Effects of SIRT1 knockdown on CML cell survival. (A) Knockdown of SIRT1 in KCL-22 cells using lentiviral vector shSIRT1-3. (B) Effect of SIRT1 knockdown on FOXO1 acetylation in KCL-22 3 days after transduction. (C) Three days after scrambled shRNA (mock) or shSIRT1-3 transduction using a multiplicity of infection of 5, cells were plated directly (without sorting) in triplicate in 24-well plates and viable cells were counted on the days indicated. (D) Three days after mock or shSIRT1-3 infection, 5 × 10⁵ cells were cultured with or without 2.5μM imatinib (IM) for KCL-22 or 0.25μM IM for K562 cells for another 2 or 5 days for apoptosis analysis. The percentage of annexin V⁺ cells was plotted. Error bars are standard deviations. (E) Soft agar colony formation assay. Three days after mock or shSIRT1-3 transduction, 500 cells per plate were seeded on standard 2-layer soft agar. The colonies were counted after 21 days. (F) Survival curves of mice receiving xenografted CML cells. After overnight infection with shRNA vectors, 3 million cells each were inoculated into NOD-SCID mice. Mice were euthanized when the tumor volume reached 1000 mm³. (G) Effect of SIRT1 knockdown on Ku70 acetylation. FLAG Ab was used as control. (H-I) The effect of Ku70 knockdown (H) on apoptosis of KCL-22 cells (I). Three days after scrambled shRNA (SCR) or shKU70 transduction, 5μM imatinib was added for another 2 or 5 days, and apoptosis was analyzed. Two sets of shRNAs showed similar results and data from one set are shown. *P < .05.