Figure 3
Suppression of the PRC1 interactor KIF4 improves the mitotic cell fate when Cdk1 activity is compromised.
(A) The ATPase activity of KIF4 is required for the RO-3306–induced cellular toxicity. Reconstitution of KIF4 knockdown U2OS cells with either WT, a phosphorylation-mutant, or an ATPase-dead KIF4 cDNA (KIF4WT, KIF4A/A, or KIF4md, respectively). (B) Western blot analysis of protein lysates corresponding to the cell lines shown in (A). shKIF4 3′UTR targets endogenous KIF4. (C) KIF4 knockout cells are resistant to Cdk1 inhibition. U2OS WT or KIF4 knockout cell clones treated with increasing concentrations of RO-3306 were examined by colony formation assay. KO, knockout. (D) U2OS WT and KIF4 KO clones were characterized by Western blot analysis. Cell lysates correspond to the colony formation assay in (C). (E) KIF4 KO cells restore chromosome alignment in mitosis when Cdk1 activity is compromised. U2OS WT, KIF4 KO clone 16 and KIF4 KO clone 16 reconstituted with GFP-KIF4WT were treated with or without 3 μM RO-3306 for 16 hours and MG132 for two hours to allow cells to arrest in metaphase. Subsequently, cells were fixed and the alignment status was determined by immunofluorescence. (Mean ± s.e.m, n = 3, at least 50 cells/experiment; n.s., not significant; **p < 0.005, Student’s t test). (F) U2OS cells stably expressing H2B-GFP and mCherry-α-Tubulin were imaged by DIC and fluorescence microscopy, 48 hours after transfection with indicated siRNAs. Scale bar, 10 μm. (G) Verification of the KIF4 depletion judged by Western blot analysis. (H) KIF4 depletion rescues the mitotic delay upon compromised Cdk1 activity. Cells shown in (F) were followed 48 hours after transfection with indicated siRNAs. Mitosis is plotted from NEB until anaphase. (Mean ± s.e.m, n = 3, 50 cells/experiment; ****p < 0.0001, Student’s t test). (I) KIF4 knockdown rescues abnormal mitotic exit and chromosome loss when Cdk1 is partially active. Analysis is performed with cells shown in (H). (Mean ± s.e.m, n = 3, 50 cells/experiment; ****p < 0.0001, Student’s t test). Western blots in panels (B,D,G) have been cropped and full-length gels can be viewed in Supplementary Fig. 6.
