Supplementary Figure 1 Separation of luminal and basal HBECs based on CD10 surface antigen expression. (a) QRT-PCR analysis of relative mRNA expression levels of luminal and basal markers in freshly separated CD10(+) (black bars) and CD10(-) (open bars) HBECs from three different donors. Three replicate samples were analyzed and normalized to TBP. Note that basal markers (ΔNp63, CK14, ITGA6) are more highly expressed in CD10(+) populations whereas luminal markers (CK18, CD24) are enriched in CD10(-) populations. (p-values; ΔNp63: 0.09, CK14: 0.14, ITGA6: 0.005, CK18: 0.11, CD24: 0.17). (b) Photomicrographs of HBECs cultured for 7 days after separation based on CD10 expression. Note, CD10(+) HBECs (left panels) are small, more rounded, and grow dispersed as characteristic of basal cells in vitro. In contrast, CD10(-) cells (right panels) are flat, show tight cell contacts, indistinct cell borders and grow as islands as characteristic of luminal cells. Scale bar: 0.2 mm. (c) Relative mRNA expression levels of basal and luminal markers, normalized to TBP, in HBECs from three different patients sorted for CD10 and subsequently cultured for 5, 7 or 11 days, respectively. (p-values; ΔNp63: 0.22, CK14: 0.14, ITGA6: 0.23, CK18: 0.17, CD24: 0.27).

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Supplementary Figure 2 HBECs express ΔNp63α isoform. (a) Protein lysates of HBECs obtained from three different patients (AF, AL and S) were immunoblotted with a pan-p63 antibody. Band is of approximate 63 kD. (b) QRT-PCR analysis of mRNA expression levels of the ΔNp63 (filled bars) and the TAp63 (open bars) isoforms in HBECs from 3 different donors. Three replicate samples were analyzed and normalized to TBP; (p-value: 0.06). (c) Relative mRNA expression levels of ΔNp63 and TAp63 in CD10(+) (10079 and 2 relative values) and CD10(-) (416 and 9 relative values) in HBECs 7 days after culturing. (d) Relative mRNA expression levels of ΔNp63 and TAp63 isoforms in HBECs 4 days after infection with control (filled bars) or ΔNp63 (open bars) shRNA expressing virus. Note, 87% decrease in ΔNp63 expression in the shRNA-infected cells. (e) Expression of p63 protein 4 days after infection with a control or ΔNp63 shRNA expressing virus.

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Supplementary Figure 3 Expression of NICD with different viral vectors leads to activation of Notch pathway to different extent. (a) QRT-PCR analysis of mRNA expression of Notch1, and its target genes HEY1 and HEY2 in four different HBECs 2 days after infection with different virus. NICD was expressed under the control of a CMV promoter with retrovirus (High-NICD, black bars) or a hPGK promoter with lentivirus (Low-NICD, open bars). Fold induction was calculated relative to corresponding control infected sample. Expression levels were normalized to TBP. (b) QRT-PCR analysis of Notch target gene HEY1 in HBECs infected with inducible NICD virus. 7 days after infection, cells were treated with ethanol (black bar) or 4-OH-tamoxifen for 4 hours (open bar) or 8 hours (lined bar) and analyzed directly after treatment. Cells were grown in selection medium starting from 2 days after infection. (CMV: cytomegalovirus, hPGK:human phosphoglycerate kinase)

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Supplementary Figure 4 RBP-jκ deficient mammary epithelial cells are found only in SMA expressing basal layer. A piece of mammary gland from MMTV-Cre.Z/EG double transgenic mice carrying two floxed alleles of RBP-jκ gene is transplanted into cleared fat pads of the immunodeficient recipient mice. 6 weeks after the transplantation the mammary glands were dissected, fixed and stained for (a) GFP and (b) Smooth Mucsle Actin (SMA). (c) Merged images show that RBP-jκ deficient cells (marked by GFP) were found only in SMA positive cells, which comprise the basal layer.