Fig 1.
In vitro cytotoxicity of adenine and AICAR on HUVECs.
Cells were treated by adenine or AICAR at different concentrations or solvent alone for 24 h. The cell viability of each condition was analyzed using XTT assay; Statistical significance was determined by two-way ANOVA followed by ad-hoc Bonferroni post hoc tests; all data are plotted as mean ± S.E.M (n = 3). *, P<0.05; ***, P<0.001.
Fig 2.
Adenine activated AMPK in HUVECs.
(A) HUVEC cells were incubated for 6 h with various concentrations of adenine and compared with 1200 μM AICAR. (B) HUVEC cells were incubated with 600 μM adenine for the times indicated. Cell lysates were used to determine the phosphorylation of AMPK and ACC by western blot using antibodies specific for the phosphorylated protein. The level of total AMPK and ACC were also assessed as controls for loading. Statistical significance was determined by one-way ANOVA followed by Tukey post hoc tests; all data are plotted as mean ± S.E.M. (n = 5). *, P<0.05; **, P<0.01; ***, P<0.001.
Fig 3.
Effects of adenine on the NF-κB targeting gene expressions in response to TNF-α.
HUVECs were treated with 10 μg/L of TNF-α in the presence or absence of adenine for 6 h. (A) The level of IL-6 in cultural media of each condition was analyzed using ELISA. Cell lysates collected from each condition were used to determine the expression of (B) COX-2, (C) VCAM and ICAM using western blot analysis. Statistical significance was determined by one-way ANOVA followed by Tukey post hoc tests; all data are plotted as mean ± S.E.M. (n = 3). *, P<0.05; **, P<0.01; ***, P<0.001.
Fig 4.
Effects of adenine on NF-κB translocation in response to TNF-α.
shNT HUVECs or shAMPK HUVECs were treated with 10 μg/L of TNF-α in the presence or absence of 600 μM adenine for 6 h. Top: representative images depicting the location of NF-κB subunit p65 as described in “Material and methods”, Scale bar: 100 μM. Bottom: quantification of NF-κB subunit p65 translocation into nuclei of HUVECs. Statistical significance was determined by one-way ANOVA followed by Tukey post hoc tests; all data are plotted as mean ± S.E.M. (n = 5). ***, P<0.001; N.S., no significance.
Fig 5.
Effects of adenine on monocyte adhesion to HUVECs in response to TNF-α.
shNT HUVECs or shAMPK HUVECs were treated with 10 μg/L of TNF-α for 6 h in the presence or absence of 600 μM adenine. Top: representative images depicting monocytes to the HUVEC cells as described in “Material and methods”, Scale bar: 200 μM. Bottom: quantification of monocyte adhesion to HUVEC cells. Statistical significance was determined by one-way ANOVA followed by Tukey post hoc tests; all data are plotted as mean ± S.E.M. (n = 5). ***, P<0.001; N.S., no significance.
Fig 6.
Effect of adenine induced AMPK activation and monocyte adhesion by APRT in HUVECs.
(A) shNT HUVECs or shAPRT HUVECs were treated with various concentration of adenine for 6 h. Cell lysates collected from each condition were used to determine the phosphorylation of AMPK and ACC by western blot using antibodies specific for the phosphorylated protein. (B) shNT HUVECs or shAPRT HUVECs were exposed to 10 μg/L of TNF-α for 6 h in the presence or absence of 600 μM adenine. Top: representative images depicting monocytes to the HUVEC cells as described in “Material and methods”, Scale bar: 200 μM. Bottom: quantification of monocyte adhesion to HUVEC cells. Statistical significance was determined by one-way ANOVA followed by Tukey post hoc tests; all data are plotted as mean ± S.E.M. (n = 5). ***, P<0.001; N.S., no significance.