Data were recorded by using a FACSort flow cytometer

al injection of thrombin. Lungs from WT and KO mice were then collected at the indicated times after thrombin administration and analyzed for expression of ICAM-1 and monocyte chemoattractant protein 1, histological examination, and lung PMN infiltration. MLCK inhibitor was administered to WT mice by i.p. injection 0.5 h prior to treatment with thrombin. RNAi Knockdown SMARTpool siRNA specific for human nmMLCK and a nontargeting siRNA control were purchased from Dharmacon. EC were transfected with nmMLCK or control siRNA using DharmaFect1 siRNA Transfection Reagent essentially as described. NF-kB Transcriptional Activity The transcriptional activity of NF-kB was measured 9850611 as described. EC were transfected with the plasmid NF-kB-LUC containing 5 copies of consensus NF-kB sequences linked to a minimal E1B promoter-firefly luciferase gene using DEAEdextran. The plasmid TKRLUC containing Renilla luciferase reporter gene MedChemExpress BIRB-796 driven by constitutively active thymidine kinase promoter was used to normalize the transfection efficiency. In experiments evaluating the effect of nmMLCK knockdown on NF-kB transcriptional activity, cells were first transfected with nmMLCK siRNA as described above. After 24 h, cells were again transfected with NF-kB-LUC and TKRLUC plasmids using DEAE-dextran essentially as described. After appropriate treatments, cell extracts were prepared and assayed for Firefly and Renilla luciferase activities using Promega Biotech Dual Luciferase Reporter Assay System. The data were expressed as a ratio of Firefly to Renilla luciferase activity. Materials and Methods Ethics Statement All mice care and treatment procedures were performed in adherence to the National Institute of Health guidelines and approved by the University of Rochester Committee on Animal Resources. Reagents Human thrombin was obtained from Enzyme Research Laboratories. ML-7 was purchased from Calbiochem-Novabiochem Corp.. Polyclonal antibodies to ICAM-1, nmMLCK, RelA/p65, b-actin, IkBa, Cu-Zn superoxide dismutase, and TATA-binding protein were from Santa Cruz Biotechnology. Antibodies to phospho–IkBa, phospho-p38 MAP kinase, phospho–RelA/p65, were obtained from Cell Signaling. RelA/p65 transcription factor assay kit was purchased from Cayman Chemical and plasmid maxi kit was from QIAGEN Inc.. All other materials were from VWR Scientific Products Corporation and Fisher Scientific. Northern Analysis Total RNA was isolated using RNeasy kit according to manufacturer’s recommendations. Quantification and purity of RNA were assessed by A260/A280 absorption and an aliquot of RNA from samples with ratio above 1.6 was subjected to Northern blot analysis as described. Immunoblot Analysis Cells were lysed in phosphorylation lysis buffer or in radioimmune precipitation buffer cultures were established by using umbilical cords collected within 48 h of delivery. Human pulmonary artery endothelial 11121575 cells were purchased from Lonza. Cells were nmMLCK Regulation of Lung Vascular Inflammation 3 nmMLCK Regulation of Lung Vascular Inflammation compared with untreated controls; Effect of RNAi knockdown of nmMLCK on thrombin-induced MCP-1 release. EC were transfected with control or nmMLCK siRNA for 2436 h. Cells were challenged with thrombin for 6 h and the conditioned media were subjected to ELISA to determine the levels of MCP-1. Data are means6S.E.. , p,0.001 compared with untreated control; ###, p,0.001 compared with thrombin-stimulated control. doi:10.1371/journal.pone.0059965.g00