Fig 2: TuNR enables orthogonal control of population mean and variance of endogenous genes.A Simplified diagram of TuNR, as shown in Fig. 1A, driving the expression of NGFR and B CXCR4 from their endogenous loci. C Kernel distributions of NGFR expression for no induction (orange) and max induction (purple) of TuNR. Also shown is the NGFR distribution in the parental cell line (green). p = 1.97E - 39. D Kernel distributions of CXCR4 expression for no induction (orange) and full induction (purple) of TuNR. Also shown is the CXCR4 distribution in the parental cell line (green). p = 9.62E - 33. E Coefficient of variation of endogenous NGFR, as a function of mean expression, following the activation of TuNR for 6 days. F Coefficient of variation of endogenous CXCR4 as a function of its mean expression, following the activation of the TuNR for 6 days. G, I Representative kernel distributions of NGFR for different TuNR induction levels that achieve the same mean (isomean) but different variability as identified by gray strips in E. Panel G p = 0.02946; Panel I p = 0.0182297. H, J Representative kernel distributions of CXCR4 for different TuNR induction levels that achieve the same mean (isomean) but different expression variability, as identified by gray strips in F. Panel H p = 0.03467; Panel J p = 0.0057441. *Padj < 0.05; **Padj « 0.05; two-sided Kolmorgorov–Smirnov test (Bonferroni corrected), NS: not significant.

Fig 3: High-risk epithelial strains with low LEp populations.Flow cytometry analysis of passage four epithelia stained for (a-b) CD271(MEp marker) and MUC1(LEp marker) or (c-d) CD271 and CD133 (LEp marker) from HR women carrying (a) BRCA1 or (b-d) BRCA2 mutations.

Fig 4: HR cultured mammary epithelial cells show luminal expansion with a basal phenotype.a–d, Flow cytometry analysis of passage 4 epithelia stained for CD271 and MUC1 from an AR 19-year-old woman and an AR 66-year-old woman (a), HR women of different ages (31, 35, 40 and 53 years) harboring BRCA1 mutations (b), an HR 52-year-old woman harboring a PALB2 mutation (PALB2mut) (c) and an HR 33-year-old woman harboring a BRCA2mut (d). e, Dot plot of the percentage of LEp populations from passage 4 epithelial strains derived from AR younger women (≤35 years, n = 17), AR middle-aged women (36–55 years, n = 9), AR older women (>55 years, n = 10) and HR women (n = 15). The median of each sample is indicated by a horizontal line. The P values were computed using the Kruskal–Wallis test adjusted for multiple comparisons with Dunn’s post-hoc test. f–j, Immunofluorescence staining of passage 4 cultured epithelial cells stained for KRT19 (green), KRT14 (red) and Hoechst (blue) from an AR 19-year-old woman (f), an AR 66-year-old woman (g), an HR 24-year-old woman harboring a BRCA1mut (h), an HR 33-year-old woman harboring a BRCA2mut (i) and a 52-year-old woman harboring a PALB2mut (j). The image on the right-hand side of each panel is a magnification of the area outlined by the white rectangle in the merged image. Scale bars, 50 μm. Experiments in f–j were repeated three times independently with similar results.

Fig 5: Derivation and characterization of H9-NCCs differentiated from H9-hESCs. (A) Phase-contrast images of H9 hESCs at passage 56 (P56) and H9-NCCs and passage 0 (P0) and passage 4 (P4). (B) Flow cytometry analysis of H9-NCCs. (C) Immunocytochemistry analysis showed that H9 hESC-derived H9-NCCs were positive for HNK1, FOXD3, AP2α, p75, SOX9, SOX10 and were negative for neural progenitor marker PAX6 and undifferentiated hESCs markers SOX2 and OCT4. (D) Real-time PCR data showed the expression of OCT4, NANOG, SOX9, SOX10, P75, PAX3, ZIC1 and AP2α in H9-NCCs relative to H9 hESCs. Data were shown as the means ± SEM of three independent experiments. Unpaired two-tailed Student's t test. *p < 0.05, **p < 0.01. Scale bars, 100 μm.