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

Figure 5. Effects of AMPKα siRNA transfection on the nuclear accumulation of GAPDH in senescent HDFs. Senescent HDFs were transfected with control scrambled siRNA duplexes (−) or siRNAs against AMPKα1/2 (+) for 48 and 72 h in DMEM with 10% FBS. (A) Cells were harvested at the indicated times after transfection, and lysates containing the same amount of protein (45 μg) were assessed by western blot analysis using polyclonal anti-AMPKα1/2 and anti-β-actin antibodies. The band densities were normalized against β-actin and the fold changes of AMPKα1/2 in AMPKα siRNA-transfected cells (+) compared to that of scrambled siRNA-treated control cells (−) are written under each band. (B) Cells were immunostained against GAPDH and analyzed by confocal laser scanning microscopy (magnification, 100×; scale bar, 50 μm). (C) 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 the mock control siRNA.