Figure 4. High CDK2 activity impairs drug-induced cell cycle arrest in MYCN-amplified neuroblastoma cells. Cells were transfected with one of four different CDK2 siRNAs or one of two control siRNAs 24 h prior to doxo treatment. (A) Western blot showing the knockdown efficacies of the four CDK2 siRNAs (%) and flow cytometric cell cycle analyses showing the CDK2 siRNA#2 with highest knockdown efficacy 48 h after doxo treatment. (C and D) IMR-32 cells stably transfected with a tetracycline-inducible shRNA targeting CDK2. Addition of tetracycline to the culture medium induced expression of the CDK2-targeting shRNA. Cells were treated with doxo and/or the RO0505124 CDK4 inhibitor 24 h after shRNA induction. Cell cycle (C) and cell death (D) were analyzed using flow cytometry 48 h after treatment. (E and F) Cell death analysis for MYCN-amplified cells transiently transfected with two siRNAs targeting CDK4 (E) or CDK2 siRNA#2 (F). Cell death was determined using flow cytometry 48 h after doxo treatment. Data in (B–F) are presented as mean ± SD of triplicates.