Figure 2

From: Par6G suppresses cell proliferation and is targeted by loss-of-function mutations in multiple cancers

Figure 2

Functional identification of genes regulating epithelial structure and cell cycle restriction. (a) Confocal images of hyperplastic and misshapen day-10 MCF10A epithelial structures. The acinar structures express shRNAs targeting MOB1B, GKS3B and DVL3 (shScra; scrambled control). The structures were immunostained for E-cadherin and nuclei to visualize acinar ultrastructure. Silencing of MOB1B and GKS3b leads to formation of large and symmetric acini whereas silencing of DVL3 to large and misshapen acini. Scale bar 20 μm. (b) Changes in acinar size (quantitated as area by Image J) of day-10 MCF10A epithelial structures expressing the indicated shRNAs. The area of control acini was set to 1, and the other area values (averages) are presented as relative to control. The averages represent at least 30 acinar structures per each shRNA. Twenty shRNA constructs resulted in 0.2-fold change (cutoff) in epithelial structure size. Data are shown as fold change relative to control and s.e.m. Dashed lines indicates the 20% fold change relative to control (cutoff limit, see Supplementary Information). (c) Changes in symmetry (circularity) of day-10 MCF10A structures. The structures were quantitated as in panel (b). Six constructs resulted in >10% decrease in symmetry (cutoff limit, see Supplementary Information). Data are shown as fold change relative to control and s.e.m. (d) Scatter plot shows the relative changes in acinar size and symmetry values corresponding to 20 shRNAs indicated in panel (b). Green dot: control shRNA-transduced acini. Red dots: shRNA-transduced acini exhibiting >20% increase in size and >10% decrease in symmetry. (e) Cell polarity in hyperplastic and misshapen DVL3-deficient MCF10A epithelial structures. The structures were immunostained for E-cadherin and nuclei to visualize the ultrastructure and for golgi marker GM130 and α6-integrin to visualize the apico-basal polarity. Scale bar 20 μm. (f) Silencing of specific hEIR genes prevents epithelial cell cycle restriction. The MCF10A acini grown in Matrigel for 18 days were immunostained with proliferation marker Ki-67. Over 70% of control shRNA-transduced acini were Ki-67 negative, indicating quiescence. The acini containing 1 Ki-67-positive cells were scored as Ki-67 positive. The acinar groups with enhanced Ki-67 positivity are grouped according to hTSG-like or nTSG-like phenotype. Mean and s.d. were calculated from three independent experiments and P-values by Student’s t-test. In 3D assays, 50 acini were counted in each experiment. *P<0.5; **P<0.05.