Fig 6. 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⁺,CD29^hi) 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 Ctnnb1^flox(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-Catenin^Δex3, a constitutively active form of β-Catenin, after Cre-mediated recombination. (F) Ctnnb1^flox(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. Ctnnb1^flox(ex3)/+ cells with Ccnys-knockdown (infected with only the shRNAs) were not able to reconstitute any outgrowth, whereas Ctnnb1^flox(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.