CDK4/CDK6 inhibition accelerates G1 arrest and enhances bortezomib killing of primary myeloma cells. Primary CD138+ BM myeloma cells isolated from individual patients (MM, referred to by the number at the bottom of each graph) were pretreated with 0.5μM PD for 4 or 24 hours in the HS-5 BMSC coculture before addition of BTZ and cultured for an additional 24 hours unless otherwise indicated. (A) BrdU uptake in MM cells treated with PD for 16 hours, with BrdU presented in the last 13 hours. (B-D) Viability of MM cells after BTZ addition. (E) Viability of MM cells after culturing as indicated (left); immediately after isolation (−24), at 24 hours after incubation with PD (0), and at 24 and 48 hours after further incubation with BTZ (4nM) and PD (middle), and ToPro-3 analysis at 48 hours of BTZ treatment (right). (F) Viability after BTZ addition in MM cells pretreated with PD for time indicated. (G) Immunoblotting of IRF4 at 12 hours after BTZ addition in MM10 (as shown in panel F) with or without 24-hour PD pretreatment. Numbers indicate the relative level of IRF4 compared with cells left untreated by PD or BTZ and corrected for loading by the actin signal. Data represent mean ± SD in triplicate. P value was determined by 2-tailed or 1-tailed (*) t test.
