lentivirus and culture in Matrigel. 200 ng ml-1 Wnt3A was added at day 1 or 3 mM LiCl was added at day 3. The colony sizes were measured at day 6. Data are presented as mean±s.e.m. Student’s t test: ***p<0.001. (D) Illustration of rescuing the regenerative capacity of Ccnys-deficient stem/progenitor cells by constitutively active β-catenin. All mammary epithelial cells were sorted out from 12-week-old Ctnnb1flox(ex3)/+ mice and virally infected with Adeno-Cre, Ccny-shRNA-mCherry and Ccnyl1-shRNA-GFP. GFP and mCherry double positive cells were FACS-isolated and transplanted. (E) Western analysis validating the generation of β-CateninΔex3, a constitutively active form of β-Catenin, after Cre-mediated recombination. (F) Ctnnb1flox(ex3)/+ mammary cells were virally infected as indicated, followed by FACS-isolation and transplantation. Recipient fat pads were harvested at 8 weeks post transplantation and analyzed for mammary outgrowth. Ctnnb1flox(ex3)/+ cells with Ccnys-knockdown (infected with only the shRNAs) were not able to reconstitute any outgrowth, whereas Ctnnb1flox(ex3)/+ cells infected with both the shRNAs and Adeno-Cre efficiently generated outgrowth, often with hyperplasia formation. Representative images are shown. Results are combined from three independent experiments.