FIGURE 3.

PD-L1⁺ basal cells can produce basal, luminal, and alveolar cells during mammary development. (A) Illustration of the lineage-tracing strategy. (B) Control, uninduced mice. (C) Whole-mount confocal imaging analysis showing that no GFP⁺ cells were present in the mammary ducts of uninduced PD-L1^{CreERT2–2A–tdTomato/+}; Rosa26^mTmG/+ mice. Scale bars, 20 μm. (D) FACS analysis indicating that no GFP⁺ cells were present in the mammary epithelium of uninduced mice. (E) Experimental design for the short-term tracing experiments; tamoxifen (TAM) was administered at the pubertal stage (5 weeks old). (F) Whole-mount confocal microscope images showing an elongated GFP⁺ basal cell. Scale bars, 10 μm. (G) Section imaging indicating the basal location of a GFP⁺ cell. Scale bars, 10 μm. (H) FACS analysis indicating that GFP⁺ cells were mainly basal cells at 48 h after tamoxifen administration. (I–L) Experimental design for the long-term tracing experiments (I), imaging (J,K), and FACS analysis (L) of the distribution of the GFP⁺ cells at 3 weeks after TAM administration. Scale bars, 10 μm. (M–P) Experimental design for the long-term tracing experiments (M), Imaging (N,O), and FACS analysis (P) of the GFP⁺ cell distribution at pregnancy day 14.5. Scale bars, 10 μm. K14⁺ cells are indicated by arrows. K8⁺ cells are indicated by arrowheads; n = 5 mice. FACS, fluorescence-activated cell sorting; PD-L1, programmed cell death ligand 1; GFP, green fluorescent protein.