Fig 1: Jagged-1 mediates signal from ECM-sensing basal cells to drive luminal cell differentiation.a JAG1 expression in basal cells from control, DDR1i, and DDR1i Rel tissues. P(DDR1iR vs Ctrl) < 2.5e−14, p(DDR1i vs Ctrl) < 7e−20, p(DDR1iR vs DDR1i) = 0.38. The likelihood ratio test (FDR) was used, not corrected for multiple testing. b JAG1 expression among epithelial clusters. c Immunofluorescent staining of Jagged-1 (green) and E-cadherin (red) in 3D-cultured patient-derived tissues. Results are representative of three independent repeats of this experiment. d, e Flow cytometry analysis of primary tissue in 3D hydrogels treated with DDR1i for 12 days. Basal cells were identified based on EpCAM and CD49f expression and further analyzed for Jagged-1 expression (d). The percentage of Jagged-1+ cells (e) was compared between control and treated groups. Data were derived from n = 3 independent patient samples. P-value = 0.035, two-sided Student’s t-test. * Indicates P ≤ 0.05. Data are presented as mean values ± SD. f–g Development of MCF10A cells (WT, overexpressing LacZ or overexpressing JAG1) in 3D collagen with or without DDR1i. Representative images shown in (f), diameter of organoids shown in (g). Organoid diameter was measured for each treatment group. n = 206 and n = 145 organoids were measured in the WT group cultured under active or inactive DDR1, respectively. n = 166 and n = 92 organoids were measured in the LacZ group cultured under active or inactive DDR1, respectively. n = 183 and n = 92 organoids were measured in the JAG1 group cultured under active or inactive DDR1, respectively. Letters over graph bars indicate statistically different groups, P = 3 × 10−13 (One-way ANOVA with Tukey’s multiple comparisons test). Data are presented as mean values ± SD. h Quantification of secondary organoid development by cells isolated from structures formed by WT, LacZ-overexpressing, or JAG1-overexpressing MCF10A cells with or without DDR1i treatment. Data were derived from n = 10 fields analyzed for each treatment group. Letters over graph bars indicate statistically different groups, P ≤ 0.001 (two-way ANOVA with Tukey’s multiple comparisons test). Data are presented as mean values ± SD. i Immunofluorescent staining of Jagged-1 (green), Notch1 (red) and Hoechst (gray) in 3D-cultured patient-derived tissues. (I) 3D reconstruction of a Z-stack captured by confocal microscopy. (II) a single 2D plane across lobule framed by rectangle in (I). (III) high-power (×60) image of lobule framed by rectangle in (I) and (II). (IV) Enlargement of area framed by rectangle in (III). j Model for the role of DDR1, Jagged-1, and Notch1 within the mammary differentiation hierarchy (left) and visual depiction of how tissue regeneration and morphogenesis is controlled by these factors through spatially regulated cell fate decisions (right). Scale bars = 50 µm. * Indicates p < 0.05; ** indicates p < 0.01. Source data are provided as a Source data file.
Fig 2: DDR1 signaling activates Notch1 to drive luminal differentiation.a Violin plot showing the distribution of DDR1 expression in epithelial clusters. b Immunofluorescent staining of DDR1 (green), E-cadherin (red) and Hoechst nuclear staining (blue) in patient-derived hydrogel-grown tissues. Arrows: basal expression of DDR1. Arrowheads: luminal expression of DDR1. Right panels depict enlargement of region in white rectangle. Results are representative of three independent repeats of this experiment. c Flow cytometry analysis of primary breast tissue cultured in 3D hydrogels for 12 days. Basal and luminal cells were identified based on EpCAM and CD49f expression and further analyzed for DDR1 expression. d The percentage of DDR1+ cells was compared between the basal and luminal populations. P = 0.03 (two-sided Student’s t-test). e DDR1 signal intensity was compared between the basal and luminal populations. P = 0.0008 (two-tailed Student’s t-test). Data were derived from n = 3 independent patient samples. Data are presented as mean values ± SD. f GSEA plots depicting enrichment of a Notch1 target gene set among genes that are overexpressed in cells from DDR1i Rel tissues compared to DDR1i tissues. Plots depict enrichment across all clusters, luminal clusters only and basal clusters only. Normalized enrichment score (NES) and false discovery rate q-value (FDR-q) are indicated. g Western blot for phospho-DDR1, total-DDR1, and cleaved Notch1 (ICN1) under the indicated conditions. β-tubulin was used as a loading control. h Graph depicts quantification of n = 3 biological repeats of western blot seen in (g), comparing DDR1i treated samples with untreated controls in the presence of collagen. P(ICN1 vs. Ctrl) = 0.0373, P(pDDR1 vs. Ctrl) = 0.0012 (two-sided Student’s t-tests). Data are presented as mean values ± SD. i Western blot for cleaved Notch1 (ICN1) and Jagged-1 in separately obtained MCF10A cell lines cultured in 3D collagen in the presence or absence of DDR1i. β-tubulin was used as a loading control. j Graph depicts quantification of n = 3 biological repeats seen in (i), comparing DDR1i treated samples with untreated controls. P(Ctrl vs. ICN1) = 0.039, P(Ctrl vs. JAG1) = 0.028. (two-sided Student’s t-test)* indicates P < 0.05. ** indicates P < 0.01. *** indicates P < 0.001. Data are presented as mean values ± SD. * indicates P < 0.05. Source data are provided as a Source data file.
Supplier Page from DNASU for JAG1 (Homo sapiens) in pDONR223 (Gateway donor/master vector)