Figure 4

NEK9 KD induces G1 arrest with senescence-like features and broad changes in gene expression profile.

(A) Seventy-two hours after transfection with 2 nM siRNA, cells were subjected to flow-cytometry analysis. Error bars indicate SD of three independent experiments. (B) SW480 cells were transfected with 5 nM siRNAs and incubated for 7 days. (C) SA-β-gal–positive cells were quantified by flow cytometry using C₁₂FDG as a fluorogenic substrate. The upper panels show data from RKO (p53 WT) and the lower panels show data from SW480 (p53 MUT). Electronic volume (EV) indicates cell size. (D) Heat map of changes in mRNA levels 72 h after transfection of SW480 cells with 5 nM siRNAs. (E) Venn diagrams indicate the number of genes up- and down-regulated in p53 MUT/KO and NEK9 KD cells. The blue circle represents genes up-regulated in p53 mutant (MUT) and knockout (KO) cell lines relative to WT cells and the orange circle represents genes down-regulated genes in NEK9 KD cells. The purple circle represents genes down-regulated in p53 MUT and KO cells relative to WT cells and the silver circle represents genes up-regulated in NEK9 KD cells. (F) Cells were transfected with the specified concentrations of siRNAs, incubated for 72 h and then subjected to immunoblot analysis with the indicated antibodies. The original full scan images are available in Supplementary Figure 7.