ABSTRACT: ECFCs and HUVECs were washed and placed overnight in EBM-2 + 2% FCS. The next morning, medium was replaced by EBM-2 + 2% FCS + 10 µg/mL LMWF or by EBM-2 + 2% FCS alone. After 6 hours or 24 hours of treatment, cells were washed with PBS, detached with accutase. After washing and centrifugation steps, the pellet was resuspended in RLT buffer (Qiagen, Venlo, Netherlands) containing beta mercaptoethanol (Sigma, St Louis, MO, USA) and stored at -80°C. Total RNA was isolated by using the RNeasy Mini Kit (Qiagen) as recommended by the manufacturer. Total RNA concentration and integrity was assessed by Nanodrop (Thermo Fisher Scientific, Waltham, USA) and by microchip electrophoresis (Agilent technologies, Santa Clara, CA, USA). Total RNA was amplified using a kit from Ambion (Carlsbad, CA, USA) and applied to Human HT-12 expression beadchips (Illumina, San Diego, CA, USA), a genome wide array with 37 804 probes, according to the manufacturer’s instructions. Data were extracted and normalized using GeneSpring GX (Per Chip: Normalize to 50th percentile; Per Gene: Normalize to median). The working lists were created, filtering probes with detection p-values<0.05 for at least half of the arrays. To control for systematic bias, unsupervised hierarchical clustering was performed, showing that samples are segregated according to the treatment groups. Differentially expressed genes with an expression fold change greater than 1.5 fold were selected using the Student test, with a significance p-value<0.05, including Bonferroni and Benjamini Hochberg false discovery detection. Differential expression of the chosen genes was assessed using supervised hierarchical clustering. The interpretation of the resulting gene lists was performed using the Ingenuity® interface. Heatmaps were generated with R. Each dataset was derived from at least four biologically independent replicate samples.
