Mammary glands from Sfrp1−/−exhibit a decrease and cell death signals in response to γ-irradiation. (A) For real-time PCR analysis of Bax and Bbc3 gene expression, total RNA was isolated from the mammary glands of control and Sfrp1−/− female mice fed a ND and HFD 6 hours following 5 Gy whole body irradiation (n = 3/geneotype). The results shown represent experiments performed in duplicate and are normalized to the amplification of β-Actin mRNA. Bars represent mean ± SEM of the difference in fold change compared with control ND fed mice. (B) Left panel, 3rd & 4th inguinal mammary gland sections were subjected to immunohistochemical analysis, stained for cleaved caspase-3 (brown chromogen), and images were captured at 400X. Inset, In addition to capturing 400X photographs of mammary gland ducts, lymph nodes were imaged as a positive control (40X). Right panel, The total number of cleaved caspase-3 positive cells was counted for each mammary gland (n = 3/genotype) and bars represent mean ± SEM cell number. (C) 3rd & 4th inguinal mammary gland sections were subjected to immunohistochemical analysis, stained for p53 (brown chromogen), and representative images were captured at 400X (scale bar 50 μm). Pictures illustrate the staining results obtained from each γ-irradiated mouse in the study. (*p < 0.05, significantly different from control mice fed a ND using Bonferroni’s t test after a two-way ANOVA).