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Fig. 5. S1 cells that form multicellular structures with incomplete tissue polarity are induced to enter the cell cycle upon incubation with NuMA antibodies. (A) Ten day 3D MatrigelTM culture of non-neoplastic S1, malignant T4-2 and reverted RT4-2 cells immunostained for tight junction marker ZO-1 (green). Apical polarization of S1 acini is indicated by the apical location of ZO-1. Arrows indicate the presence of ZO-1 in the nucleus of RT4-2 cells. (B) Immunostaining for collagen IV (green), 
6-integrin (green), 
-catenin (red) and ZO-1 (red) in non-neoplastic S1 cells cultured in collagen I for 10 days (Coll I) or cultured in collagen I for 9 days and replated in Matrigel for 24 hours (Coll I + MG). Multicellular structures in collagen I display alterations in all markers tested, as shown by the absence of continuous peripheral staining of collagen IV, the overlap of 6-integrin and -catenin staining at cell-cell junctions – see yellow – and the diffuse location of ZO-1. By contrast, only ZO-1 staining remains altered in multicellular structures replated in MatrigelTM, as shown by the presence of staining at the basal side of cells (see arrows). (C) Non-neoplastic S1 cells were cultured in MatrigelTM for 10 days to produce basoapically polarized acini (MG) or in collagen I for 9 days before replating in MatrigelTM for 24 hours (Coll I + MG) to produce a population enriched with only basally polarized multicellular structures. Acini and multicellular structures were then permeabilized with digitonin, incubated with NuMA antibodies (NuMA mAb) or nonspecific immunoglobulins (IgGs) for 3 days and immunostained for Ki67 (red) and mouse immunoglobulin (green). Immunostaining for mouse immunoglobulins highlights the nuclear location of NuMA mAb and the cytoplasmic location of nonspecific mouse IgGs. Arrows indicate Ki67-positive cells. Nuclei are counterstained with DAPI (blue). Bars, 10 µm.
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