Journal: bioRxiv

Article Title: Less is More: Nek2A Unclusters Extra Centrosomes and Induces Cell Death in Cancer Cells via KIF2C Interaction

doi: 10.1101/2023.11.21.567992

Figure Lengend Snippet: Proximity labelling and Co-IP reveals interaction between Nek2A and KIF2C regulating centrosome clustering. ( A ) Turbo-ID proximity labelling system to identify interaction partners of Nek2A. Image created with BioRender. ( B ) Cellular localization of TurboID-Nek2A verified by IF staining. ( C ) Plot showing enriched biotinylated proteins identified by Mass-Spec. Data was generated by MaxQuant LFQ analysis. Targets selected for further analysis were marked with blue colour, known interaction partners of Nek2A was colored green. ( D ) Cellular Component analysis on identified peptides in both WT and KD Nek2A interactome indicating sub-cellular localizations. FDR: false discovery rate. ( E, F ) Percentage of multipolar metaphases observed in varying conditions for selected targets, NuMa and KIFC1. Experiments were performed using nocodazole and Plk4 CA models in U2OS and endogenously extra centrosome harbouring cell line SU86.86. ( G ) Pan-cancer Analysis of Advanced and Metastatic Tumors data retrieved from cBioPortal demonstrates positive correlation between NEK2 and KIF2C expressions. ( H ) Co-IP assay to verify physical interaction between Nek2A and KIF2C. ( I ) IF staining demonstrating the co-localization of Nek2A and KIF2C in centrosomes and spindle poles. Arrowheads point to spindle poles and centrosomes where KIF2C and Nek2A co-localize. ( J ) Percentage of multipolar spindles observed when KIF2C is silenced. Experiments were performed as two biological repeats with at least 200 metaphases with CA scored per experiment. . Error bars show standard deviations. Statistical significance was shown as * : p<0.05, **: p<0.01, ***: p<0.001, **** : p<0.0001. KD: kinase-dead, WT: wild type, OX: overexpression

Article Snippet: Oligos for dox-inducible shRNA expression targeting KIF2C were cloned into Tet-pLKO-neo as previously described [ ] and provided in ( Supp.

Techniques: Co-Immunoprecipitation Assay, Staining, Mass Spectrometry, Generated, Over Expression

Journal: bioRxiv

Article Title: Less is More: Nek2A Unclusters Extra Centrosomes and Induces Cell Death in Cancer Cells via KIF2C Interaction

doi: 10.1101/2023.11.21.567992

Figure Lengend Snippet: KIF2C is required for Nek2A to exert centrosomal unclustering activity. ( A ) Competition experiment result displays that suppression of KIF2C promotes cell survival in cells with supernumerary centrosomes. ( B ) WST-1 cell viability assay shows that suppression of KIF2C expression increases cell survival and attenuates the effect of Nek2A overexpression on survival of cells harbouring extra centrosomes induced by Plk4 overexpression. ( C ) Annexin-V staining shows that suppression of KIF2C reverts apoptotic phenotype in Nek2A overexpressing cells harbouring Plk4-induced extra centrosomes. ( D ) Schematic representation of the data demonstrating that Nek2A and KIF2C interaction in centrosomes and spindle poles regulate centrosome clustering and affect cancer cell survival. Image created with BioRender. Experiments were performed as two biological repeats. Error bars show standard deviations. Statistical significance was shown as * : p<0.05, **: p<0.01, ***: p<0.001, **** : p<0.0001. OX: overexpression

Article Snippet: Oligos for dox-inducible shRNA expression targeting KIF2C were cloned into Tet-pLKO-neo as previously described [ ] and provided in ( Supp.

Techniques: Activity Assay, Viability Assay, Expressing, Over Expression, Staining