ERK activation by CSF-1 and cAMP opposes cAMP’s growth-inhibitory and apoptosis-promoting effects. (A) Exponentially growing 32D/CSF-1R cells were thoroughly washed and then seeded into medium without CSF-1 or IL-3 in the presence or absence of 50 μmol/L PD98059 (PD) and allowed to incubate for 1 hour and 40 minutes. btcAMP (1 mmol/L) was added, followed by 5 nmol/L CSF-1 (left) or 100 U/mL of rIL-3 (right) 20 minutes later. DMSO was present in the same amounts in all samples. Cell counts were performed in duplicate daily. Duplicate cell counts showed less than a 5% error in most cases. (B) Cells were treated as described in (A) using either 1 mmol/L btcAMP or 30 μmol/L forskolin. Cytoplasmic DNA was isolated from a fixed number of starting cells as described in Materials and Methods and analyzed by agarose gel electrophoresis and ethidium bromide staining after 3 days (left) or 24 hours (right). Relative numbers of viable cells remaining after 24 hours under the different conditions for the experiment on the right are as follows: CSF-1 (100%), CSF-1+PD (67%), CSF-1+btcAMP (46%), CSF-1+btcAMP+PD (35%), CSF-1+forskolin (67%), and CSF-1+forskolin+PD (43%).