Fig. 1

Preliminary whole-mount and histological analysis showed differences between vehicle, E2, and E2 + PBDE-treated mice. a Overall scheme of experimental design. BALB/cj female mice (9 weeks old) were ovariectomized. Mice were treated (n = 10 per treatment) at 19 weeks of age and were euthanized one week after treatment. b Toluidine blue stained whole-mounted mammary glands showed that vehicle mice had regressed branching trees as characterized by thin ductal structure by comparing to mammary gland structure of intact mice. Treatment of OVX mice with E2 or E2 + PBDE showed a regrowth of gland structures, characterized by thicker and larger ductal structures and greater prevalence of TEB-like structures. Scale bars = 0.4 cm. c TEB-like structures had multiple layers of cells with Ki67-positive staining. d The TEB count was significantly increased in mice fed with E2 + PBDE compared with the mice with E2 only. The data are expressed as mean ± SEM of the mean using duplicate assays. Tukey’s multiple comparisons test was performed. *P < 0.05. e Duct quantification by KRT18 immunohistochemistry staining. The number of large, small, and total ducts were counted among vehicle treated (n = 4), PBDE treated (n = 6), E2 treated (n = 5), and E2 + PBDE treated (n = 5) mice. A threshold of 1906 µm² was used to classify ducts as large or small. Ducts that fell below this threshold were classified as small, and ducts that had areas larger than this value were classified as large. f Overview of scRNAseq approach using mouse mammary gland samples. Cells from two separate experiments were sequenced on the 10X Genomics platform and then pooled together for analysis