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. 2021 May 19;12:2939. doi: 10.1038/s41467-021-23269-8

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© The Author(s) 2021

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 2 — a Representative carmine-stained mammary gland whole mounts (WMs) showing pubertal morphogenesis of Bad mutants. Shown are the #4 mammary glands of 5wk mice of Bad^+/+ (n = 6), Bad^−/− (n = 6), Bad^S155A (n = 6), and Bad^3SA (n = 7). For comparison, a control Bad^+/+ gland at 4wk (left, n = 7) representing early pubertal onset is included. The epithelial tree (dark pink branched structure) is embedded within the mammary fat pad (surrounding light pink stroma). N, nipple; LN, lymph node. Arrows highlight front of migrating epithelial tree. Scale bars = 5 mm. b, c Quantitation of epithelial tree morphological features for Bad^+/+, Bad^−/− Bad^S155A, and Bad^3SA at different developmental times (5wk, 8wk, >40wk). b) Quantitation of epithelial tree extension shows delay in Bad^S155A (gray) and Bad^3SA (blue) at 5wk. c Quantitation of epithelial tree area shows decreased area in Bad^S155A (gray) at 5wk and Bad^3SA (blue) at 5wk and 8wk. Data are mean ± SD. Number of animals: For pubertal onset (4wk), Bad^+/+ n = 7. For puberty stage (5wk), towards end of puberty (8wk) and aged virgins (>40wk), Bad^+/+ n = 6, 10, 6; Bad^−/− n = 6, 11, 6; Bad^S155A n = 6, 11, 3; and Bad^3SA n = 7, 12, 3, respectively. d Transplant assay schematic showing fat pad clearance in recipient mouse and subsequent donor tissue transplant. Donor tissue growth was assessed 6 or 7 weeks post transplant surgery. e Representative carmine-stained WMs of negative control “recipient” mice (n = 5 mice per genotype) that did not receive transplant shows the efficiency of fat pad clearance. Scale bars = 5 mm. f Representative carmine-stained WMs for experimental transplant series (n = 5 mice per transplant condition). Red text represents the donor tissue genotype and blue text represents the recipient genotype. Scale bars = 5 mm. g Percentage of cleared fat pads with successful transplant repopulation show failure of Bad^3SA epithelium to repopulate fat pad (red asterisk). The ratio of successful repopulation per total transplants is shown on top of the bars. n = 10 for each group. Computed Chi-square (χ²) p-value = 0.0235. h Quantitation of area occupied by transplanted Bad^+/+ epithelium in either Bad^+/+ or Bad^3SA fat pads show similar repopulation areas. Data are mean ± SD. n = 5 for each group. For all p-values, ***P < 0.001, **P < 0.01, *P < 0.05. Statistical test details and exact p-values are provided in Supplementary Data 4. Source data are provided in the Source data file.