Figure 4

Aging/aged and GL TM cells and TM cells exposed to oxidative-stress displayed elevated cell senescence markers, p16, p21 and SA-β-gal activity with reduced hTERT expression and activity. (A) Western analysis showing increased expression of senescence markers, p16 and p21 in old normal and GL cells compared with younger normal TM cells. (B) Western analysis revealed that normal TM cells facing oxidative-stress showed further increases in levels of p16 and p21 in an age-dependent manner. (C) Lysate samples containing equal amounts of protein from normal TM cells of different ages and GL TM cells were incubated with SA-β-gal substrate, and SA-β-gal activities were measured and presented in the form of histograms. Values are mean±S.D. of three independent experiments. A significant age-dependent increase in the SA-β-gal activity was observed in aging/aged, and GL TM cells (*P<0.001). (D) Oxidative-stress increased levels of SA-β-gal activity in TM cells. Lysate was prepared from normal TM cells of different ages after exposure to H₂O₂, and was processed for SA-β-gal activity assay. Histogram values are mean±S.D. of three independent experiments, each with triplicate wells. An age-dependent significant increase in levels of SA-β-Gal activity was observed (*P<0.001), suggesting that oxidative-stress promotes cell senescence. (E) Expression analyses showing reduced expression of Prdx6 was related to reduced expression of hTERT (telomerase) expression in GL cells (Ea) Cellular extracts from normal (4M old subject) and glaucomatous (56Y old subject) TM cells having equal amounts of protein were immunoblotted with Prdx6 or hTERT antibodies. β-actin was used as loading control. (Eb) Total RNA was isolated from the same subjects’ TM cells, and processed for qPCR for hTERT mRNA expression by using specific probe. Histogram values are mean±S.D. of three independent experiments (*P<0.001). (F) Relative telomerase activity was evaluated using quantitative telomerase detection kit (QTD kit, Allied Biotech, Inc.,) in normal aging/aged and GL TM cells as indicated. Expression of telomerase activity was found to be age-dependent, and was highly reduced in GL TM cells as indicated. Histogram values represent mean±S.D. of three independent experiments (*P<0.001).