(a)
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Figure 2: The elevation of cellular ROS levels in replicative aged HDFs through activation of PI3K, PKC, and NADPH oxidase. (a) Blockage of aging-induced increase of ROS by incubating 55 PD HDFs with medium alone or with 100 nM wortmannin or 100 nM calphostin for 30 min. Blockage was measured by flow cytometry using DCF-DA. (b) Blockage of aging-induced increase of NADPH oxidase activity by wortmannin and calphostin C. NADPH oxidase activity was measured by lucigenin activity with medium alone or with 100 nM wortmannin or 100 nM calphostin C in the presence of 500 μM NADPH. (c) Aging-induced increase of PKCζ and Akt phosphorylation. Various PDs of HDFs were lysed, and western blots for phospho-Akt, phospho-PKCζ, and β-actin were performed with the cell lysates (see Methods). (d) Inhibition of PKCζ phosphorylation by wortmannin. 35 PD HDFs were treated with 100 nM wortmannin for the indicated times and the cells were analyzed with western blots for phospho-PKCζ and β-actin. (e) Inhibition of gp91phox and p67phox expression by wortmannin. 55 PD HDFs were treated with 100 nM wortmannin for the indicated times. The cells were analyzed by western blotting for gp91phox, p67phox, and β-actin. Error bars, SD; in each group. , compared with 55 PD.