Figure 4.

Nfatc1 reporter-marked basal epithelial cells significantly contribute to lobuloalveolar development during pregnancy (see also Figure S5)

(A) Co-immunofluorescence assay of GFP (green), K14 (red), and K8 (blue) in mammary ducts from female Nfatc1^CreERT2;R26^mTmG mice at pregnancy day 5.5. Scale bar, 50 μm. Quantification analysis showing the percentageof GFP⁺ basal cells among total basal cells. A total of 2369 basal cells from three mice were quantified. Quantification analysis showing the percentages of single-cell, two-cell, and multicell basal clones. n = 3 mice. A total of 290 basal GFP⁺ clones were quantified

(B) Co-immunofluorescence assay of GFP (green), K14 (red), and Ki67 (white) in mammary glands from female Nfatc1^CreERT2;R26^mTmG mice at pregnancy day 5.5. Scale bar, 50 μm. Quantification analysis showing the percentage of GFP⁺Ki67⁺ cells among GFP⁺ cells and the percentage of GFP⁻Ki67⁺ cells among GFP⁻ cells. A total of 175 GFP⁺ cells and 1,444 GFP^- cells were quantified from three mice

(C) Co-immunofluorescence assay of GFP (green), K14 (red), and K8 (blue) in mammary ducts from female Nfatc1^CreERT2;R26^mTmG mice at pregnancy day 17.5. Scale bar, 50 μm. Quantification analysis showing the percentages of single-cell, two-cell and multicell basal clones. n = 3 mice for each timepoint. The total number of basal GFP⁺ clones was 70

(D) Quantification analysis showing the numbers of single-cell, two-cell, and multicell basal clones for Nfatc1^CreERT2;R26^mTmG mice at indicated timepoints. n = 3 mice for each timepoint

(E and G) Wholemount confocal image for Nfatc1^CreERT2;R26^mTmG mice at the first pregnant day 17.5 (E) and third pregnant day 17.5 (G). High-magnification images were shown in the middle. Optical serial sections were shown on the right. Scale bar, 50 μm

(F and H) Co-immunofluorescence assay for GFP (green), K14 (red), and K8 (blue) in sections from Nfatc1^CreERT2;R26^mTmG mammary alveoli at the first (F) and third (H) pregnant day 17.5,respectively. Scale bar, 50 μm. Quantification analysis showing the percentage of GFP⁺ basal cells among total basal cells. A total of 1,949 basal cells in (F) and 988 basal cells in (H) from three mice were quantified. Quantification analysis shows the percentages of single-cell, two-cell and multicell basal clones for Nfatc1^CreERT2;R26^mTmG mice at the indicated timepoints. n = 3 mice for each timepoint. The total number of basal GFP⁺ clones was 197 and 174 at the first (F) and third (H) pregnant day 17.5, respectively

(I) Quantification analysis showing the numbers of single-cell, two-cell and multicell basal clones for Nfatc1^CreERT2;R26^mTmG mice at the indicated timepoints. n = 3 mice for each timepoint. Data represent the mean value ±SD ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001