Figure 5

Involvement of the cAMP signaling pathway in NED promoted by high cell density. Western blot analysis of changes in expression of selected members of the cAMP signaling pathway in response to high cell density. A, Phosphorylation of PKA RII (Ser 96), phosphorylation of CREB (Ser 133), and total level of CREB were detected in cells cultivated in complete media with androgens (FBS) for 2 to 16 days. B, Analysis of p-(Ser/Thr) PKA substrates in cells cultivated in the presence of androgens for 2 to 16 days. C, Analysis of the p-Ser/Thr PKA substrate in LAPC-4 cells in response to treatment with the MDL-12330A, an inhibitor of cAMP signaling. Cells were treated with 2 μM MDL-12330A for 8 days. D, Analysis of the expression of the NED marker γ-enolase in response to treatment with MDL-12330A. E, qRT-PCR analysis of mRNA levels of ENO2 in response to inhibition of cAMP signaling. The data represent means ± SD of three experiments. “*” and “^#” denote statistical significance compared with control cells harvested on day 0 or cells cultivated for 8 days and treated with vehicle, respectively. F, Schematic illustration of the proposed mechanism for the promotion of NED by high cell density.