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. 2017 Dec 15;9(5):5562–5577. doi: 10.18632/oncotarget.23726

Figure 7. Aging and senescence affect eNOS levels due to increased GM1 levels in short-term 1 ng/ml TNFα-treated HAECs.

Figure 7

(A) At day 0, aged/senescent HAECs were reseeded with 1 ng/ml TNFα in HAEC medium with or without AMP-dNM and incubated for 3 days. At day 3, cells were harvested and analyzed (B–E). (F) At day 0, non-aged HAECs were reseeded with or without 25 nM GM1 in HAEC medium. At day 1, cells were treated with 1 ng/ml TNFα with or without 25 nM GM1 in HAEC medium and incubated for 3 days. At day 4, cells were harvested and analyzed (G and H). Control cells were grown in HAEC medium. (B, D and G) Real-time PCR analysis of eNOS performed using cDNA derived from short-term (3 days) 1 ng/ml TNFα-treated senescent HAECs with or without AMP-dNM treatment (B), short-term (3 days) 1 ng/ml TNFα-treated aged HAECs with or without AMP-dNM treatment (D), and short-term (3 days) 1 ng/ml TNFα-treated GM1-overexpressing HAECs (G). Results shown were normalized against values obtained for control HAECs (value = 1). (C, E and H) Western blot analysis of eNOS performed for short-term (3 days) 1 ng/ml TNFα-treated senescent HAECs with or without AMP-dNM treatment (C), short-term (3 days) 1 ng/ml TNFα-treated aged HAECs with or without AMP-dNM treatment (E), and short-term (3 days) 1 ng/ml TNFα-treated GM1-overexpressing HAECs (H). Histograms below Western blot panels show mean eNOS densitometric readings ± SD normalized to those of the loading controls (β-ACTIN). All values were obtained from three independent experiments. *P < 0.05; **P < 0.01. Control (Ctr): untreated cells.