Figure 1

(a) Summary of the changes in gene expression in untransformed and v-Src-transformed MDCK cells grown in 2D and 3D environments at various temperatures to confluency or cell clusters. The mRNA was harvested from the specimens, converted to cDNA and used to probe the Affymetrix Canine arrays as described in Materials and Methods. We took three specimens with epithelial phenotype in 2D and 3D environment: (1) untransformed MDCK cells, (2) v-Src-transformed MDCK cells grown at non-permissive temperature, +40.5°C and (3) v-Src-transformed MDCK cells grown at +35°C with Src activity inhibited by pp2 and compared the changes in gene expression to v-Src-transformed MDCK cells grown at permissive temperature, +35°C without inhibitor. The changes which took place at the same direction in all three comparisons were considered specific to Src activation. The results for 2D environment are shown in Supplementary Table 1 and 3D environment in Supplementary Table 2 in the supplement part of this work and the highlights are indicated in (a). (b) The upper panel shows immunoblot analysis of expression of Src in cell lysates from v-Src-transformed MDCK cells grown to confluency at 2D at +35°C (lanes 1, 3 and 5) or +40.5°C (lanes 2, 4 and 6) and the lower panel shows expression of Src in v-Src-transformed MDCK cells grown to confluency at 2D at +35°C (lanes 1, 3 and 5) and untransformed MDCK cells grown to confluency at 2D at +37°C (lanes 2, 4 and 6) visualized with a set of antibodies to Src: 36D10 towards total c-Src, pY416 towards Src phosphorylated on tyrosine 416, EC10 towards avian Src. A 60 kDa band corresponding to total c-Src was observed in both untransformed and v-Src-transformed MDCK cells grown at 2D (lanes 1 and 2), to activated v-Src in v-Src-transformed MDCK cells grown to confluency at 2D at +35°C (lane 3) and faintly in v-Src-transformed MDCK cells grown to confluency at 2D at +40.5°C (lane 4 in upper panel) and to avian Src in v-Src-transformed MDCK cells grown to confluency at 2D at +35°C (lane 5 upper and lower panel) or at +40.5°C (lane 6 upper panel).