Figure 5
Effect of A1AT-0 and A1AT-LA on LPS-induced caspase-1 expression and activity. Human neutrophils (5 × 106) were incubated for 5 h in either medium alone or medium containing LPS (20 ng/mL) in the absence or presence of A1AT-0 (1 mg/mL) or A1AT-LA (1 mg/mL). mRNA expression of the CASP1 gene (A) was analyzed by real-time qPCR as described in Materials and Methods. GAPDH and HPRT were used as housekeeping genes. Box plots represent data from 4 individual experiments; n = number of replicates for each experiment. P value indicates significant differences compared with the values seen in LPS-activated cells. Total cell lysates were analyzed for the caspase-1 protein profile characterization by western blotting (B). For loading control, blots were reprobed with antibodies to β-actin. The blot is representative of 3 independent donors with similar results. Caspase-1 activity (C) was analyzed in vitro by using caspase-1 fluorogenic tetrapeptide substrate (YVAD-AFC). Data represents caspase activity (in relative fluorescence units) in the presence of either 1 mg/mL A1AT-0 or A1AT-LA or 20 µM z-VAD-FMK (generic caspase inhibitor). Measurements were taken every 10 min. Each curve represents data from 2 independent experiments, each performed in triplicate.