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Fig. 4. Alteration of NuMA induces apoptosis in non-neoplastic S1 cells and entry into the cell cycle in reverted T4-2 cells. Non-neoplastic S1 cells and malignant T4-2 cells treated with or without the reverting agent AG1478 were cultured in 3D Matrigel^TM for 10 days, followed by digitonin permeabilization and incubation with antibodies against NuMA (NuMA mAb) or nonspecific mouse immunoglobulins (IgGs) for 3 days. (A) Immunostaining with a FITC-tagged antibody against mouse IgG reveals the location of IgGs in the cytoplasm and NuMA mAb in the cell nucleus of permeabilized RT4-2 and/or T4-2 cells. Arrowheads indicate the location of NuMA at the poles of mitotic spindles in malignant cells. (B) Immunostaining for collagen IV (green, see arrowhead) in RT4-2 and T4-2 multicellular structures. Arrows indicate the lack of continuous collagen IV staining in RT4-2 cells treated with NuMA mAb. (Note that fluorescent staining for NuMA mAb can be seen in the nuclei; both collagen IV and NuMA antibodies are mouse IgG1, thus they are recognized by the same secondary antibody.) Nuclei are counterstained with DAPI (blue). (C) Histograms of the percentage of apoptotic cells. (D) Histograms of the percentage of cells in the cell cycle (Ki67-positive). Immunostaining for NuMA (green) shows evidence of mitotic spindle formation in NuMA mAb-treated RT4-2 cells (see image and arrowhead). A minimum of 300 cells were scored per replicate in three independent experiments. ^*P<0.05. Bars, 5 µm.

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