For dose response experiments (BRET and western blot), drugs were prepared in 100% DMSO at 10 mM. Cells were treated with Abd-2 or 3344 compounds at concentration of 5, 10 or 20 μM for 3 hr (short-term incubation) or 20 hr (long-term incubation). The compounds were diluted in the BRET medium: OptiMEM no phenol red (Life Technologies) supplemented with 4% FBS and with a final concentration of 0.2% DMSO.
For serum starvation studies with the BRET assay, cells were grown 24 hr in the presence of OptiMEM no phenol red supplemented with 1% FBS and stimulated with 50 ng/mL EGF (Life Technologies) for 5 min at 37°C. For serum-starvation studies of cancer cell lines, cells were grown 24 hr in the presence of DMEM without FBS and stimulated 10 min with 50 ng/mL EGF. The compound was incubated for 3 hr before the EGF stimulation at 2, 5, 10 and 20 μM.
For serum starvation studies with the BRET assay, cells were grown 24 hr in the presence of OptiMEM no phenol red supplemented with 1% FBS and stimulated with 50 ng/mL EGF (Life Technologies) for 5 min at 37°C. For serum-starvation studies of cancer cell lines, cells were grown 24 hr in the presence of DMEM without FBS and stimulated 10 min with 50 ng/mL EGF. The compound was incubated for 3 hr before the EGF stimulation at 2, 5, 10 and 20 μM.