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Fig. 6. TIN2 controls growth arrest in mammary epithelial cells. (A) Schematic of wild-type TIN2 (WT TIN2), and N-terminally (TIN2-13) and C-terminally (TIN2-15) truncated forms of TIN2. (B) Expression of TIN2 and TIN2-13 in control and TIN2-13 expressing cells shown on western blots. Lanes contained cells used for infection (control), cells expressing TIN2-13 (TIN2-13), cells infected with empty vector (control vector), cells overexpressing wild-type TIN2 (TIN2), control HT1080 fibroblasts expressing exogenous TIN2 and TIN2-13 (TIN2 +TIN2-13 mixture in HT1080). Arrows indicate the location of the respective bands for TIN2 and TIN2-13. ß-catenin was used as a loading control. (C) TIN2-15 expression in monolayer and 3D culture shown by anti-myc immunostaining (green). Nuclei were counterstained with DAPI. Magnification x1200. (D) Vector control, TIN2-13 and TIN2-15 infected S1 cells were cultured in 3D for 10 days. Shown are phase-contrast images of acini formed by vector control S1 cells and abnormal looking colonies formed by TIN2-13 and TIN2-15 S1 cells. The arrows indicate enlarged and/or irregular multicellular structures. (E) Non-infected S1 cells (control) and vector control, TIN2-13, or TIN2-15 infected S1 cells were cultured in 3D for 10 days. Acini were classified according to six diameter ranges (6-15 µm, 16-25 µm, 26-35 µm, 36-45 µm, 46-55 µm, 56-65 µm). Shown is the percentage of acini in each diameter range out of a total of 400 acini observed in each independent experiment. Three experiments were performed. (F) Immunostaining for the endogenous basement membrane component collagen IV (red) and Ki-67 (green) in vector control or TIN2-15 S1 cells cultured in 3D for 10 days. When proper morphogenesis occurs, acini are surrounded by a continuous basement membrane and >90% of the cells arrest proliferation. One nucleus positive for Ki-67 is seen out of ten nuclei in vector control; five nuclei positive for Ki-67 are seen out of 14 nuclei in TIN2-15. Arrows indicate the absence of collagen IV around part of the TIN2-15 colony. (G) GFP-S1 cells organized in an acinus (left panel). Immunostaining for the endogenous basement membrane component collagen IV (red) (central panel) and 6-integrin (green) and ß-catenin (red) (right panel) in hTERT-expressing S1 cells cultured in 3D for 10 days. When proper morphogenesis occurs, in addition to the continuous basement membrane, acini display the localization of 6-integrin at the basal cell membrane (against the basement membrane) and ß-catenin at cell-cell junctions. (H) Immunostaining for TIN2 (red) in control or TIN2-15 S1 cells. In the control acinus, eight of nine nuclei, identified by DAPI staining, have a large TIN2 domain (arrowheads). In the acinar structure formed by TIN2-15-expressing cells, four of thirteen nuclei show one or two large TIN2 domains (arrowheads) and one nucleus shows completely fragmented TIN2 domains (arrow). Bar, 25 µm.

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