Fig. 7. TGFβ1 treatment inhibits the formation of Rad51-containing nuclear complexes upon repair of DNA damage. Rad51-containing nuclear complex formation in wild-type Mv1Lu cells (A and B) or in Mv1Lu cells stably transfected with Rad51 or empty vector (R51 or Mv1Lu cells, respectively) (C) was evaluated. Mv1Lu cells were treated with TGFβ1 (5 ng/ml) for 24 h, then MMC (500 ng/ml) was added, as indicated. After 1 h treatment with MMC, cells received fresh medium and were cultured for another 5 h, and immuno fluorescence staining with Rad51-specific antibodies was then performed. Cells with more than five nuclear foci were scored as positive (A). Quantification of positive wild-type Mv1Lu cells, Mv1Lu vector-transfected and R51 cells, are shown in (B) and (C). Two hundred cells were analysed for each condition, and the percentage of positive cells is presented. *, p <0.01, cells treated with TGFβ1 and MMC, compared with cells treated only with MMC. #, p <0.01, R51 cells compared with vector-transfected Mv1Lu cells treated with MMC and TGFβ1. (D) The expression level of Rad51 protein in Mv1Lu vector-transfected and R51 cells after TGFβ1 treatment was investigated by western blotting using Rad51 antibodies. (E) MMC does not affect inhibition of Rad51 protein expression after TGFβ1 treatment for 24 h, as evaluated by western blotting of Mv1Lu cell extracts. Cells were treated with MMC (500 ng/ml) for 1 h. To monitor equal sample loading, membrane was probed with anti-α-tubulin antibodies. The migration positions of Rad51 and α-tubulin are indicated. (F) Proteasome inhibitors MG-132, lactacystin and ALLN block TGFβ1-dependent inhibition of the formation of Rad51 nuclear complexes. Mv1Lu cells were treated with TGFβ1 and proteasome inhibitors, as indicated in Figure 5D. Treatment with MMC and detection of Rad51 nuclear complexes were performed as indicated in (A), (B) and (C) above. *, p <0.05, cells treated with MMC and TGFβ1 compared with cells treated with MMC only.