Figure 1: Identification and characterization of binucleated luminal cells in lactating glands.
From: Essential role for a novel population of binucleated mammary epithelial cells in lactation
(a) Whole-mount 3D confocal image of a mammary ductal portion (FVB/N mouse) at 4 dL (left) and optical section of the enlarged region showing an alveolar unit (right). The gland was stained for DAPI (white), E-cadherin (Ecad; green), F-actin (red) and milk (blue, see Supplementary Fig. 1a) (n=3 mice). DAPI staining shows that the vast majority of luminal cells are binucleated. Scale bars: 200 μm (whole-mount); and 40 μm (optical section). (b) Whole-mount 3D confocal image of a mammary ductal portion (FVB/N mouse) at 16.5 dP (left) and optical section showing a representative alveolar unit (right). The gland was stained for DAPI (white), E-cadherin (green) and keratin 5 (red) (n=3 mice). No binucleated cells could be detected. Scale bars: 20 μm. (c) Bar graph showing the percentage of cells containing 2N or 4N DNA content in the luminal (Lin−CD29loCD24+), basal (Lin−CD29hiCD24+) and stromal cell (Lin−CD24−) compartments in late pregnancy (16.5 and 18.5 dP) or early lactation (2 dL). Error bars represent mean±s.e.m. (n=3). (d) Representative FACS plots of DNA ploidy in the luminal, basal and stromal populations at 2 dL, and confocal images of sorted cells from the luminal population with 2N or 4N DNA content (far right panels). (e) Optical section from a 3D image of a mammary gland at 4 dL to illustrate the first step in volume quantification. Using ImageJ software, the cell membranes were outlined (yellow) based on E-cadherin immunostaining. Scale bar: 20 μm. (f) 3D image of representative bi- and mononucleated cells used for volume quantification performed with Imaris software. Scale bar: 20 μm. (g) Bar graph showing volume for luminal cells with either one or two nuclei at 18.5 dP, 2 dL, 4 dL and 6 dL (n=3 samples per time-point). Error bars represent mean±s.e.m. **P<0.01, ***P<0.001, ****P<0.0001.
