Caspase inhibitor

U-251 MG Cells were plated in 96 well plates as described above and left to adhere overnight. Cells were pre-treated for 1 h with either zVAD-FMK; caspase inhibitor (BD Bioscience, Oxford, England), SP600125; JNK inhibitor, SB203580; p38 MAP Kinase Inhibitor (Sigma Aldrich, Arklow, Ireland), U0126; MEK1 and MEK2 Kinase Inhibitor (Sigma Aldrich, Arklow, Ireland), chloroquine or 3-methyladenine (3-MA), autophagy inhibitors (Sigma Aldrich, Arklow, Ireland) after which the UA standard (IC50) was added to each well. Cell viability was assessed 48 h later using Alamar Blue cell viability assay.

HaCaT cell were grown to confluency as describe above. Two days after reaching confluency (to insure formation of strong intercellular adhesion structures), cells were treated with the respective anti-Dsg3 or irrelevant control antibodies for the indicated time periods before harvesting the cells by trypsinization. In some experiments, Fas Ligand (BD Pharmingen) was added 48 h before harvesting the cells. In experiments including Fas Ligand neutralizing antibody (BD Pharmingen) or caspase inhibitor (BD Pharmingen), these agents were added 30 min prior to adding Fas Ligand or the anti-Dsg3 antibody. Trypsinized cells were washed in PBS and combined with dead cells from their conditioned supernatants. To detect apoptotic and/or dead cells, HaCaT cells were re-suspended in staining buffer, and annexin V antibody and/or propidium iodide were added as per manufacturer's instructions (Annexin V-FITC apoptosis detection kit, BD Pharmingen). Apoptosis and cell death was analyzed by FACS on a Vantage SE (BD Biosciences) flow cytometer.