Inhibition of CDK4/CDK6 induces early G₁ arrest and cell-cycle synchronization. (A) Schema for reversible inhibition of CDK4/CDK6 by PD 0332991 (PD). D indicates cyclin D. (B) MM1.S cells were cultured with PD for 24 hours (h) and released into fresh media for the time indicated. *BrdU was added 30 minutes before cell harvest for FACS analysis of BrdU uptake and DNA content per cell. Number in the FACS profile indicates the percentage of BrdU-positive cells. (C) Immunoblotting and (D) quantitative RT-PCR analysis of cells cultured as in panel B. pSRb indicates Rb phosphorylated on serine 807/811. (E) Hierarchical clustering analysis of gene expression in MM1.S cells cultured with PD for 12 hours (12hPD) or 24 hours (24hPD), or released into fresh medium for 4 hours (4h_po24hPD) or 18 hours (18h_po24hPD) after 24 hours of PD treatment. The 2797 genes included in this analysis have a log₂(treated/control) value > 1 in at least 1 of 3 independent arrays and a P value < .05. (F) Scatter plot of Log₂ (12hPD/Cntl) and Log₂(24hPD/Cntl) after normalization to the expression of ACTB (β-actin). The middle line indicates no variation, and the outer 2 lines indicate a 2-fold variation between the log ratio values. (G) Heat maps of genes from panel E. Data are representative of 6 independent experiments.