Fig. 1. Upregulation of TG2 expression in fibroblasts is linked to cell senescence and is independent of culture conditions. (A) Neonatal foreskin, senescent HCA2 and hTERT immortalised HCA2 fibroblasts were seeded into 0.5 mg/ml collagen type I lattices and diametric contraction was measured over a 14-day period. Values are the mean±s.d. (n=3). (B,C) Senescent and hTERT immortalised fibroblasts were grown in monolayer culture (Mono) or seeded into either attached (Attach) or unrestrained (Float) collagen I lattices for 2 days prior to RNA isolation. HCA2 fibroblasts were shown to express 4 iso-enzymes with TG activity (TG1, TG2, TG5 and TG7) by RT-PCR (B). mRNA levels were quantified with the 5' nuclease assay using the TaqMan system and are presented as a mean±s.d. (C). (D) hTERT immortalised HCA2, neonatal foreskin (from two individuals) and senescent HCA2 fibroblasts were extracted with 1% SDS/4 M urea-containing buffer. Cell extracts were separated in 4-20% SDS-PAGE gels and analysed for TG2 expression by immunoblotting with mAb CUB7402. Mol mass standards are indicated on the left. Re-probing of the membrane with antibodies to ß-tubulin (ß-tub) shows equal sample loading (bottom panel). Note, the comparison of pre-senescent to senescent HCA2 cells demonstrates the senescence-associated upregulation of TG2 for an individual fibroblast strain while the comparison of the pre-senescent HCA2 cells to neonatal cells from other individuals shows the level of variability in TG2 expression between individuals.