Fig. 5

TRAF6 promotes G1/S transition and cell survival during pregnancy. a Real-time RT-qPCR analysis of Cyclin D1 mRNA (Ccnd1) expression in luminal and basal cells isolated from outgrowths developed from TRAF6-He and TRAF6-KO epithelia in recipient mice. Values are means ± SD (Virgin, n = 3; P14, n = 4; L1, n = 3; *p < 0.05). b Immunostaining of the mammary gland in #2 fat pads of recipient wild-type mice and the outgrowths derived from transplanted TRAF6-He and TRAF6-KO epithelia in cleared fat pads at P14 with antibody specific to Cyclin D1 (red) and E-cadherin (green). Nuclei were stained with Hoechst 33342 (blue). Insets show higher magnification views of the area indicated by dotted boxes. Scale bars, 100 μm. c Western blotting analysis of Cyclin D1, p-RB, and RB in luminal and basal cells isolated from outgrowths in recipient WT mice (#1) at P14. Tr: Transplanted fat pad; Re: Recipient’s fat pad. d S/G2/M phase population was analyzed in luminal and basal cells isolated from outgrowths in recipient mice at P14. Values are means ± SD (n = 4; *p < 0.05 and **p < 0.01). e Real-time RT-qPCR analysis of Birc2, Birc3, and Tnfaip3 expression in luminal and basal cells isolated from outgrowths developed from TRAF6-He and TRAF6-KO epithelia in recipient mice. Values are means ± SD (Virgin, n = 3; P14, n = 4; L1, n = 3; *p < 0.05 and **p < 0.01). f TUNEL staining of outgrowths in cleared fat pads of recipient mice. Tissues were stained with anti-E-cadherin antibody (E-cad, red) after TUNEL staining. Arrows indicate TUNEL-positive cells (green). Nuclei were stained with Hoechst 33342 (blue). Insets show higher magnification views of the area indicated by dotted boxes. Scale bars, 100 μm. g Population of TUNEL-positive epithelial cells in f. Values are means ± SD (virgin, n = 3; P14, n = 3; L1, n = 3; *p < 0.05 and **p < 0.01)