Fig. 4: 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.