Fig. 6 | Nature Communications

Fig. 6

From: Sfrp3 modulates stromal–epithelial crosstalk during mammary gland development by regulating Wnt levels

Fig. 6

Sfrp3 modulates Wnt signalling in the mammary gland. a Representative images of immunofluorescence staining for β-catenin (red), Troma (green) and nuclei (blue), on 5-weeks-old MG. Arrow denote localisation of β-catenin at cellular junctions in controls (Sfrp3+/+). Scale bar: 50 µm. b Quantification of the number of β-catenin positive cells in mammary ducts of 5 weeks old Sfrp3+/+ (n = 2, 5 mice) and Sfrp3−/− (n = 2, 8 mice) females. Data represent mean ± SD (****p < 0.0001). c Relative mRNA level of Wnt2 (left), Wnt5a and Wnt7b (middle) and Wnt16 (right) in whole MG from Sfrp3+/+ and Sfrp3−/− females at 10 weeks of age. n = 6. d Representative images of immunofluorescence staining for Wnt2 (red), K8 (green), on 5-weeks-old MG. Arrow denote localisation of Wnt2 at stromal-epithelial boundary. Images are representative of 3 MG sections analysed in 3 different experiments. Scale bar: 20 µm. e Relative mRNA level of Wnt2 (left) and Wnt5a (right) in de-epithelialised cleared fat pad from Sfrp3+/+ and Sfrp3−/− females at 8 weeks of age. n = 4. f Left, schemes of wild-type myc-tagged SFRP3 and truncated variants. Numbers indicate the aa position of each conserved domain according to UniProtKB database. SP, signal peptide; CRD, cysteine-rich domain; NTR, netrin-like domain. Right, western blot detection of SFRP3-myc in cell lysates and supernatants of MDCKII expressing different variants. The lack of SP domain abolishes protein expression. SFRP3-NRT variant is expressed and secreted, while SFRP3 CDR is expressed but not secreted in the medium. g Immunoprecipitation of SFRP3-myc followed by immunoblot with anti-myc, anti-V5 and anti-HA antibody in HEK293 cells transiently co-transfected with wild-type human SFRP3 construct and Wnt2-V5, Wnt5a-V5 and Wnt7b-HA constructs. αcont, cell lysate incubated with negative antibody conjugated beads; αmyc, cell lysate incubated with anti-myc antibody conjugated beads. h Pull-down of GST-tagged forms of SFRP3 followed by immunoblot with anti-V5 and anti-HA antibody in HEK293 cells transfected with empty, Wnt2-V5, Wnt5a-V5 and Wnt7b-HA constructs. αcont, cell lysate incubated with negative antibody conjugated beads; αmyc, cell lysate incubated with anti-myc antibody conjugated beads. For c, d, e, data represent mean ± SD (***p < 0.001, ****p < 0.0001, ns, not significant). fh blots are representative of 3 different experiments. Source data are provided as a Source Data file

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