Egm 2

Human coronary artery EC (HCAEC, Cambrex, Verviers, Belgium) proliferation was determined using a BrdU incorporation assay kit (cell proliferation ELISA, Roche, Mannheim, Germany). Briefly, HCAEC were seeded on 1 % gelatin-coated on 96-well plates at a seeding density of 3 × 10³ cells per well in endothelial growth medium (EGM-2, Cambrex) for 24 h, followed by serum starvation and pre-treatment with different concentrations of thalidomide between 50 and 250 μg/ml for 24 h. Afterwards cells were stimulated with EGM-2 for 18 h again in the presence or absence of thalidomide in same concentrations. BrdU labeling solution was applied for another 6 h and BrdU incorporation was detected according to the manufacturer’s instructions. Number of viable cells in growth factor enriched buffer alone was set to a value of 100 %. Number of viable cells resulting from different thalidomide concentrations was compared to the number of cells in DMSO-enriched buffer.

DU145 WT (passages 5–20, ATCC HTB-81, certified by STRA) is a PCa cell line with high constitutive expression of mPGES-1 (Hanaka et al. 2009 (link)). DU145 mPGES-1 knockdown (mPGES-1^KD cells, passages 8–20) and non-target shRNA (mPGES-1^SC cells, 8–20) cells were obtained and cultured as described (Hanaka et al. 2009 (link)). PC-3 WT (passages 8–20, ATCC CRL-1435, certified by STRA) and LNCaP WT (passages 5–15, ATCC CRL-1740, certified by STRA) PCa cells were from ATCC. Cells were grown in RPMI (Euroclone, Pero Milano, Italy) and supplemented with 10% FBS (v/v). Human umbilical vein endothelial cells (HUVEC, passages 3–10) were from Lonza (Basel, Switzerland) (C2519A, certified by expression of CD31/105, vWFVIII, and positivity for acetylated low-density lipoprotein uptake). Cells were grown in endothelial growth medium (EGM-2) (Clonetics, Lonza) and supplemented with 10% FBS (v/v).