Figure 5 | Scientific Reports

Figure 5

From: N-Octanoyl-Dopamine inhibits cytokine production in activated T-cells and diminishes MHC-class-II expression as well as adhesion molecules in IFNγ-stimulated endothelial cells

Figure 5

NOD inhibits the expression of CD74, CIITA and HLA-DR. (A) CIITA and CD74 mRNA expression was assessed by qPCR. Total RNA was isolated from HUVECs cultured in normal medium or stimulated by IFNγ (50 ng/ml). IFNγ stimulation occurred in the absence (filled bars) or presence (open bars) of 50 µM of NOD. The results of three independent experiments are expressed as the mean fold change (2−ΔCT) + SD. Data were analyzed using two-way ANOVA followed by Sidak´s multiple comparison test on the normalized ΔCT values. Significance was defined as p-value < 0.05 (*p < 0.05; ***p < 0.001) for +IFNγ/−NOD compared to +IFNγ/+NOD. (B) CIITA protein expression was assessed by Western-Blotting using cell-lysates obtained from HUVECs cultured for various times as described in A. The results of a representative blot are depicted, a total of 3 independent experiments with different HUVEC cultures were performed. CIITA protein expression was quantitated by means of densitometry of all independent blots. Data were analyzed using two-way ANOVA followed by Sidak´s multiple comparison test. Significance was defined as p-value < 0.05 for +IFNγ/−NOD compared to +IFNγ/+NOD. Equal protein loading was demonstrated by staining for β-actin. Displayed are the cropped blots. The scanned full-length blots are provided in Supplementary Fig. 3. Densitometry graphs with significance marks are provided in Supplementary Fig. 4. (C) HLA-DR expression was assessed by FACS analyses of HUVECs cultured for 24 or 72 hours as described in (A). A total of 3 independent experiments using 3 different HUVEC donors were performed. Since each donor differed in the extent by which HLA-DR was up-regulated by IFNγ, for each donor the mean HLA-DR fluorescence intensity (MFI) was normalized for IFNγ stimulation. The results are expressed as mean normalized ratio + SD, i.e. (MFI HLA-DR +NOD/MFI HLA-DR −NOD) with (MFI HLA-DR −NOD/MFI HLA-DR −NOD) as 1. (*p < 0.05; ns: not significant). (D) The results of a representative experiment are shown as histograms. Red histogram: HUVEC stimulated with IFNγ only, blue histogram: HUVEC stimulated with IFNγ and NOD, black histogram: HUVEC not stimulated.

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