Figure 5. Evidence that circadian components of the estrogen-regulated circuit are implicated in breast acinar morphogenesis. (A) Immunocytochemistry followed by confocal microscopy showing control HME1 acini after 12 d of three-dimensional (3D) culture on reconstituted basement membrane (Matrigel). In the top panel, the organization of the DAPI-stained nuclei (blue) indicates whether the acini are hollow, the presence of an outer layer of integrin (green) indicates baso-lateral polarity, while the presence of the Golgi apparatus (red) in the inner portion of the acinus is a sign of apico-basal polarity. The percentage of acini with the displayed morphology is indicated in the upper left corner. In the bottom panel, immunostaining with anti-ERA (green) shows ERA expression both in the cytoplasm and, to a lesser extent, in the DAPI-stained nuclei (blue). The lower inset shows an enlargement of the framed area. (B) The top panels show the aberrant morphology of the HME1-shERA (left), HME1-shPER2 (middle) and HME1-shBMAL1 (right) acini, with the relative percentage. The bottom panels show that HME1-shERA, HME1-shPER2 and HME1-shBMAL1 aberrant acini differ in ERA expression.