Priority Research Paper Volume 14, Issue 1 pp 4—27

Figure 4. Effects of AICAR and CompC on AMPK activation and GAPDH distribution in senescent HDFs. (A) Subconfluent young (Y, PD 12), middle (M, PD 48), and senescent (S, PD 86) HDFs were incubated with SFM or 10% FBS medium for 2 days. (B) Subconfluent young (PD 20) and senescent (PD 74) HDFs were treated with vehicle (−) or 1 mM AICAR and/or 10 μM CompC (+) for 2 days. Cells in A and B were lysed in a lysis buffer, and 45 μg of protein from each lysate was assessed for the levels of phosphorylated AMPKα1/2 on Thr172 (P-AMPKα1/2), total AMPKα1/2, and β-actin by western blot analysis. The band densities were normalized against β-actin and the fold changes compared to that of young cells (Y/SFM) in A and vehicle treated control cells (−/−) in B are written under each band. (C) Subconfluent senescent (PD 72) HDFs were treated with vehicle (−) or 1 mM AICAR and/or 10 μM CompC (+) for 2 days. Cells were immunostained against GAPDH and analyzed by confocal laser scanning microscopy. The number of cells with cytosolic GAPDH alone (Cytosol) and cells having nuclear GAPDH with or without cytosolic GAPDH (Nuclear +/− Cytosol) was counted, and the percentage distributions were calculated (n = 40 for total replicates) and plotted as means ± standard deviations. ^***p < 0.001 (Cytosol) and ^###p < 0.001 (Nuclear +/− Cytosol), compared with vehicle-treated control cells (−/−).