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Fig. 2. AG1478-induced phenotypic reversion of malignant cells is accompanied by partial tissue polarity and the re-establishment of the organization of markers of higher order nuclear structure. Malignant T4-2 cells were treated either with the reverting agent AG1478 or vehicle DMSO for 10 days in 3D culture in the presence of MatrigelTM and labeled for differentiation markers collagen IV, 
6-integrin, 
-catenin, mucin-1, and Ki67, and for nuclear proteins H3K9m, H4K20m, NuMA, SC35 and lamin B. (A) Immunostaining for basal polarity markers collagen IV (green) and 6-integrin (green), lateroapical polarity marker -catenin (red), and apical polarity marker mucin-1 (red) in malignant T4-2 cells (top panel) and reverted RT4-2 cells (bottom panel). Drawings represent the organization of T4-2 and RT4-2 cells after 10 days of 3D culture. Nuclei are represented in black. (B) Immunostaining for cell cycle marker Ki67 (green). Nuclei are counterstained with DAPI (blue). Bar graph shows the percentage of cells in the cell cycle based on Ki67 expression. (C) Organization of heterochromatin markers H3K9m (red) and H4K20m (red) in the nuclei of malignant (T4-2) and reverted (RT4-2) cells. Arrows indicate the concentration of H3K9m and H4K20m around the nucleolus (*) in differentiated cells. (D) Organization of coiled-coil protein NuMA (green), splicing factor speckle marker SC35 (green), and lamin B (red) in the nuclei of malignant (T4-2) and reverted (RT4-2) cells. The arrowhead indicates the formation of enlarged NuMA foci in mid-nucleus in RT4-2 cells. SC35 is organized into large domains in the nucleus of RT4-2 cells (a dotted line delineates the cell nucleus). The arrows point to the twisted shape, as shown by lamin B staining, of the nucleus in malignant and reverted cells. (E) Left histogram: Percentage of nuclei in S1 acini, T4-2 tumor nodules, and RT4-2 spheroids with nuclear proteins displaying a distribution characteristic of acinar differentiation. Drawings show the distributions of H4K20m, H3K9m and SC35 representative of acinar differentiation. Right histogram: ratios of cells with differentiation-like distribution of NuMA over cells with nondifferentiation-like distribution of NuMA in the S1, RT4-2 and T4-2 populations.*P<0.01. Bars, 5 µm.
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