Substitution of Ala for Asp at 188 abrogates AE catabolism and confers oridonin resistance to some extent in U937 cells. (A) Schematics for mutations introduced into the AE fusion protein. (B) D188A mutant in AE abrogates AE degradation or ΔAE generation upon oridonin treatment. Wild type, D171A-AE, D188A-AE, and D192A-AE are transfected into U937 cells that are then treated without or with oridonin. Western blot is performed using an anti-ETO antibody to analyze the effects of oridonin on AE mutants. (C) Oridonin induces a much lower inhibition rate of U937 cells expressing D188A-AE compared with U937 cells expressing wild type or D192A-AE, suggesting D188A-AE confers oridonin resistance to U937 cells. In this experiment, U937 cells expressing AE, D188A-AE, and D192A-AE are treated without or with oridonin, and trypan blue dye exclusion assay is done. Inhibition rate is determined by viable cells in oridonin treatment group compared with those in vehicle control group. Error bars represent SD of experiments.