Fig 2: Unc5B regulates BBB Wnt/ß-catenin signaling.a qPCR analysis of P67 brain mRNA extracts, n = 4 Unc5Bfl/fl and n = 5 Unc5BiECko brains for quantification of LEF1 and PLVAP mRNA levels, n = 9 Unc5Bfl/fl and n = 9 Unc5BiECko brains for quantification of CLDN5 mRNA levels. Each dot represents one mouse. One control mouse was set as 1. Western blot (b) and quantification (c) of Wnt/ß-catenin signaling components in brain protein extracts, n = 7 Unc5Bfl/fl and n = 8 Unc5BiECko brains. Each dot represents one mouse. One control mouse was set as 1. Immunofluorescence (d) and quantification (e) of LEF1 staining on adult brain sections. Each dot is the mean of several images, n = 6 Unc5Bfl/fl and n = 6 Unc5BiECko brains. One control mouse was set as 1. f CTRL IgG and Unc5B immunoprecipitation on cultured brain endothelial cells, reproduced on n = 3 independent experiment. g Schematic of Unc5B adenoviral constructs. CTRL IgG or GFP immunoprecipitation in Unc5B siRNA knockdown ECs infected with siRNA resistant Unc5B adenovirus (h), and quantification of LRP6 pulldown (i), n = 4 independent experiment. Each dot represents one independent experiment. j Unc5B and Ctnnb1 gene deletion strategy using tamoxifen injection in adult mice. k Quantification of cadaverine content in P67 brains, 30 min after i.v. cadaverine injection, n = 4 Unc5Bfl/wt, n = 3 Unc5Bfl/wtiECko, n = 4 Ctnnb1fl/wt, n = 4 Ctnnb1fl/wtiECko, n = 6 Unc5Bfl/wt;Ctnnb1fl/wt and n = 6 Unc5Bfl/wt;Ctnnb1fl/wtiECko brains. Each dot represents one mouse. l Unc5B gene deletion and Ctnnb1flex/3 gene overexpression strategy using tamoxifen injection. Western blot (m) and quantification (n) of P67 brain protein extracts, n = 4 Unc5BiECko and n = 5 Unc5BiECko;Ctnnb1flex/3 brains. Each dot represents one mouse. One control mouse was set as 1. o Quantification of P67 brain cadaverine content, 30 min after i.v cadaverine injection, n = 5 Unc5BiECko and n = 5 Unc5BiECko;Ctnnb1flex/3 brains. Each dot represents one mouse. All data are shown as mean ± SEM. NS non-significant. Two-sided Mann–Whitney U test was performed for statistical analysis between two groups. ANOVA followed by Bonferroni’s multiple comparisons test was performed for statistical analysis between multiple groups. Source data are provided as a Source Data file.

Fig 3: FAM13A reduces the adipogenic differentiation of BM-MSCs by activating the Wnt/β-catenin signaling pathway. (A) AA BM-MSCs were transfected with OENC or OE-FAM13A. FAM13A protein expression levels were analyzed via western blotting. (B) Total β-catenin and non-phospho β-catenin protein expression levels were analyzed via western blotting. (C) The nuclear and cytoplasmic protein expression levels of non-phospho β-catenin were analyzed via western blotting. (D) The protein expression levels of LRP6 and DVL3 were analyzed via western blotting analysis. (E and F) AA BM-MSCs were transfected with OENC or OE-FAM13A with or without DKK1 treatment. (E) Non-phospho β-catenin protein expression levels were determined via western blotting. (F) Lipid droplet accumulation was analyzed by Oil Red O staining. Data are presented as mean ± SD. **P<0.01 vs. OENC; ##P<0.01 vs. shNC or OE-FAM13A. FAM13A, family with sequence similarity 13, member A; AA, aplastic anemia; BM-MSCs, bone marrow-mesenchymal stem cells; sh, short hairpin; LRP6, low density lipoprotein receptor-related protein 6; DVL3, disheveled segment polarity protein 3; OE, overexpression; NC, negative control; DKK1, Dickkopf-related protein 1.

Fig 4: β‐catenin pathway activity was mediated by Wnt2, LRP6, and GSK3β in Mycoplasma hyorhinis‐infected gastric cancer cells. A, Co‐immunoprecipitation assay to validate the physical association of LRP6, GSK3β, and the p37 protein of M hyorhinis. Lysates from M hyorhinis‐infected gastric cancer cells (500 μg) were immunoprecipitated with 1 μg of PD4 antibody or pre‐immune IgG. Input, 50 μg of lysates. B, M. hyorhinis upregulated LRP6 phosphorylation in gastric cancer cells. Cells were serum‐starved for 24 h and then infected with M hyorhinis or stimulated with serum. Cell lysates were harvested at the indicated time points and analyzed by Western blotting. C, Validation of GSK3β silencing efficiency. Cells were transfected with 50 nmol/L GSK3β‐specific siRNAs or NC siRNA for 48 h. D, Knocking down GSK3β expression downregulated M hyorhinis‐induced LRP6 phosphorylation. Cells were transfected with the indicated siRNAs for 48 h and then infected with M hyorhinis for 30 min. E, Validation of LRP6 silencing efficiency. Cells were transfected with 50 nmol/L LRP6‐specific siRNAs or NC siRNA for 48 h. F, Validation of Wnt2 silencing efficiency. Cells were transfected with 50 nmol/L Wnt2‐specific siRNAs or NC siRNA for 48 h. G, Effect of GSK3β, Wnt2, or LRP6 ablation on M hyorhinis‐induced nuclear entry of β‐catenin. Cells were transfected with the indicated siRNAs for 24 h and infected with M hyorhinis for 24 h, followed by cytoplasmic and nuclear protein fractionation

Fig 5: Generation of Ccny and Ccnyl1 mutant mice.(A) qPCR analysis of mouse Ccny and Ccnyl1 mRNA levels in different tissues isolated from a 6-week-old CD1 mouse. (B) Membrane localization of Ccnys. Mouse mammary epithelial EpH4 cells were fractionated into cytosol and membrane fractions. GAPDH and Lrp6 serve as cytosol and membrane loading control, respectively. (C) Ccny gene targeting strategy. Exon 4 was flanked by two loxP sites. Ccnyflox/+ mice were crossed with EIIa-Cre, which can induce recombination in germ cells and transmit the genetic alteration to progeny Ccny+/- mouse. (D) Western analysis to confirm the whole-body knockout of Ccny in Ccny-/- mouse. Lysates of different organs were prepared from a 12-week-old Ccny-/- mouse and its littermate wildtype control mouse. The lysates were analyzed by western with anti-Ccny and anti-Ccnyl1 antibody. β-actin served as loading control. (E) Ccnyl1lacZ/+ reporter mice gene targeting strategy. (F) Western analysis to confirm the knockout efficiency of Ccnyl1 in Ccnyl1lacZ/lacZ mouse. Lysates of mammary gland, brain and lung were prepared from a 5-week-old female Ccnyl1lacZ/lacZ mouse and its wildtype female littermate. Testis lysates were prepared from an 8-week-old male Ccnyl1lacZ/lacZ mouse and its wildtype male mice. The lysates were analyzed by western with anti-Ccnyl1 and anti-Ccny antibodies. β-actin served as loading control. (G) No double knockout mice (Ccny-/-;Ccnyl1lacZ/lacZ) were born from indicated cross, while the other genotypes displayed the expected Mendelian ratios. (H) Representative images of a Ccny-/-;Ccnyl1lacZ/lacZ embryo and its control littermates at E14.5. The double knockout embryo has smaller body size (n = 3 embryos per group). (I) Embryonic lethality of the double knockout embryo at E16.5 (n = 3 embryos per group).