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. 2022 Feb 26;25(3):103982. doi: 10.1016/j.isci.2022.103982

Figure 3.

Figure 3

Nfatc1 reporter-marked basal epithelial cells have restrictive contribution to mammary epithelium during homeostasis (see also Figures S3 and S4)

(A) Experimental strategy for successive lineage tracing experiments at different timepoints

(B) Flow cytometry for GFP+ epithelial cells from 8-week-old female Nfatc1CreERT2;R26mTmG mice 4, 12, and 24 weeks post-TAM induction (p.i.). Another approach to analysis was also shown. Total GFP+ cells were applied to CD24 and CD29 gates, showing the distribution of GFP+ cells in the basal and luminal layers. Quantification analysis showing the percentages of GFP+ basal cells per total basal cells and GFP+ luminal cells per total luminal cells for each timepoint. n = 4, 3, and 4 mice at 4, 12, and 24 weeks post-induction, respectively

(C) Quantification analysis of the percentages of GFP+ basal cells per total basal cells in Nfatc1CreERT2;R26mTmG mice 48 h (n = 3), 4 weeks (n = 4), 12 weeks (n = 3), and 24 weeks (n = 4) after TAM induction

(D) Co-immunofluorescence assay for GFP (green), K14 (red), and K8 (blue) in Nfatc1CreERT2;R26mTmG mice at 4, 12, and 24 weeks post-TAM induction. Scale bar, 50 μm

(E and F) Quantification analysis shows the numbers (left panel of (E)) and the percentages (right panel of (E)) of single-cell, two-cell, and multicell basal clones in Nfatc1CreERT2;R26mTmG mice at indicated timepoints (E). Another statistical result is also shown in panel (F). The total numbers of basal clones were 80, 137, and 147 and 122 at 48 h, 4, 12, and 24 weeks post-induction, respectively. n = 3, 3, 4, and 4, respectively

(G and H) Co-immunofluorescence assay for GFP (green), K14 (red), and K8 (blue) from ProcrCreERT2-IRES-tdTomato;R26mTmG mice 48 h (G) and 4 weeks (H) post-induction. Scale bar, 50 μm

(I–K) Quantification analysis shows the numbers (I) and the percentages of single-cell, two-cell, and multicell clones in ProcrCreERT2-IRES-tdTomato;R26mTmG mice at the indicated timepoints (J). The percentages of basal-only and bilineage two-cell clones was also quantified, shown on (K). n = 3 for each timepoint. A total of 61 and 149 clones were quantified at 48 h and 4 weeks post-induction, respectively. Data represent the mean value ±SD