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Fig. 2. TG2 expression level modulates collagen lattice reorganisation by fibroblasts. hTERT immortalised HCA2 fibroblasts were stably transfected with mock, TG2 sense, TG2 antisense and TG2 C277S mutant expression constructs. (A) Transfected fibroblasts were extracted with 0.25 M sucrose/1% Triton X-100 to harvest the soluble TG fraction (S) and subsequently with 1% SDS/1 mM EDTA/1 mM DTT-containing buffer to solubilise the particulate TG fraction (P). 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 right. Note, 5% and 25% of total protein extracted was applied for soluble and particulate fractions, respectively. (B) TG activity expressed by transfected fibroblasts was determined by incorporation of [³H]putrescine into N,N-dimethylcasein for 30 minutes at 37°C. The results from four independent measurements are presented as the mean±s.d. (C,D) Transfected 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). (D) Macroscopic images of lattices at day 3.

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