The ATF6 branch of unfolded protein response and apoptosis are activated to promote African swine fever virus infection

Galindo, I; Hernáez, B; Muñoz-Moreno, R; Cuesta-Geijo, M A; Dalmau-Mena, I; Alonso, C

Caspase activity assays. Using fluorimetric caspase 3, 8 and 9 assay kits (Sigma-Aldrich), the activity of caspases 3, 8 and 9, respectively, were measured at a range of times post-infection in Vero cells and in WSL-R cells infected with ASFV at a multiplicity of 1 pfu/cell. Caspase 12 activity was determined using a fluorimetric caspase 12 assay kit (Biovision, Mountain View, CA, USA), following the manufacturer's instructions. FACS analyses. Vero cells were pretreated with inhibitors at the indicated concentrations in growth medium for 1 h at 37 1C, followed by infection with ASFV B54GFP-2 recombinant virus at a moi of 1 pfu/ml. After 24 hpi cells were washed with PBS, harvested by trypsinization, and then washed with FACS buffer (PBS, 0.01% sodium azide, and 0.1% bovine serum albumin. In order to determine the percentage of infected cells per condition, 10 000 cells/time point were scored and analyzed in a FACSCalibur flow cytometer (BD Sciences, Franklin Lakes, NJ, USA). Untreated control infected cultures yielded 25-30% of infected cells from the total cells examined. Infected cell percentages obtained after drug treatments were normalized to values found in control plates.