Figure 3

Aging TM cells exposed to H₂O₂ displayed reduced Prdx6 expression, increased TGFβs and accumulation of ECM proteins with elevated ROS levels and reduced cell viability. (a) Total RNA were isolated from TM cells of different ages exposed to H₂O₂ and processed for real-time PCR with specific primers as indicated. Histogram values are mean±S.D. of three independent experiments, showing significant reduction of Prdx6 expression in aging TM cells, in age-dependent manner (*P<0.001). (b) TM cells obtained from human subjects of different ages were seeded and exposed to H₂O₂ (100 μM) and then processed for cellular extraction. Equal amounts of protein from each sample were resolved onto SDS-PAGE and immunoblotted with specific antibodies as indicated. The same membranes were probed with different antibodies followed by stripping and restripping. (c) Oxidative-stress induced by H₂O₂ increased the oxidative load more in aging TM cells than in younger ones. TM cells were exposed to H₂O₂, and 8 h later processed for intracellular ROS measurement by using 10 μM H2-DCF-DH dye at Ex485/Em530 nm. Plotted values are mean±S.D. of three independent experiments, showing significant increase of ROS in aging TM cells (*P<0.001 compared with respective controls). (d) Viability assay showing that aging TM cells were more sensitive to H₂O₂-induced oxidative-stress, which leads to significant increase in cell death. Cells of different ages were cultured and exposed to 100 μM of H₂O₂ as indicated. Plotted values are mean±S.D. of three independent experiments, showing significantly increased cell death in aging TM Cells (*P<0.001).