αB-Crystallin overexpression disrupts mammary acinar morphology. (A) Immunoblot of day-12 acini formed by parental MCF-10A cells and MCF-10A pools (pLXSN, αB-WT, and αB-3XSE). αB, αB-crystallin. (B) Phase contrast images of day-12 acini formed by parental MCF-10A cells, MCF-10A pools, or an MCF-12A pool stably expressing αB-WT. (C) Day-12 acini formed by parental MCF-10A cells or pools were scored for abnormal morphology (enlarged structures with filled lumens), diameter, and cell number per cross section (mid-acini). (D) Silencing αB-crystallin in MCF-10A–αB-WT cells by RNAi suppressed the abnormal morphology of αB-WT acini. (E) Day-12 pLXSN, αB-WT, or αB-3XSE acini were immunostained with antibodies to αB-crystallin (green) and GM130, an apical marker of polarized epithelium (red, upper panels), or the integrin β4 subunit, a basolateral marker (red, lower panels). Confocal images of mid-acini cross sections are shown. Inset: Higher magnification of the same acinus (magnified ×2.5 of original). (F) Percentage of day-12 acini with polarized epithelium. (G) pLXSN, αB-WT, and αB-3XSE acini were immunostained with antibodies to αB-crystallin and active caspase-3 (upper panels, day 8) or Ki-67 (lower panels, day 12). (H) Percentage of acini that contained ≥2 cells positive for active caspase-3 (day 8) or Ki-67 (day 12). In A–H, data are mean ± SD (n = 3, except RNAi in D and laminin-5 polarity in F, which were n = 2, each in duplicate). *P < 0.05 versus control; ***P < 0.001 versus pLXSN. Scale bars: 100 μm (B and D); 50 μm (E and G).