ROBO1 up-regulation and pFAK397 FA enrichment occurs in high-stiffness 3D collagen systems. (A) Representative images of NMuMG aggregates in HD 3D collagen gels treated with Mock or SLIT2 and stained for pFAK397 (red), phalloidin (green), and Hoechst (blue). (B) Area of pFAK397 puncta located at the end of phalloidin-labeled fibers in acini (3+ nuclei; n = 15+ acini). (C) Representative images of NMuMG acini in LD and HD 3D culture and stained for ROBO1 (green) and Hoechst (blue). (D–F) Analysis of ROBO1 protein (D) and mRNA (E and F) of mammary cells cultured in LD and HD collagen matrices for 7 d. (D, top) Quantification of ROBO1 protein normalized to GAPDH from NMuMG cells (n = 3 experiments). (bottom) Representative blots for ROBO1 and loading control GAPDH. (E and F) Expression of Robo mRNA in NMuMG (E) or MDA–MB-231 (F) cells from 7 d, 3D cultures (n = 3 experiments). (G) MCF10A acini cultured in 3D SAP gels were analyzed for Robo1 (n = 3 experiments). (H) Robo1 expression in NMuMG cells cultured in either LD detached (Dtch), LD attached (Atch), or HD (n = 3 experiments). For mRNA analysis, expression was normalized to Gapdh of total mRNA. Bars, 30 µm. White asterisks denote individual nuclei. Black asterisks denote significance by t test: *, P < 0.05; **, P < 0.01; ***, P < 0.001. ns, not significant.