Several studies have reported JNK1 activation following Influenza A infection

von Willebrand factor, VEcadherin and PECAM-1 were analyzed by fluorescence-activated cell sorter. Cell Adhesion Assay Cells were washed with PBS, and then gently detached with 0.25% trypsin/EDTA. After centrifugation and resuspension with serum-free medium, equal cell numbers were seeded onto either plastic or culture surfaces coated with different ECM proteins, such as fibronectin, collagen I and laminin, and incubated for 1 h at 37uC. After non-adherent cells were removed by washing, adherent cells were counted independently in six random highpower microscope fields /well by three observers unaware of the treatments. Fluorescent Staining of Cytoskeleton Late EPCs were fixed with 4% paraformaldehyde in PBS for 10 min and blocked in PBS containing 1% BSA for 30 min at room temperature. F-actin was stained with FITC-Phalloidin for 45 min, and the images were acquired by using a fluorescence microscope. FACS Analysis of the Expressions of Integrins b1 and b3 The surface expressions of integrins b1 and b3 present on late EPCs were determined by FACS. Cells were trypsinized, incubated with CD29-FITC and CD61-PE antibodies for 1 h. 20,000 cells were measured for fluorescent intensity per experiment. In addition, isotype controls were performed for each sample condition. FACS Analysis of Apoptosis Approximately 16106 late EPCs were double-stained with AnnexinV-FITC and propidium iodide by using the Annexin V and propidium iodide apoptosis detection kit according to the manufacturer’s instructions. Following staining, the cells were washed twice with binding buffer. Apoptotic cells were detected by FACS. Fluorescence parameters were gated using unstained and single-stained cells, and 20,000 cells were counted for each sample. The apoptotic percentage analysis was performed using CellQuest software. Wound Healing Assay Late EPCs were seeded in a 12-well plate until they reached,80% cell density. The cell layer was scratched in each well to create a cleared line using a 10 ml pipette tip. It was photographed both before, and at different time points after creating the scratch. The cell-free area was determined using an automatic softwareassisted image analysis. Caspase-3 Activity Assay The activity of caspase-3 in whole cell lysates was determined using the Caspase-3 Fluorescent Assay Kit according to the manufacturer’s instructions. Briefly, 16106 cells were resuspended in 50 ml chilled cell lysis buffer, and incubated on ice for 10 min. Lysates were centrifuged and the supernatants transferred to a new tube with 50 ml 26reaction buffer/DTT mix. In each reaction, 5 ml of 1 mmol/L caspase-3 substrate was added to a final concentration of 50 mmol/L. After incubation for 1 h at 37uC, the AFC liberated from the Ac-DEVD-AFC was measured by a spectrofluorometer using 400 nm excitation and measurement of 480520 nm emission. Modified Boyden Chamber Analysis of the Migration of Late EPCs The migratory function of late EPCs was evaluated by a modified Boyden chamber assay. Briefly, a total of 16105cells late EPCs were placed in the upper chamber, while the medium without serum and cytokines was placed in the lower chamber. The assays were conducted over a 16 h incubation period at 37uCin an incubator purchase (-)-Blebbistatin equilibrated with 5% CO2. The membrane was then washed gently with PBS, and fixed with 4% paraformaldeyde. Non-migrating cells were gently removed with cotton balls from the upper side of the membrane, and the membrane was then stained by using 49,6-diamidi