Apoptosis assays

1.0 × 10⁶ P3724-R4 cells were allowed to attach overnight to a T25 flask (BD Falcon) in DMEM/F-12 medium supplemented with 10% FBS, PenStrep, 5 μg ml⁻¹ insulin, 200 ng ml⁻¹ hydrocortisone and 20 ng ml⁻¹ EGF and were then washed and cultured for 48 h on DMEM/F-12 medium only supplemented with PenStrep (starved). Cells were released by EDTA-free trypsin-250 (Gibco) diluted in DMEM/F-12 medium and immediately stained with Annexin V and propidium iodide (PI) from the Annexin V-FITC Apoptosis Detection Kit (Abcam), following manufacturers' instructions. 20,000 PI-negative cells per replicate were analysed for Annexin V-staining using a Beckton Dickinson LSRII FACS analyser.