Figure 4

(a) Confocal microscopy images of E-cadherin double stained with α-catenin, β-catenin and p120catenin through a central part of the monolayer of v-Src-transformed MDCK cells grown at +40.5°C in cell culture dishes to confluency. E-cadherin is shown in green, catenins in red and nuclei blue. The cells have an epithelial morphology, cadherin is at lateral membranes co-localizing with all catenins and occasionally it is seen at Golgi area. (b) Confocal microscopy images of E-cadherin double stained with α-catenin, β-catenin and p120catenin through a central part of the monolayer of v-Src-transformed MDCK cells grown at +35.0°C in cell culture dishes to confluency. E-cadherin is shown in green, catenins in red and nuclei blue. The cells have a fibroblastic morphology, cadherin is at Golgi area colocalizing with β-catenin and p120catenin, but not with α-catenin. Bar: 20 μm. (c) Confocal microscopy images of E-cadherin double stained with actin and β-catenin and caspase-3 double stained with ZO-1 through a central part of the cell cyst of untransformed MDCK cells grown at +37°C in a mixture of collagen I and Matrigel. E-cadherin and caspase-3 are shown in green, actin, β-catenin and ZO-1 in red and nuclei blue. The cells have formed a spherical cyst with a lumen inside. Cadherin is at lateral membranes (arrow head) co-localizing with β-catenin, actin is delineating the apical surfaces (arrow), caspase-3 is showing the remnants of apoptotic cells captured within the lumen (arrow head) and ZO-1 is at tight junctions close to the apical surface (arrow). Bar: 20 μm. (d) Confocal microscopy images of E-cadherin double stained with actin and β-catenin and caspase-3 double stained with ZO-1 through a central part of the cell cluster of v-Src-transformed MDCK cells grown at +35°C in a mixture of collagen I and Matrigel. E-cadherin and caspase-3 are shown in green, actin, β-catenin and ZO-1 in red and nuclei blue. The cells have formed a spherical cluster filled with cells. Cadherin is at lateral and basal membranes (arrow head) co-localizing with β-catenin, actin is delineating the cell membranes of cells captured inside the cluster (arrow), caspase-3-staining is not visible and ZO-1 is in short pieces at cell membranes through the cell cluster (arrow head). (e) Immunoblot analysis showing expression of cadherin in Triton soluble (s) and cytoskeletal (p) fractions of untransformed and v-Src transformed MDCK cells grown to confluency at 2D cell culture dishes or in 3D Matrigel visualized with E-cadherin antibody to extracellular domain (rr1, lanes 1 and 2), E-cadherin antibody to the cytoplasmic domain (E-cadherin, lanes 3 and 4) and pan-cadherin antibody to the cytoplasmic domain (lanes 5 and 6). A 120 kDa band corresponding to E-cadherin was observed in all specimens with all three cadherin antibodies, the majority of cadherin being in the Triton-soluble fraction. A second band with a slightly higher molecular weight was detected with E-cadherin antibody and two extra bands with pan-cadherin antibody. (f) Immunoblot analysis showing expression of survivin in cell lysates from untransformed and v-Src transformed MDCK cells grown to confluency at 2D cell culture dishes or in 3D Matrigel visualized with survivin antibody diluted to 1:500 (lanes 2, 4, 7 and 9) or 1:1000 (lanes 3, 5, 8 and 10). A 16 kDa band corresponding to survivin was observed in both untransformed and v-Src-transformed cells grown at 2D (lanes 2, 3, 7 and 8). There was hardly anything visible in the lanes from untransformed cells grown in 3D Matrigel (lanes 4 and 5) and a slight band in v-Src-transformed cells grown in 3D Matrigel at +35°C (lanes 9 and 10). Lanes 1 and 6 show the 15.2 kDa standard.