Compromised epithelial integrity functionally interacts with Myc. (
a) Confocal images of day-10 MCF10A structures expressing chronically activated Myc and shRNAs to DVL3, RHOA or MOESIN. Stainings for E-cadherin and nuclei visualize the acinar ultrastructure. Scale bar 20 μm. ( b) Changes in the size of acinar structures were quantitated as in Figure 2b. The area value for control shRNA plus Myc expressing acini was set to 1, and the other area values (averages) are shown as relative to control. Data are shown as fold change relative to control and s.e.m. Dashed lines indicates the 20% fold change relative to control (cutoff). ( c) Changes in the acinar symmetry (circularity) quantitated as in Figure 2c. Data are shown as fold change relative to control and s.e.m. Dashed lines indicates the 10% fold change relative to control (cutoff). ( d) Scatter plot shows the relative changes in acinar size and symmetry values corresponding to 22 shRNAs indicated in Figure 3b. Green dot: Myc plus control shRNA. Red dots denote acini expressing Myc plus hEIR shRNA and exhibiting 20% increase in size and 10% decrease in symmetry values. Blue dots indicate hEIR shRNAs resulting in 20% decrease in size and 10% decrease in symmetry. ( e) Synthetic lethal interaction between Myc and knockdown of RHOA. The acini were grown on Matrigel in the presence of chronic Myc activation and shRNA for RHOA or CDH1 for 10 days. Active caspase-3 antibody visualizes apoptosis and the basal α6-integrin acinar borders. Note the active caspase-3 positivity in RHOA-deficient but not in CDH1-deficient acini. Scale bar 20 μm. ( f) Quantification of active caspase-3 (apoptosis) in RHOA-deficient acini with or without Myc activation. The acini were grown as in panel ( e). Mean and s.d. were calculated from three independent experiments and P-values by Student’s t-test. In 3D assays, at least 50 acini were counted in each experiment. ** P<0.05. NS, not significant.