Figure 2: Oridonin triggers proteasomal degradation of BCR-ABL.

(a) Distinct effects of oridonin and imatinib on BCR-ABL. Immunoblotting showing the total and phosphorylated Bcr-Abl protein levels in K562 cells after a 24 h drug treatment. Ctrl, DMSO; Ori, 20 μM oridonin; IM: 10 μM imatinib. (b) Cychloheximide chase assay showing the half-life of BCR-ABL protein in the absence or presence of 20 μM oridonin. (c) Proteasome inhibitors prevented oridonin-induced degradation of BCR-ABL. Cells were pretreated with proteasome inhibitors MG132 (M), ALLN (A) or lactacystin (L) for 2 hours, followed by treatment with oridonin (20 μM for 2 hours for K562; 10 μM for 2 hours for KU812; 10 μM for 6 hours for SUP-B15) and subsequent immunoblotting with an anti-c-ABL antibody. (d) Immunoblotting showing the effect of 2-hour of oridonin treatment on the expression of chaperons. (e) K562 cells were treated with 20 μM oridonin for 24 hours and subjected to immunoblotting analysis of downstream targets of BCR-ABL. (f) Silence of HSP70 by siRNA partially prevented oridonin-induced degradation of BCR-ABL and MYC proteins. K562 cells were transfected with negative control siRNA (NC), hsc70 siRNA (C70) and hsp70 siRNA (P70). Forty-eight hours later, cells were treated with 20 μM oridonin for 2 hours and subjected to immunoblotting. (g) K562 cells treated with DMSO or oridonin (20 μM) for 1 h were washed and lysed with IP lysis buffer and subjected to immunoprecipitation with an anti-c-Myc or anti-c-ABL antibody. The levels of CHIP protein in the IP products were examined by immunoblotting. (h) Protein dot blot showing the total levels of ubiquitin in Ph⁺ leukemia cells after a 2-hour treatment with oridonin. The relative expression of ubiquitin to GAPDH is quantified and shown. (i) K562 cells pretreated with MG-132 for 2 hours were then exposed to oridonin (20 μM) for 1 hour and lysed for IP.