Cell proliferation assay

Proliferation assays were performed by cell counting and colony formation assays. Total cell numbers were counted using the LUNA-II™ cell counter. Briefly, 10,000 OVCAR-4 or OVCAR-5 cells and 7500 OVCAR-8 cells were plated in 6-well plates and treated with 10–100 ng/ml apelin-13 and/or 15–50 μM ML221. After incubation for 24–96 h, cells were trypsinized, and suspended in equal volumes of media. An average of two replicates per sample was used for the analysis. For colony formation assays, 2500 cells/well were seeded in 6-well plates and cultured for ~11 days with media replacement every 3–4 days. Apelin-13 and drugs were added at the time of plating as indicated in the text. On day ~11, the colonies were fixed using 70% ethanol and stained using 0.4% crystal violet (CV). Images were taken using a bright field microscope (Leica, CFD365-FX). The assay was analyzed by counting colonies using ImageJ or measuring absorbance at 570 nm.