Figure 1: Dnmt1 expression during mammary gland development

(a) Dnmt1 gene transcript levels were analysed in the mammary glands harvested from virgin (V), pregnant days 10 and 15 (P10 and P15), lactation days 0, 5 and 10 (L0, L5 and L10), and involution days 1, 2, 3, 4 and 8 (I1, I2, I3, I4 and I8) mice. Hprt1 was used as a loading control. (n=3 mice in each time point). (b) Real-time PCR showing relative expression of Dnmt1 from virgin, pregnancy and involution stage (n=3). (c) Representative immunofluorescence (IF) staining for Dnmt1 (red), CD49f (green) and DAPI (4′-6-diamidino-2-phenylindole; blue) in mammospheres generated with cells from virgin mice and pregnant mice at 10th day of gestation (n=3). Scale bar, 100 μm. (d) Representative contour plots of FACS gating showing a fourfold increase in basal myoepithelial stem cells in mammary glands of pregnant mice at 10th day of gestation compared with mammary glands of virgin mice (n=3–5). (e) Bar diagram showing per cent positive basal and luminal cells in virgin and pregnant mice. Data represents mean±s.d. of four independent mice in each group. (f) Carmine staining of inguinal mammary glands from 7-week-old wild-type (Dnmt1^fl/fl) and Dnmt1-knockout (Dnmt1^Δ/Δ) littermates shows severe delay in TEB development in Dnmt1-knockout mice (n=3). LN, for lymph node. Scale bar, 100 μm. (g) Quantification for per cent fat pad filled in the mammary gland (n=3). (h) Haematoxylin and eosin (H&E)-stained image of wild-type and Dnmt1^Δ/Δ mouse mammary gland sections show severe defect in mammary gland development. Images were taken at × 4 and × 20 magnifications (n=3). Scale bar, 100 μm. (i) Ki67-stained section and histogram of wild-type and Dnmt1^Δ/Δ mammary glands show significantly reduced Ki67 staining in Dnmt1^Δ/Δ mice. Images were taken at × 20 and × 40 magnifications. Scale bar, 100 μm. Statistical analysis was performed using unpaired Student’s t-tests. Error bars represent s.e.m. of independent experiments.