2.9. ABCG2 ATPase assay

A colorimetric ABCG2 ATPase assay was carried out as described previously with minor modifications31. Briefly, crude membranes isolated from ABCG2-overexpressing MCF7/FLV1000 cells (100 g protein/mL) were incubated with olmutinib (0–5 μmol/L) at 37 °C in the presence or absence of 1.2 mmol/L sodium orthovanadate in an assay buffer (50 mmol/L KCl, 5 mmol/L sodium azide, 2 mmol/L EDTA, 10 mmol/L MgCl₂, 1 mmol/L DTT, pH 6.8) for 5 min. 5 mmol/L Mg-ATP (concentration in a final volume of 60 μL) was then added to start the ATP hydrolysis reaction and the reaction mixture was allowed to incubate for 10 min. The reaction was terminated by the addition of SDS solution (30 μL of 10% SDS). Absorbance was subsequently measured at 750 nm after the addition of a detection reagent (35 mmol/L ammonium molybdate, 15 mmol/L zinc acetate, 10% ascorbic acid) and incubation at 37 °C for 20 min. The amount of inorganic phosphate released was quantified by reading from a standard curve. Olmutinib-stimulated ABCG2 ATPase activity (i.e. vanadate-sensitive) was determined as the difference between the amounts of inorganic phosphate released from ATP in the absence and presence of sodium orthovanadate.