FIGURE 8.

A, PRDX6 delivery to Prdx6^−/− cells restored the Prdx6 promoter activity by attenuating Smad3 binding to RSBE. Prdx6^−/− and Prdx6^+/+ cells were cultured in the presence (4 μg/ml) or absence of PRDX6 (4). Nuclear extract was isolated and incubated with radiolabeled RSBE-1 probe, and a gel shift assay was performed. Binding affinity of nuclear extract from Prdx6^−/− cells supplied with recombinant PRDX6 (lane 3, Cm1; see Figs. 1 and 4 for RSBE-1) was compared with nuclear extract of Prdx6^−/− cells (lane 2) to radiolabeled oligonucleotide probe containing RSBE. B, gel shift assay showing binding of nuclear extract from Prdx6^−/− cells to radiolabeled probe containing NF-κB binding site or its mutant probe (NF-κB-2; see Fig. 7); binding activity of nuclear extracts isolated from cells treated with PRDX6 (lanes 2 and 3) compared with that of cells where TAT-HA-PRDX6 was not added (lane 1). C, Prdx6^−/− cells showing higher expression of ROS. An H2-DCF-DA assay was conducted to monitor the effect of NAC on levels of ROS (C, gray bars). D and E, cells were transiently transfected with different Prdx6-CAT constructs and were cultured with or without PRDX6 for 3 consecutive days (4 μg/ml) or treated with NAC (1 mm), an antioxidant. After 72 h, cells were harvested, and CAT activity was monitored (D, gray bars) or NAC (E, gray bars).