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Fig. S1. Analysis of mammary gland development in MMTV-Cre;NRP2loxP/loxP, MMTV-Cre;NRP2+/loxP and MMTV-Cre glands. Carmine-stained whole-mounts obtained from the fourth gland of each genotype at 5, 7, 9 and 12 weeks of age.
Fig. S2. NRP2 functions in TGFβ-induced epithelial-mesenchymal transition (EMT) of mouse mammary epithelial cells. An EMT in NMuMG cells was induced by the presence of TGFβ (5 ng/ml, 2-21 days) and reversed (mesenchymal-epithelial transition, MET) by withdrawal of TGFβ (7-14 days) after EMT (7 days). (A) Parental (−), EMT (+, 2 days) and MET (+/−, 7 or 14 days) cells. Scale bars: 10 µm. (B) Expression of NRP1, NRP2, E-cadherin (E-Cad), N-cadherin (N-Cad), and β actin was determined by immunoblotting (50 µg cell extracts) after the indicated number of days of TGFβ treatment (a) and/or withdrawal (b). (C) NMuMG cells were transfected with siRNA (100 nM siCTL or siNRP2) for 3 days before TGFβ treatment (5 ng/ml) for 2 days. Extracts from these cells were immunoblotted for NRP2, E-cadherin and actin. (D) The cell populations in C were photographed using phase contrast optics. Note that the NRP2 siRNA inhibited the induction of NRP2 that occurs during the EMT and prevented the acquisition of a mesenchymal morphology in comparison with cells transfected with a control siRNA. Scale bars: 10 µm. (E) NMuMG cells were treated with either VEGF165, bFGF (10 ng/ml) or TGFβ (5 ng/ml) for 15 days and photographed. Scale bars: 100 µm. Cell extracts were immunoblotted for E-cadherin and actin and the data obtained from six independent experiments were quantified by densitometry. Only TGFβ had a significant effect on E-cadherin expression. Error bars indicate s.d., *P<0.05.
Fig. S3. NRP2 does not regulate E-cadherin in the pubertal mammary gland. E-Cadherin immunostaining in terminal end buds (TEBs) of MMTV-Cre, MMTV-Cre;NRP2+/loxP and MMTV-Cre;NRP2loxP/loxP mice are shown. Note the defined cell surface staining in all of the glands shown. Immunostaining was quantified by scoring the intensity of staining in the ducts and TEBs of each gland on a scale of 1-5. Only scores of 4 and 5 were considered positive staining. The following number of ducts and TEBs were quantified: MMTV-Cre (102 ducts, 86 TEBs); MMTV-Cre;NRP2+/loxP (108 ducts, 121 TEBs); MMTV-Cre;NRP2loxP/loxP (100 ducts; 127 TEBs). Scale bar: 10 µm.