Fig. 1: TAT-TVTSP effectively reduces Der f 2-induced PLD activation and IL-13 production in BEAS-2B cells.

a PLD1-specific inhibitory peptide (TVTSP) was fused with the cell-penetrating peptide delivery system (TAT). b BEAS-2B cells were labeled with 2 µCi/ml [³H]-palmitic acid and then pretreated with TAT or TAT-TVTSP at the indicated concentrations for 2 h, followed by stimulation with 20 µg/ml Der f 2 for 30 min. PLD activities were determined by estimating [³H]-PBt in the presence of 1-butanol. The results are expressed as the means ± SD of three independent experiments. **P < 0.01 compared with TAT-TVTSP (10 µM) + Der f 2/TAT + Der f 2 or TAT-TVTSP (20 µM) + Der f 2/TAT + Der f 2. c BEAS-2B cells were treated with TAT-TVTSP-FAM and examined over time. FAM fluorescent signals were examined with a fluorescence microscope. Scale bar, 20 µm. d, e Total RNA was isolated using Trizol reagent, and the mRNA levels of IL-13 and GAPDH were determined by RT-PCR (d) and real-time RT-PCR (e). The results are expressed as the means ± SD of three independent experiments. *P < 0.05 compared with TAT-TVTSP (10 µM) + Der f 2/TAT + Der f 2. **P < 0.01 compared with TAT-TVTSP (20 µM) + Der f 2/TAT + Der f 2. f Cell supernatants were collected after Der f 2 treatment for 30 min, and the concentrations of IL-13 were measured with ELISA. The results are expressed as the means ± SD of three independent experiments. *P < 0.05 compared with TAT-TVTSP (10 µM) + Der f 2/TAT + Der f 2 or TAT-TVTSP (20 µM) + Der f 2/TAT + Der f 2. g An MTT assay was performed after 30 min of treatment with Der f 2. The results are expressed as the means ± SD of three independent experiments. All differences were assessed by Student’s t-test