Figure 8.

PP-1 and PP-2A protect lens epithelial cells from oxidative stress-induced apoptosis. A: western blot analysis confirms the overexpression of PP-1α (a) and PP2Aα (b) in αTN4–1 cells. The stable clones expressing the pCI-neo vector, pCI-PP-1α, or pCI-PP-2Aα were obtained through G418 screen (400 µg/ml). The expression levels of PP-1α and PP-2Aα were determined by western blot analysis using 100 µg of total protein extracted from pCI-neo or PP-1α-tranfected αTN4–1 cells (a) and from pCI-neo or PP-2Aα-transfected αTN4–1 cells (b). B: Results of the MTT assay is shown in the chart. The MTT assay is described in the Methods section. Note that PP-1α displayed a stronger ability against oxidative stress-induced cell death than PP-2Aα did. C: Hoechst staining analysis of the pCI-neotransfected cells (a, d), PP-1α-tranfected αTN4–1 cells (b, e), or PP-2Aα-transfected αTN4–1 cells (c, f) without treatment (a, b, c) or treated by 85–95 µM H2O2 (d, e, f) for 3.5 h is pictured. Hoechst staining was conducted as previously described [14,17]. Note that after treatment, the apoptotic cells became either dissociated from the culture plate (thus leaving empty space in the culture dish) or condensed. D: Western blot analysis of the p53 hyperphosphorylation at Ser-15 and Bak expression. The western blot analysis was conducted as described before [23]. Note that in both PP-1α and PP-2Aα-transfected cells, hydrogen peroxide-induced hyperphosphorylation of p53 at Ser-53 and Bak upregulation were obviously attenuated.