Immortalization by retroviral transduction. (A) Immunoblot analysis of human telomerase reverse transcriptase (hTERT), simian virus 40 large T antigen (SV40LTag) and P53 expression in cell lysates from mortal and immortal cells at extended culture periods (passage 20 (P20) for mortal and P40 for immortal cells). Cells were immortalized at P5. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as a loading control. (B) Telomeric repeat amplification protocol assay demonstrating telomerase activity in immortalized donor 4 (D4) and D5 annulus fibrosus (AF) and nucleosus pulposus (NP) cells (lanes 4 and 6, respectively) compared to control cultures (primary cultures (P5), lanes 3 and 5). Lanes 1 and 2 represent positive controls for PCR amplification (TSR8 oligonucleotide synthesis reagent in the TRAPeze kit) and TERT, respectively. Lane numbers are indicated below the images. Fragment size is indicated in base pairs (bp). #Internal PCR standard. *Aspecific PCR products. (C) Spectrometric quantification of Crystal violet staining assays reflecting relative proliferation of immortalized and primary cells at each indicated time point (compared to baseline (t = 0)). P20 cultured primary cells had a negligible proliferative index as they approached 50 population doublings (PDLs). The immortalized cells had undergone approximately 160 PDLs (corresponding to ±40 passages) postimmortalization at this time point. hT/LT denotes cells transduced with hTERT (hT) and SV40LTag (LT).