Extended Data Fig. 7: RAP2 prevents aberrant acinus growth in MCF10A cells on soft matrices.
a, RAP2 deletion selectively enhances HEK293A cell growth on soft matrices. Wild-type and RAP2-KO HEK293A cells were seeded on stiff or soft matrices, and cell numbers were recorded as mean ± s.e.m. every day. Two-way ANOVA test, ****P < 0.0001, wild-type versus RAP2-KO cells cultured at 1 kPa, n = 3 biologically independent samples. b, Model of 3D culture of MCF10A cells. c, RAP2 deletion causes abnormal acinus growth and cell polarity defects in MCF10A cells. Immunofluorescence staining of acini for cell polarity markers, Laminin V and GM130, and YAP and TAZ in wild-type and RAP2-KO MCF10A cells cultured for 6 days. Scale bars, 25 µm. Images are representative of three independent experiments with similar results. d, Western blot showing deletion of MST1 and MST2 in wild-type and RAP2-KO cells. The arrow indicates the specific band for MST1. The image is representative of two independent experiments with similar results. e, Representative images showing acinus formation by various genetically engineered MCF10A cells at 1 kPa. The red arrow indicates aberrant acini. The blue arrow indicates invasive cell morphology. Scale bars, 100 µm. The results are representative of two independent experiments with similar results. f, Quantification of aberrant, oversized, and invasive acini in e. The percentage of the cells is presented as mean ± s.e.m. Two-tailed t-tests were used for statistical analyses of aberrant and oversized clones in n = 3 biologically independent samples. Aberrant: ****P = 0.0000062; ***P = 0.0012; oversized: *P = 0.017 (wild-type versus RAP2-KO) or 0.046 (MST1/2-dKO versus RAP2-MST1/2-KO). For analysis of invasive clones, one-tailed t-test was used as no invasive clones were observed in either wild-type or MST1/2-dKO cells. *P = 0.047 (wild-type versus RAP2-KO) or 0.029 (MST1/2-dKO versus RAP2-MST1/2-KO). n = 3 biologically independent samples. g, Representative images showing acinus formation by various genetically engineered MCF10A cells at 150 Pa. Scale bars, 100 µm. Images are representative of two biologically independent experiments with similar results. h, Quantification of aberrant acini in g. The results are from three biologically independent samples. Two-tailed t-tests were used for statistical analyses of aberrant clones in n = 3 biologically independent samples. ****P = 0.000045; ***P = 0.00011.
