RNA-Seq analysis of CDK4/6 activity-regulated genes in MCF10A cells (related to Fig. 1). (A) Number of up-regulated genes in DMSO-treated cells compared with palbociclib treatment (log₂[fold-change] > 1, adjusted P value < 0.1). (B) Volcano plot from differential expression analysis. Differentially expressed genes (|log₂[fold-change]| > 1, adjusted P value < 0.1 at any time point) are highlighted in red. Genes targeted in CRISPR screen are highlighted in blue. Triangles indicate genes that lie outside the maximum y-axis range. (C) GO term analysis of CDK4/6–dependent genes. Processes are sorted by false discovery rate (FDR)–adjusted P value. Redundant processes are omitted. (D) Gene expression time course for CDK4/6–dependent genes that are also E2F1, FOXM1, or MYBL2 targets. For each gene, average expression of DMSO-treated cells at each time point was normalized to maximum expression of that gene. Line plots are medians of genes; shaded error bars indicate 25th to 75th percentile (n = 208, 150, and 142 targets for E2F1, FOXM1, and MYBL2, respectively; n = 3 independent experiments). (E) Same experiment as Fig. 1 H. Rate of EdU incorporation was measured in S-phase cells as the total EdU fluorescence in the nucleus and normalized to control cells (sgCNTL). Error bars are population medians with 95% confidence intervals (n ≥ 8,725 cells per condition). RPKM, reads per kilobase of transcript per million mapped reads.