Effect of CDK6 silencing on apoptosis and leukemogenesis of BV173 cells. BV173 cells were transduced with scramble (SCR), CDK4, or CDK6 (82, 86, 88, 73) shRNA vectors and selected with puromycin or treated with palbociclib (2 µM). (A) Cell cycle analysis by propidium iodide staining of shRNA-transduced or palbociclib-treated cells. (B) Apoptosis detected by Annexin V staining after 7 days of puromycin or palbociclib treatment. (C) Representative immunoblot for CDK4/6 and phospho-RB expression. (D) Apoptosis detected by Annexin V staining of BV173 cells transduced with TET-ON shCDK6-88 and treated with DOX (1 µg/mL) or palbociclib (1 µM) for 7 days. (E) Leukemia load (peripheral blood flow cytometry analysis of CD19⁺mCherry⁺ cells performed 2 weeks after treatment cessation) of NSG mice injected with BV173 TET-ON shCDK6-88 cells and left untreated or treated with DOX (2 g/L in the drinking water) or palbociclib chow for 4 weeks starting 7 days post–cell injection. (F) Kaplan-Meier survival plot of NSG mice injected with BV173 TET-ON shCDK6-88 and left untreated or treated with DOX (2 g/L in the drinking water) or palbociclib chow for 4 weeks starting 7 days post–cell injection. (G) Serial bioluminescence images (left) and survival (right) of NSG mice injected with shCDK6-BV173-Luc cells and treated continuously with DOX in the drinking water or palbociclib chow.