Fig. 3.

Nck1 and Nck2 have opposing effects on TEB number at 5 and 8 weeks of age, with no effect on TEB size. A Representative high magnification images of TEBs acquired from mammary gland wholemounts prepared from WT, Nck1KO and Nck2KO mice at 5 weeks (left) and 8 weeks (right). Scale bar 100 μm. B Quantification of total TEB number per mouse at 5-week and 8-week timepoints. TEB minimum size was set at 100 μm to ensure consistency across samples (n = 5–10 mice of each genotype). TEBs undergoing bifurcation were excluded from the analysis. C Quantification of average TEB size per mouse in each genotype at 5-week and 8-week timepoints. D Representative image showing area selected for quantitation of branching rate. Scale bar 1 mm. E Quantification of branching rate (average distance between branches) in 8-week old mice (n = 4–5 mice of each genotype), counted as the total length of the ductal system divided by the number of branch points in the area measured in (D). B,C,E, 1KO: Nck1KO, 2KO: Nck2KO, * adjusted P < 0.05; at each time point by one-way ANOVA with post-hoc Dunnett’s test of Nck1KO and Nck2KO compared to WT