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 1. Nuclear accumulation of GAPDH in senescent HDFs. (A and B) Young (Y-HDFs, PD 16) and senescent (S-HDFs, PD 72) cells were maintained in DMEM containing 10% FBS for 2 days and then serum-depleted by incubation with SFM for the indicated times (1–5 days). Cells were immunostained with monoclonal anti-GAPDH antibody and FITC-conjugated anti-mouse secondary antibodies, and analyzed by confocal laser scanning microscopy (magnification, 100×; scale bar, 50 μm). Immunostained young and senescent cells with serum (10% FBS) and without serum for 5 days (SFM-5d) are shown in A. The number of young and senescent HDFs having nuclear GAPDH with or without cytosolic GAPDH was counted, and the percentage distribution was calculated (n = 10 for total replicates) and plotted as means ± standard deviations in B. ***p < 0.001 (Y-HDFs), ##p < 0.01 and ###p < 0.001 (S-HDFs), compared with 10% FBS-treated control cells. (C) The levels of GAPDH in the back skin cells from young (6 months, a and c) and aged (24 months, b and d) rats were detected by immunohistochemistry and the epithelial layers containing mainly keratinocytes (a and b) and fibroblasts (c and d) were photographed by light microscopy and the ×200 magnified photos with 50 μm scale bar are shown. Each experiment was performed at least three times with similar results. (D) The number of keratinocytes and fibroblasts with nuclear GAPDH was counted in young and aged skin (C), the percentage of cells with nuclear GAPDH was calculated (n = 8 for total replicates) and plotted as means ± standard deviations. ***p < 0.001, compared between young and aged skin.