Fig. 3

TRAF6 is required for maintaining mammary stem cells (MaSCs) and luminal progenitor cells (LPCs). a In vitro limiting-dilution assay of luminal and basal cells isolated from outgrowths of transplanted TRAF6-He and TRAF6-KO epithelia in recipient mice in the virgin stage. Isolated cells were cultured in ultra-low adherent plates at densities of 1000 to 31 cells/well. At 16 days after seeding, spheres (>50 μm) were counted to calculate the sphere-forming unit (TRAF6-He and TRAF6-KO, n = 4/group). b Morphology of the spheres in a. c Size distribution of the spheres in a. d Immunostaining of the spheres in a with antibodies specific to Keratin 5 (Krt5, red) and E-cadherin (E-cad, green). Nuclei were stained with Hoechst 33342 (blue). Insets show higher magnification views of the area indicated by dotted boxes. Arrowheads indicate Krt5^highE-cad^low basal cells. Scale bars, 50 μm. e In vitro limiting-dilution assay of luminal and basal cells isolated from mammary tissue of 10-day-old (prepubertal) TRAF6-WT and TRAF6-KO female mice. Isolated cells were cultured in ultra-low adherent plates at densities of 1000–30 cells/well for luminal cells and 700–20 cells/well for basal cells. At 16 days after seeding, spheres (>50 μm) were counted to calculate the sphere-forming unit (TRAF6-WT, n = 4; TRAF6-KO, n = 3). f Morphology of the spheres in e. g Size distribution of the spheres in e. h Immunostaining of the spheres in e with antibodies specific to Keratin 5 (Krt5, red) and E-cadherin (E-cad, green). Nuclei were stained with Hoechst 33342 (blue). Insets show higher magnification views of the area indicated by dotted boxes. Arrowheads indicate Krt5^highE-cad^low basal cells. Scale bars, 50 μm