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. 2014 Apr 25;3(5):397–407. doi: 10.1242/bio.20147005

Fig. 6. Hydra (hy) and viral Myc hybrid proteins display cell transforming activity.

Fig. 6.

(A) Schematic depiction of the v-myc (blue), and of hy/v-myc and v/hy-myc hybrids' coding regions (Hydra myc1 and myc2 sequences shown in yellow and orange, respectively). The coding regions were inserted into the unique ClaI site of the Bluescript (pBS) vector for in vitro transcription/translation, or into the replication-competent retroviral pRCAS vector used for DNA transfection into primary quail embryo fibroblasts (QEF). (B) In vitro translation of [35S]methionine-labeled proteins encoded by the pBS constructs shown in panel A. (C) Growth of QEFs transfected with the RCAS constructs shown in panel A. After several passages, equal numbers (Y0 = 7.5×105) of cells were seeded onto 60-mm dishes and cell numbers (Y) were determined after 3 and 6 days. Standard deviations are indicated by vertical lines. (D) Immunoprecipitation of endogenous c-Myc and ectopic Myc proteins using 1.0×107-cpm aliquots of lysates from [35S]methionine-labeled QEFs transfected with the pRCAS constructs shown in panel A, and antibodies directed against amino-terminal [N] or carboxyl-terminal [C] segments of v-Myc, or normal rabbit serum (NRS). Proteins were resolved by SDS-PAGE (10%, wt/vol). (E) Top: QEFs on 60-mm dishes were transfected with 4-µg aliquots of DNA from the pRCAS constructs shown in panel A, kept under agar overlay for 2 weeks, and then stained with eosin methylene blue. Numbers of foci per dish are indicated. Middle: QEFs were transfected with the pRCAS constructs shown in panel A and passaged several times. The doubling times (Td) of the cell populations, which are indicated below the phase-contrast micrographs were determined from the average proliferations rates (k) obtained in panel C according to Y = Y0(ek×t) and Td = ln2/k. Bottom: equal numbers (1.0×105) of transfected and passaged cells were seeded into soft agar and incubated for 2 weeks. Numbers of colonies per 1,000 cells seeded are shown below the bright-field micrographs. Standard deviations are indicated by ± signs. Scale bars: 1 mm (E, top), 50 µm (E, middle), 200 µm (E, bottom).