FIGURE 9.

A, photomicrograph of Smad3^+/+ and Smad3^−/− lens epithelial cells cultured in vitro and validation of their integrity. Smad3^+/+ (a) and Smad3^−/− (b) LECs were isolated from their corresponding mouse lenses and maintained in DMEM plus 10% fetal bovine serum (1). B, integrity of these cells was validated using αA-crystallin antibody (middle), a specific marker of LECs, and Smad3-specific antibody (top) using Western analysis. Bottom, β-actin, an internal control. C–E, unlike Prdx6^−/− cells, Smad3^−/− cells displayed enhanced Prdx6 promoter activity and increased expression of PRDX6 protein. C, a representative of transactivation assay experiments showing CAT activities in Smad3^−/− and Smad3^+/+ cells cultured in serum-depleted media. Cells were transfected with equal amounts of Prdx6 promoter linked to CAT. CAT activity was measured as described under “Experimental Procedures.” CAT activity of deletion mutant constructs in Smad3^−/− cells (dotted bar) and Smad3^+/+ cells (black bars) was monitored. Western analysis (D) and RT-PCR (E) showing expression of PRDX6 protein and mRNA in Smad3^−/− cells, respectively.