Fig 2: Human endothelial cells (cell line TIME, ATCC® number CRL-4025) were evaluated following stimulation with 5 ng/mL each of IL-1β, TNF-α, and IFN-γ for 24 h and compared to untreated controls. Three biological replicates were tested in each group. Transcriptome data obtained by next generation sequencing (NGS) were gathered into a gene set enrichment analysis (GSEA). Shown are 5 enrichment plots, which correspond to defined gene sets. (A) GO-Cellular-Response-To-Cytokine-Stimulation, (B) GO-Inflammatory-Response, (C) GO-Regulation-Of-Cytokine-Production, (D) GO-Homophilic-Cell-Adhesion-Via-Plasma-Membrane-Adhesion-Molecules, (E) GO-Cell-Adhesion-Via-Plasma-Membrane-Adhesion-Molecules. Each plot shows the running Enrichment score (ES) for the gene set, which reflects the degree to which a gene set is over-represented at the top or bottom of a ranked list of genes. A positive ES indicates gene set enrichment at the top of the ranked list (=upregulation); a negative ES indicates gene set enrichment at the bottom of the ranked list (=downregulation).

Fig 3: Human endothelial cells (cell line TIME, ATCC® number CRL-4025) were evaluated following stimulation with 5 ng/mL each of IL-1β, TNF-α, and IFN-γ for 24 h, and compared to unstimulated controls. Absolute quantification of miRNAs from isolated total RNA was performed using ddPCR analysis. The QX 200 Droplet Digital PCR System (Bio-Rad) and specific LNA PCR primers (Qiagen) were used according to the manufacturer’s instructions. Based on the number of measured positive droplets, the system calculates the copy number of the target RNA in the total mixture, assuming a Poisson distribution. Since a defined amount of RNA sample is used in each ddPCR assay, the copy number per ng RNA can be determined. The box plots depict the median of determined miRNA expression levels, the lower and upper quantile, and the two extreme values. Each group included six biological replicates and two technical replicates. An unpaired t-test was used to identify significant differences. Statistical analysis was performed utilizing GraphPad Prism 9 (GaphPad Software, La Jolla, CA, USA). In all cases, p < 0.05 was assumed to indicate significant differences.

Fig 4: The cellular impedance of human endothelial cells (cell line TIME, ATCC® number CRL-4025) was investigated in real-time using the xCELLigence® RTCA DP instrument (ACEA Bioscience Incorporation). Changes in impedance are reported by the dimensionless parameter “cell index”. The blue and red data points symbolize cells cultured in basal cell culture medium versus medium containing 5 ng/mL each of IL-1β, TNF-α, and IFN-γ for a defined time, respectively. The arrows indicate the time points of the performed medium changes. Three biological replicates and two technical replicates were tested in each group. Data are shown as means ± standard deviation.