Figure 4.

Combined inhibition of STAT3 and STAT5 with shRNA and targeted drugs results in synergistic growth inhibition in chronic myeloid leukemia (CML) cells. (A) KCL22 and K562 cells were incubated with CDDO-Me (1 μM), ponatinib (1 μM) or a combination of both drugs for 4 hours (h). Thereafter, cells were subjected to Western blot analysis using antibodies against p-STAT5, STAT5, p-STAT3, STAT3, p-CRKL, or CRKL, as indicated. (B–D) K562 and KCL22 cells were transfected with control shRNA, with an shRNA-construct directed against STAT5, or shRNA-constructs directed against STAT3 (#1 = #V3LHS_376016; #2 = #V3LHS_641818) as indicated. Protein knockdown was confirmed by western blotting using antibodies against STAT3 or STAT5. β-Actin served as loading control (B). (C) K562 cells (upper panels) and KCL22 cells (lower panels) were treated with control-shRNA (black bars) or with shRNA directed against STAT3 (construct #2, gray bars) and were then incubated with control medium (control), with BCR-ABL1 TKI (as indicated), or with the STAT5 inhibitor AC-3-019 for 48 h. Results are expressed in % of control and represent the mean±Standard Deviation (S.D.) of triplicates. *P<0.05. (D) K562 cells (left panel) and KCL22 cells (right panel) were treated with control-shRNA (black bars) or shRNA against STAT5 (gray bars) and were then incubated in control medium or in CDDO-Me for 48 h. Results are expressed in % of control and represent the mean±S.D. of triplicates. *P<0.05.