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

AMP-activated protein kinase-dependent nuclear localization of glyceraldehyde 3-phosphate dehydrogenase in senescent human diploid fibroblasts


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.