Cell Proliferation Assay

Cell proliferation assays, including cell viability and clonogenic assay, were performed according to previous reports (28, 29). Cell viability was determined by the MTS assay (CellTiter 96 Aqueous One Solution reagent). The absorbance of optical density was measured with a microplate reader (Sunrise, Tecan) at wavelength 490 nm. For the clonogenic assay, A375 and SKMEL28 cells were treated with vemurafenib and bilirubin alone or combination, and then suspended in 30% agarose supplemented with 20% FCS and 50% DMEM medium. The resuspended cells were then cultured in 60 mm dishes for 10 days, and then stained with 0.3% crystal violet solution. The colonies > 60 μm were counted under a light microscope. All experiments were done in triplicate.