Fig. 2

Lack of YAP activation with enhanced 3D culture stiffness. a Effects of 3D culture stiffness. b MCF10A cells encapsulated in 3D hydrogels. c Encapsulated cells treated with Leptomycin B (LepB). Arrows indicate nuclei with YAP. Bars: 20 µm. d YAP quantification from 3D and 2D (control) culture conditions. **p < 0.05, one-way ANOVA followed by Tukey post hoc comparison tests, symbols represent each cell, n = 6–24 cells per hydrogel, bars represent mean ± SEM. Measurements were taken from distinct samples. e Western blot analysis of p-YAP (S127) from 3D culture. p38 was used as a loading control. Quantification of bands (p-YAP/total YAP/p38) below each lane. f RNA-seq of YAP target genes (as identified by Dupont et al., 2011) in 3D culture. g RNA-seq of canonical YAP target genes in 3D culture. n.s. not significant, unpaired two-sided t test, symbols represent each hydrogel, n = 2 hydrogels, bars represent mean ± SD. Measurements were taken from distinct samples. h Western blot analysis of dox-inducible MCF10A::Cas9/sgGAL4 or sgYAP cells. Quantification of bands (YAP/p38) below each lane. i CRISPR/Cas9 cells encapsulated with dox. Bars: 10 µm. j Proliferation of cells from i. k Invasiveness of cells from i as measured by cell cluster circularity. Only cells verified by IF for KO were assayed. l Set of genes regulated by enhanced stiffness in IPNs also upregulated in DCIS patient samples. Symbols represent individual genes. Most highly enriched gene (S100A7) highlighted in red. m Western blot analysis of cells from 3D culture for S100A7. Quantification of bands (S100A7/p38) indicated below each lane. n S100A7 staining in primary tissue. Bars: 50 µm