Figure 8

S1P ₅ mediates immune quiescence of brain endothelial cells by NF-κB. (a) Protein content of p65 in S1P₅ knockdown and control cells was assessed by western blot. The protein expression of control cells was set as 100%. The S1P₅ knockdown cells show an increased expression of about 250% compared to control. The expression of S1P₅ was corrected for actin. (b) Mock cells and S1P₅ knockdown cells were left unexposed or were exposed to NF-κB inhibitor BAY 11–7085 and ICAM-1 gene expression levels were determined by qPCR. Upon exposure to BAY 11–7085 ICAM-1 RNA expression in S1P₅ knockdown cells decreased to approximately the same level as in BAY 11–7085 exposed mock cells. The ratio of reduction in ICAM-1 gene expression by inhibition of NF-κB in S1P₅ knockdown cells increased 2.3-fold compared to 1.6-fold in mock cells. (c) Mock cells and S1P₅ knockdown cells were left unexposed or were exposed to NF-κB inhibitor BAY 11–7085 and VCAM-1 gene expression levels were determined by qPCR. Upon exposure to BAY 11–7085 VCAM-1 RNA expression in S1P₅ knockdown cells decreased to approximately the same level as in BAY 11–7085 exposed mock cells. The ratio of reduction in VCAM-1 gene expression by inhibition of NF-κB in S1P₅ knockdown cells is increased to 10.3-fold compared to 7.3-fold in mock cells. Data are expressed as the mean ± SEM (n = 3). Statistical significance (t-test) is indicated with asterisks: *P < 0.05, **P < 0.002 and ***P < 0.001.