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. 2002 Sep 4;87(6):645–653. doi: 10.1038/sj.bjc.6600544

Figure 4.

Figure 4

(A) Effect of ShcY317F on TRK-T3 induced PC12 differentiation. PC12 cells were cotransfected with TRK-T3 and ShcWT (a), ShcY317F (b) or pCGN (c) plasmids, together with VGF8-luc and Renilla plasmids, as described in Materials and Methods and scored for the presence of neurites 3 days later. As control, untreated (d) and NGF-stimulated (50 ng ml−1) (e) PC12 cells are shown. After morphological analysis, transfected PC12 cells were lysates and subjected to immunoblot using anti-TRK and anti-HA antibodies to detect the level of transfected proteins. To quantify the TRK-T3 differentiating activity both luciferase activities were measured using the Dual-Luciferase reported assay system (Promega). The values were expressed relative to Renilla luciferase activity for normalisation. The results presented are an average of three experiments. (B) Microfocus formation assay. One hundred cells from focus NF797 (NIH3T3 cells transformed by TRK-T3 oncogene) transfected with pCGN vector, ShcWT or ShcY317F constructs were combined with 1×105 NIH3T3 cells in 10-cm dishes, and cultured for 2 weeks in medium containing 5% calf serum. The foci were counted after GIEMSA staining. (C) Transforming activity of TRK-T3 in NIH3T3, NWT (NIH3T3 cells stably expressing ShcWT) and NY317F (NIH3T3 cells stably expressing ShcY317F) cells. Transfection and foci selection were performed as described in Materials and Methods. Foci and G418 resistant colonies were either fixed and GIEMSA stained or isolated for further analysis after two weeks of selection. (D) Effect of hygromycin on TRK-T3 foci formation in NWT and NY317F cell lines. Foci selection was performed in 5% serum medium supplemented or not with 400 μg ml−1 of G418 (selectable marker contained in the TRK-T3 expression vector) in the absence or presence of 25 μg ml−1 hygromycin (selectable marker contained in the Shc expression vector). Foci were scored after 2 weeks of selection. For each cell line, the number of foci per μg DNA selected in the absence of hygromycin was set at 100%.