Figure 2

Depletion of NEK9 inhibits proliferation of p53-inactivated cancer cells.

(A) Cells were counted 3 days (or 4 days, for Ma2 and H2009 cells) after transfection with either 2 nM NEK9 siRNA (#1 or #2) or non-targeting control (siNC). Error bars indicate SD (n = 3). (B) HCT116 cells in which p53 was stably knocked down were transfected with 5 nM siRNAs and the cell number was determined. (C) H1299 transfectants expressing WT p53 or one of four p53 mutants were depleted of NEK9 and then subjected to proliferation assays. Error bars indicate SD (n = 3). (D) Colony-formation assay on culture dishes was conducted using RKO (p53 WT) and SW480 (p53 MUT) cells, transfected with 2 nM NEK9 siRNAs or NC siRNA and then allowed to grow for 9 days. Representative images of the colonies, stained with crystal violet, are shown on the left; colony counts are shown in the graph on the right. Three independent experiments yielded similar results; error bars indicate SD (n = 3). (E) Images of colonies of NEK9 KD cancer cells are shown at left and the graph indicates the number of colonies. Error bars indicate SD (n = 3). (F) Expression of NEK9 protein after knockdown of endogenous NEK9. SW480 cells were co-transfected with NEK9 siRNAs (#3 and #4) targeting the 3′ UTR region of its mRNA and a siRNA-resistant NEK9 ORF expression vector. KD or expression of Halo-tagged NEK9 was determined by IB. WT and MUT (T210A) indicate wild-type and T210 mutant (kinase-inactivated) of NEK9, respectively. The original full scan images are available in Supplementary Figure 7. (G) Cell proliferation assay for NEK9 ORF–expressing cells. Cell counts are expressed relative to the count in the negative control (i.e., co-transfected with NC siRNA and vector control), which was normalized to 1.