Fig. 1: CRISPR screen in 3D mammary epithelial cell culture for regulators of stem cell self-renewal.
a Development of primary breast cells (single MECs), primary organoids (Organoid MECs), and MCF10A cells in 3D hydrogels. Phalloidin-stained Type I and II lobules that developed from organoids are shown. Results are representative of at least 3 independent experiments. Scale bars = 100 µm (Single MECs and MCF10A), 200 µm (Phalloidin stained lobules) or 500 µm (Organoid MECs). b Schematic of pooled CRISPR screening strategy. c Phalloidin-stained secondary organoids from MCF10A cells with or without sgRNAs (top), and quantification of organoids (bottom). P = 0.0007 (two-sided Student’s t-test). Data were derived from n = 4 independently analyzed gels. Scale bar = 100 µm. d Screened kinases scored by significance relative to a null distribution using RIGER (y-axis) and by comparing the mean differential abundance of sgRNAs targeting the kinase (x-axis). Adjacent histograms indicate p-value distribution (right y-axis) and mean differential (top x-axis). RIGER’s p-values are adjusted for multiple tests. Significant genes previously implicated in cellular differentiation indicated in green. e Western blot for total DDR1 in parental MCF10A cells or after knocking out DDR1 with two independent sgRNAs. f Phalloidin-stained secondary organoids from parental or DDR1 knocked-out MCF10A cells. Scale bar = 100 µm. g Quantification of organoids from (f). P(Ctrl vs. sg1) = 2.28 × 10−6; P(Ctrl vs. sg2) = 7.56 × 10−7 (ordinary one-way ANOVA with Dunnett’s multiple comparisons test). Data were derived from n = 4 independently analyzed gels for each treatment group with n = 3 fields sampled per gel. h Quantification of secondary organoid development with or without DDR1 inhibition. P = 8.2 × 10−5 (two-sided Student’s t-test). Data were derived from n = 4 independently analyzed gels. For all bar graphs in this figure, data are presented as mean values ± SD. *** indicates P ≤ 0.001; **** indicates p ≤ 0.0001. Source data are provided as a Source data file.
