Fig. 6. Partially differentiated acini enter the cell cycle upon NuMA alteration regardless of the method used to induce incomplete polarity. (A) Histogram of the percentage of S1 multicellular structures obtained following 9 days in collagen I and 4 days in Matrigel^TM (black bars) that possess intact collagen IV and 6-integrin staining, and apical location of ZO-1 compared with S1 cells cultured in Matrigel^TM (shaded bars) for 13 days. (B) Non-neoplastic S1 cells were cultured in Matrigel^TM for 10 days to produce basoapically polarized acini (shaded bars) or in collagen I for 9 days before replating in Matrigel^TM for 24 hours (black bars) 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. Shown is the percentage of cells positive for cell cycle marker Ki67. 300 cells were scored in each replicate in three independent experiments. (C) Histogram of the percentage of S1 multicellular structures with ZO-1 apically located after treatment with EGTA for 24 hours. (D) Acini and multicellular structures were treated with EGTA for 1 hour, then briefly permeabilized with digitonin and incubated with NuMA antibodies (NuMA mAb) or nonspecific immunoglobulins (IgGs) for 3 days. EGTA was only left in the culture medium during the first 24 hours of antiNuMA antibody treatment. Shown is the percentage of cells positive for cell cycle marker Ki67. 300 cells were scored in each replicate in three independent experiments. ^*P<0.05. (E) Dual staining for NuMA (red) and Ki67 (green) in acini formed by cells transfected with 50 nM siRNA NuMA or 50 nM nontargeting siRNA and cultured in 3D for 8 days. Nuclei are counterstained with DAPI (blue). Arrowheads indicate strong Ki67 staining in nuclei that lack NuMA staining.