In vitro model of acquired resistance used to identify the mechanisms of resistance to the EZH2 inhibitor GSK126. (A) Schematic for the generation of GSK126-resistant DLBCL cell lines. (B) The parental (P) and GSK126-resistant (R) DLBCL cell lines were treated with the EZH2 inhibitor GSK126 at the indicated concentrations and analyzed for cell viability 72 hours after treatment using an MTT assay. (C) The parental and GSK126-resistant cells were treated with GSK126 (5 μM) for 3 or 10 days and apoptosis was analyzed by annexin V–fluorescein isothiocyanate (FITC) staining. (D) The parental and GSK126-resistant DLBCL cells were analyzed by immunoblotting for the indicated proteins. The densitometric quantitation for phosphorylated ERK1/2 (p-ERK1/2), p-AKT, p-IGF-1R, and cyclin D1 is presented below the respective blots and were normalized to total ERK1/2 (T-ERK1/2), T-AKT, T-IGF-1R, and ACTINB. AUC was calculated to allow for the comparison between the 2 curves and the P values were calculated using a Student t test. Data are presented as mean ± SEM. ***P < .001.