Chemotaxis: to determine the kinases required for Id1 mediated HMVEC chemotaxis, cells were incubated with chemical signaling inhibitors. Human dermal microvascular endothelial cells (HMVECs) were pre-incubated with chemical signaling inhibitors for one hour prior to the assay, and the inhibitors were present in the lower chamber with the HMVECs during the assay. The following inhibitors were purchased from and used at concentrations recommended by Calbiochem (La Jolla, CA): PD98059 (10 μM Erk1/2 inhibitor), PDTc (100 μM NFκB inhibitor), LY294002 (10 μM PI3K inhibitor), SB203580 (10 μM p38 MAPK inhibitor) and PP2 (1 μM Src inhibitor). HMVEC migration assays were performed by placing cells in a 48-well neuroprobe microchemotaxis chamber. A) HMVECs were chemotactic for Id1 at physiologically relevant concentrations of Id1 (1, 10 and 100 nM, n = number of independent experimental replicates). B) We examined HMVEC signaling pathways to Id1 using signaling inhibitors, and performed HMVEC chemotaxis assays at the peak concentration of Id1 chemotactic activity (10 nM). We found that PDTc (NFκB inhibitor) and Ly (PI3K inhibitor) significantly reduced HMVEC migration towards Id1. The other inhibitors used had no effect upon Id1 HMVEC chemotaxis (n = number of independent experimental replicates).
Isozaki et al. Arthritis Research & Therapy 2014 16:R68 doi:10.1186/ar4507