Fig. 3. LY294002 induced-phenotypic reversion of malignant cells is accompanied with the re-establishment of basal polarity and higher order nuclear structure. Malignant T4-2 cells were treated either with the reverting agent LY294002 or vehicle DMSO for 10 days in 3D culture in the presence of Matrigel^TM and labeled for differentiation markers collagen IV, 6-integrin, mucin-1 and Ki67, and nuclear proteins H4K20m, NuMA and SC35. (A) Immunostaining for basal polarity markers collagen IV (green) and 6-integrin (green), apical polarity marker mucin-1 (red), and proliferation marker Ki67 (green) in malignant (T4-2) cells (small tumor nodules are shown) and reverted (RT4-2) cells. Nuclei are counterstained with DAPI (blue). (B) Organization of heterochromatin marker H4K20m (red), coiled-coil protein NuMA (green; arrowheads indicate the presence of enlarged foci in mid-nucleus) and splicing factor speckle marker SC35 (green) in the nuclei of malignant (T4-2) and reverted (RT4-2) cells. The dotted line delineates the cell nucleus. Bars, 5 µm.