Figure 4. RANK protects from radiation-induced epithelial apoptosis and controls mammosphere formation.

a, γ-Irradiation (5 Gy) induced mammary epithelial cell apoptosis in control and RANK^Δmam female littermates either sham operated or implanted with a pellet of MPA. Apoptosis was detected by immunostaining for active caspase-3. Apoptotic nuclei of epithelial cells (arrows) are shown for representative mammary-gland sections. Original magnifications ×40. b, Quantification of mammary epithelial apoptosis. A minimum of 5,000 nuclei were counted for each mouse. Results shown are means±s.e.m. (n = 3 mice per group). Two asterisks, P < 0.02 (Student’s t-test). c, Self-renewal of mammary cancer stem cells (TICs) requires RANK expression. Mammary tumour cells from RANK^Δmam and control littermate females treated with MPA and DMBA were cultured for 7 days; a small percentage of primary cells formed mammospheres (first passage). Primary mammospheres were then digested into single cells and assayed for their ability to form secondary mammospheres (second passage). Tumour cells from control littermate but not from RANK^Δmam females could form secondary mammospheres determined 7 days after replating. d, Quantification of primary and secondary passage (F1 and F2) mammospheres from RANK^Δmam and control littermate females treated with MPA and DMBA. Results shown are means±s.e.m. (n = 3 mice per group). Asterisk, P < 0.05 (Student’s t-test).