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Figure 4: ROS generation in replicative aged HDFs is induced by the activation of PI3K signaling through aging-induced downregulation of PTEN and activation of PI3K/PKC. (a) The inhibition of aging-induced increases in PIP3 levels by PTEN gene transfer. Replicative aged HDFs (55 PD) were cultured in standard medium and infected with Ad/LacZ and Ad/PTEN. After 48 h, the cells were fixed with 4% paraformaldehyde, incubated with anti-PIP3 antibody, and stained with TRITC-labeled anti-mouse IgM antibody. Images were acquired with an Axiovert S100 fluorescence microscope (Zeiss, Germany) equipped with a DP70 digital camera (Olympus, Japan). (b) Inhibition of p91phox and p67phox expression by PTEN gene transfer. Replicative aged HDFs (55 PD) were cultured in standard medium and infected with Ad/LacZ or Ad/PTEN. After 48 h, the cells were lysed and blotted with antibody for PTEN, gp91phox, p67phox, and β-actin. (c) Inhibition of ROS generation by PTEN gene transfer. Replicative aged HDFs (55 PD) were cultured under standard medium and infected with vector alone (Ad/LacZ) or PTEN viral vector (Ad/PTEN). After 48 h, ROS was measured by flow cytometry using DCF-DA. Error bars, SD; in each group. , compared with 55 PD.