Figure 7

PDE6D siRNA knockdown inhibits cGMP hydrolyzing PDE activity, increases cGMP levels and inhibits serum stimulated ERK phosphoprylation in A549 AECs. (A) cGMP hydrolyzing PDE activity in PDE6D siRNA transfected cells 24 h post serum stimulation. cGMP PDE activity in PDE6D knockdown cells was significantly decreased as compared to control siRNA (csiRNA) and no siRNA transfected (only lipofectamine (Lf)) cells (*P < 0.05 versus csiRNA 100 nM transfected cells, ^† P <0.01 versus Lf treated cells). (B) Intracellular cGMP levels in PDE6D siRNA transfected cells 24 h post serum stimulation. Intracellular cGMP levels were significantly increased as compared to csiRNA transfected and Lf treated cells (*P < 0.01 versus csiRNA 100 nM transfected cells, ^† P <0.01 versus Lf treated cells). (C, D, E) Immunoblotting of ERK (~42/44 kDa) and p38α/β (~38/41 kDa) phosphorylation in PDE6D siRNA transfected cells 1 h, 12 h and 24 h post serum stimulation, respectively. GAPDH (~37 kDa) was used as a loading control for PDE6D expression and total ERK (~42/44 kDa) for ERK phosphorylation. (F, G) Bar graph presentation of [³ H]-Thymidine uptake in U 0126 (10 μM, 20 μM) and SB 203580 (10 μM, 20 μM) treated cells 12 h and 24 h post serum stimulation, respectively. Serum stimulation was significant ^# P < 0.001 versus 0.1% FBS stimulated cells. [³ H]-Thymidine uptake of U 0126 (10 μM, 20 μM) treated cells was significantly decreased as compared to control (no DMSO) and DMSO treated cells, data were present as mean ± S.E from two independent experiments, *P < 0.001 versus DMSO treated 10% FBS stimulated cells. SB 203580 (10 μM, 20 μM) exerted no effect.