Figure 3.

PDE8A knockdown in human airway smooth muscle (HASM) cells. (A) Immunoblot analysis of PDE8A and β-actin in HASM cells after 72-hour incubation with recombinant lentivirus expressing scrambled or PDE8A shRNA. The PDE8A antibody detected multiple nonspecific bands. The genuine PDE8A immunoreactivity was identified on the basis of the expected molecular weight of 93 kD, so only this band is shown (representative images of n = 3 experiments are shown). Full immunoblot of PDE8A is shown in Figure E1. (B) Immunoreactive bands were analyzed by densitometry using volume plus density, and PDE8A density was normalized to the β-actin loading control. Data are expressed as the mean ± SEM (n = 3 experiments). **P < 0.01 by paired t test as compared with control. The effect of PDE8A knockdown on cAMP accumulation in HASM cells in (C) basal or (D) 1 μM Fsk-stimulated conditions. HASM cells were incubated with recombinant lentivirus expressing scrambled or PDE8A shRNA for 72 hours and recombinant adenoviruses expressing either lacZ, AC2, or AC6 for 18 hours. cAMP accumulation was measured over 10 minutes in the presence of 200 μM 3-isobutyl-1-methylxanthine. Data are expressed as the mean ± SEM (n = 5 experiments). *P < 0.05 by paired t test as compared with lacZ; ^#P < 0.05 by paired t test as compared with control. cAMP stimulated by various concentrations of forskolin was measured in HASM cells overexpressing either (E) AC2 or (F) AC6. Data are expressed as the mean ± SEM (n = 4 experiments). Nonlinear regression analysis was used to fit each dataset. The logarithmic half-maximal effective concentration for forskolin in AC6 cells was −4.97 ± 0.232 M, and the maximal effect value was 4.96 ± 0.785 fold. The logarithmic half-maximal effective concentration for forskolin in AC6 cells treated with PDE8A shRNA was −6.24 ± 0.481 M, and the maximal effect value was 9.069 ± 0.609 fold. ns = not significant.