Figure 6

Overexpression of Prdx6 repaired GL TM cell integrity by blocking accumulation of ROS, LPO contents and oxidative DNA damage and reducing SA-β-gal activity. (A) Photomicrograph of GL TM cells expressing empty vector showing more abnormal morphology and slow growth rate (a and c) than GL-TM cells overexpressing Prdx6 (b and d). GL TM cells were transfected with GFP-vector or GFP-Prdx6 and enriched with antibiotic. (Ae) represents Western analysis of transfectants. (B) Levels of ROS intensity in GL-TM cells overexpressing Prdx6 and GFP vector (Panel B, black bar versus gray bar). ROS intensity was quantified with CellRox. Histogram values are mean±S.D. of two independent experiments (*P<0.001). (C) Prdx6 blunted lipid peroxidation in GL TM cells. Cultured GL TM cells overexpressing Prdx6 or GFP vector for 72 h were processed for LPO assay as described elsewhere.²⁸ A significantly reduced level of LPO contents was observed in GL TM cells overexpressing Prdx6 compared to GFP vector expressing ones (black bar versus gray bar). Histogram values are mean±S.D. of two independent experiments, (*P<0.001). (Da) Delivery of transduction domain (TAT)-linked Prdx6 internalized in GL-TM cells (D, ai), and blunted the process of oxidative DNA damage (Da). 8-OHdG level was measured in normal and GL TM cells pre-transduced with TAT-HA-Prdx6 or TAT-HA-Vector protein (10 μg/ml) using OxiSelect TM oxidative DNA damage ELISA, as described in ‘Materials and Methods’ section. 8-OHdG levels were significantly reduced in GL TM cells supplied with Prdx6 compared with control (black bar versus gray bar) in 5 days. (Db and c) GL or aged TM cells overexpressing Prdx6 showed resistance against oxidative-stress-induced DNA damage. GL or aged TM cells overexpressing Prdx6 or its empty vector were subjected to H₂O₂-induced oxidative-stress and DNA oxidation was measured. Overexpression of Prdx6 offered more protection against DNA damage induced by H₂O₂ than did empty vector in GL (Db; open bars versus gray bars) and normal aged (Dc; gray bars versus black bars) TM cells (*P<0.001). Values represent the mean±S.D. from two experiments, and are presented as histograms (*P<0.001). (Ea and Eb) GL TM cells overexpressing Prdx6 showed significantly diminished levels of SA-β-gal activity. GL TM cells overexpressing Prdx6 or vector counterpart were cultured and processed for SA-β-gal ELISA assay (Ea), as well as SA-β-gal staining (Eb). GL TM cells overexpressing Prdx6 displayed significantly lower SA-β-gal activity compared to vector transfected control (black bar versus gray bar), Histogram values are mean±S.D. of two independent experiments (*P<0.001). Photomicrographs are representative of SA-β-gal staining in GL TM cells overexpressing Prdx6 versus cells expressing vector only (Eb; left panel versus right panel).