A, Western blot analysis of CDK4 and RB upon IPTG-inducible CDK4 knockdown in Rh30 cells. Intensity ratios of phospho-RB to RB are normalized to the ratio in vehicle-treated cells expressing NT control shRNA. B, Unsupervised clustering of genes containing the 8-nucleotide consensus E2F-binding site categorizes Rh30 cells expressing NT control shRNA or vehicle-treated (−) cells expressing IPTG-inducible CDK4 shRNAs #2 and #3 versus IPTG-treated (+) cells expressing IPTG-inducible CDK4 shRNAs #2 and #3 into two distinct clusters. IPTG-inducible depletion of CDK4 (C) diminishes CDC25A and CCNE2 mRNA expression, (D) abrogates cell proliferation, (E) inhibits focus formation, and (F) arrests cells in G1-phase of the cell cycle. Data represent the mean ± SD of three independent experiments performed in triplicate. * p < 0.005, ** p < 0.00005 by Student’s t-test.