Research Paper Volume 11, Issue 18 pp 7492—7509

PTEN loss regulates alveolar epithelial cell senescence in pulmonary fibrosis depending on Akt activation

Figure 4. Change of PTEN expression affects cell senescence through AKT pathway activation. Genetic overexpression of PTEN in A549 cells was achieved by a transduction of a lentiviral vector followed by bleomycin (10 μg/ml) stimulation for 72 hours and transfer to fresh medium for 24 hours. (A, B) Cellular senescence was detected by SA-β-Gal staining (original magnification, 200×). (CG) Western blot analysis was performed to confirm alteration of P21WAF1 and targets of the PTEN/AKT pathway. Lentiviral vector was used to knockdown PTEN expression in A549 cells. Bleomycin was then added to the culture medium to stimulate cells for 72 hours followed by transfer to fresh FBS-free medium for another 24 hours. (H, I) SA-β-Gal staining was applied to detect cellular senescence (original magnification, 200×). (JN) Change of P21WAF1 and expression of PTEN/AKT targets were determined by western blotting. Data are shown as the mean ± SEM, n ≥ 3 per group. *p < 0.05, **p < 0.01, ***p < 0.001. One-way ANOVA followed by Dunnett’s Multiple Comparison Test.