Figure 2.
Early adaptation to CDK4/6 inhibition is mediated by noncanonical cyclin D1–CDK2 interaction. The addition of PI3K inhibitors reduces expression of G1–S-phase regulators and induces apoptosis. A and H, Western blot analysis of cell lysates from MCF-7 and T47D cell treated for 96 hours with vehicle, 500 nmol/L palbociclib (Palbo), 250 nmol/L GDC-0941 or combination and blotted with the indicated antibodies. B and C, cells transfected for 4 days with individual siRNAs or SMARTpool targeting CDK2, cyclin D1, or cyclin E2 and treated with vehicle or palbociclib for 72 hours. B, BrdUrd staining and cell area quantified with Columbus software (***, P < 0.001; *, P < 0.05, Student t test). C, BrdUrd incorporation-ELISA assay (***, P < 0.001, one-way ANOVA with Tukey multiple comparisons test). D–G, Western blot analysis of MCF-7 cells lysates. D and F, cells transfected for 5 days with individual siRNAs or pools siRNAs targeting CDK2 or cyclin D1 and treated with vehicle or palbociclib for 96 hours. E, cells treated with vehicle, 500 nmol/L palbociclib, 10 μmol/L CYC202, or the combination for 72 hours. G, CDK2 immunoprecipitation (left) and cyclin D1 immunoprecipitation (right), or control normal IgG, blotted for CDK2 and cyclin D1. I, relative caspase-3/7 activation and survival fraction in MCF-7 cells treated as in A (*, P < 0.05; ***, P < 0.001, one-way ANOVA with Tukey multiple comparisons test).