Fig. 2

Identification of candidate proteins by SILAC MS proteomic analysis of the endothelial secretome. a The protein levels in confluent and subconfluent HUVEC cells were accurately measured with SILAC using the MaxQuant software. The proteins are depicted in a scatter plot. Highlighted in the square are the proteins with a SILAC ratio ≥ 1.5 SD from the mean of the calculated ratio in each replicate experiment, A and B. These proteins were secreted at higher levels by confluent HUVEC. b The candidate proteins identified by SILAC MS analysis were silenced with siRNAs in HUVECs kept under confluent growth conditions. The migratory potential of SK-BR-3 cells exposed to conditioned medium of HUVECs with silenced proteins was evaluated using a Boyden chamber migration assay and compared to controls (*p ≤ 0.05 versus ctrl by unpaired student’s t-test). c The efficiency of siRNA knockdown was analysed for each candidate gene that was identified by SILAC. The mRNA expression levels were evaluated by quantitative RT-PCR analysis of confluent HUVECs after siRNA-mediated knockdown of the respective gene. d Gene expression of NID1 and NID2 was evaluated by quantitative RT-PCR analysis of HUVECs plated in a confluent or subconfluent monolayer. e Gene expression of NID1 and NID2 was determined by quantitative RT-PCR analysis of HUVECs plated in a confluent monolayer after siRNA-mediated knock-down of NID1