Figure 7.

Ectopic expression of EPO enhances growth in Hep2 cells. (A) The 614-bp full-length ORF of EPO was cloned in pCMV-Tag1, and 1 µg of plasmid DNA and vector control were transfected in the HNSCC cell line Hep2. Cells were grown under normoxia or hypoxia and selected with 1.25 mg/mL G418 for 3 weeks. Colonies were stained with Giemsa, and colony numbers were determined in triplicates. (B) Three clones re-expressing EPO and 3 control clones were picked, and the growth rate was analyzed. Therefore, 50,000 cells/mL (= 1) each were plated in 6-well plates with 1 mg/mL G418, and cell numbers were counted after 48 hours of normoxia or 72 hours of hypoxia. (C) Anchorage-independent growth was analyzed in 0.3% soft agar. Three stable transfected clones were each seeded in 0.3% agarose. Experiments were performed in triplicates with 5,000 cells per plate under selection with 1.25 mg/mL G418. The colony sizes of 50 clones were measured using a microscope after 4 weeks.