Figure 2

pVD inactivates ERK1/2 and downregulates NPRA expression. (A) A549 cells were transfected with pVD, pKP2 or pVAX1 control plasmids. Cells were collected 24 hrs after transfection. Expression of ERK1/2 and phospho-ERK1/2 was detected by western blot. (B) HEK293 cells grown on 96-well plates were cotransfected with 0.5 μg of pNPRA-Luc and 1 μg pVAX1 or pVD. Cells were lysed 48 hrs later and luciferase activity was measured in the lysates (p < 0.01). (C) Effect of VD on NPRA expression in vitro. HEK-GCA cells were transfected with pVAX1, pVD or pMut. NPRA expression was detected by western blot. Non-transfected cells were used as control. (D). Effect of VD on NPRA expression in vivo. NPRA mRNA expression was detected by RT-PCR in the lungs of mice intranasally treated with chitosan nanoparticles containing 20 μg of pVAX1 (n = 4), pVD (n = 4) or pMut (n = 4). Mice from the naïve group (n = 4) served as mock controls. All experiments were repeated, and the results of a representative experiment are shown.