Figure 1.

Immortalization of primary human tongue keratinocytes. (A) Two batches of primary human tongue keratino-cyte (HTK), termed HTK1 and HTK3, were transduced with retroviruses expressing HPV 16 E6 and E7 for the HPV-positive OSCC model. After selection, cells were harvested and subjected to SDS-PAGE. Western blotting confirmed expression of the two transgenes in the resultant cell populations and showed endogenous expression of p53. (B) HTK1 and HTK3 were infected with lentiviruses expressing mutant CDK4, cyclin D1 and TERT for the HPV-negative OSCC model. Western blotting confirmed expression of the two transgenes in the resultant cell populations and showed endogenous expression of p16^INK4a. (C) Telomerase activity of primary and immortalized HTK cells was measured by the TRAP assay. Hela cells, eight tandem repeats of the teiomeric sequence (TSR8) and CHAPS buffer alone (NC) were used as controls. (D) Growth curves of HTK1 and HTK3 cell lines. Day 0 is the time when the immortalizing genes (mutant CDK4, cyclin D1 and TERT) or HPV 16 E6E7 were transduced.