Fig 1: Effects of RAGE blockade on S100A8/9 stimulation of the PI3K/Akt/mTOR and mTORC2 signaling pathways. (A) Human umbilical vein endothelial cells transfected with si-RAGE, si-Rictor or control, as indicated, were subjected to serum starvation for 24 h. Total protein was harvested and subjected to western blotting. (B) Total RNA was harvested and subjected to reverse transcription-quantitative PCR. (C) Viability was assessed by Cell Counting Kit-8 assays. The relative cell viability ratios are expressed as a percentage of the 48 h control group. (D) RAGE and Rictor knockdown by siRNA can partially reverse the S100A8/9-induced increase in cell migration. (E) RAGE and Rictor knockdown by siRNA can partially prevent the S100A8/9-induced angiogenesis. Data and error bars represent the mean ± SEM (n=3). *P<0.05, **P<0.01 vs. control group; ##P<0.01 vs. S100A8/9 group. RAGE, receptor for advanced glycation end products; S100A, S100 calcium binding protein A; Rictor, rapamycin-insensitive companion of mTOR; siRNA, small interfering RNA; NC, negative control.
Fig 2: S100A8/9 stimulates the PI3K/Akt/mTOR pathway, Bcl-2 and Rictor in HUVECs. (A) S100A8/9 administration stimulates the PI3K/Akt/mTOR pathway by increasing the expression ratios of p-PI3K/t-PI3K, p-Akt/t-Akt and p-mTOR/t-mTOR. (B) The changes in Rictor and PKCa expression were detected by western blotting. (C) S100A8/9 increased protein and mRNA expression of Bcl-2. Data and error bars represent the mean ± SEM (n=3). *P<0.05, **P<0.01 vs. control group. HUVECs, human umbilical vein endothelial cell; S100A, S100 calcium binding protein A; Rictor, rapamycin-insensitive companion of mTOR; PKCa, protein kinase Ca; p-, phosphorylated; t-, total.
Fig 3: S100A8/9-induced HUVEC activation is RAGE-dependent. HUVECs were pretreated with 10 µg/ml RAGE blocking antibody or 100 nM rapamycin for 1 h prior to treating with S100A8/9 for 48 h. Total protein was harvested and subjected to western blotting. (A) RAGE antibody pretreatment blocked the PI3K/Akt/mTOR pathway, whereas rapamycin only reduced mTOR phosphorylation. (B) RAGE antibody pretreatment blocked Rictor and PKCa, whereas rapamycin made no difference. (C) The change in Rictor mRNA expression was measured by reverse transcription-quantitative PCR. Data and error bars represent the mean ± SEM (n=3). *P<0.05, **P<0.01 vs. control group; #P<0.05, ##P<0.01 vs. S100A8/9 group. RAGE, receptor for advanced glycation end products; S100A, S100 calcium binding protein A; Rictor, rapamycin-insensitive companion of mTOR; PKCa, protein kinase Ca; p-, phosphorylated; t-, total.
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