Figure 8.
Aurora A regulation of KIF4A is required for efficient chromosome congression. (A) A schematic of the chromosome congression assay. (B) HeLa cells transfected with siControl, siKIF4A, or siKID for 48 h were treated with 10 µm monastrol for 2 h. Monastrol was removed and replaced with inhibitors for either Aurora A (0.5 µM MLN8237) or Aurora B (0.2 µM ZM447439) for 1 h. Cells were then washed into fresh medium and after a further 1 h fixed and then stained for tubulin and DNA. (C) The percentage of cells with unaligned chromosomes is plotted in the graph (Aurora A inhibition: siControl n = 77, siKIF4A n = 149, and siKID n = 74; Aurora B inhibition: siControl n = 109, siKIF4A n = 101, and siKID n = 132 from two independent experiments). Error bars indicate the SD. (D) HeLa cells transfected with siKIF4A for 24 h and transfected with GFP-tagged KIF4A rescue constructs for a further 24 h were treated with 10 µm monastrol for 2 h. Monastrol was removed by washing in fresh growth medium and replaced with medium containing Aurora A inhibitor (0.5 µM MLN8237) for 1 h. Cells were then washed into fresh medium and after a further 1 h, fixed, and then stained for tubulin and DNA. (E) The percentage of cells with aligned chromosomes is plotted in the graph (siKIF4A n = 93, KIF4A WT n = 96, FF1154AA n = 96, FF1220AA n = 145, T799A/S801A n = 82, and T799D/S801D n = 60 from two independent experiments). Error bars indicate the SD.

Aurora A regulation of KIF4A is required for efficient chromosome congression. (A) A schematic of the chromosome congression assay. (B) HeLa cells transfected with siControl, siKIF4A, or siKID for 48 h were treated with 10 µm monastrol for 2 h. Monastrol was removed and replaced with inhibitors for either Aurora A (0.5 µM MLN8237) or Aurora B (0.2 µM ZM447439) for 1 h. Cells were then washed into fresh medium and after a further 1 h fixed and then stained for tubulin and DNA. (C) The percentage of cells with unaligned chromosomes is plotted in the graph (Aurora A inhibition: siControl n = 77, siKIF4A n = 149, and siKID n = 74; Aurora B inhibition: siControl n = 109, siKIF4A n = 101, and siKID n = 132 from two independent experiments). Error bars indicate the SD. (D) HeLa cells transfected with siKIF4A for 24 h and transfected with GFP-tagged KIF4A rescue constructs for a further 24 h were treated with 10 µm monastrol for 2 h. Monastrol was removed by washing in fresh growth medium and replaced with medium containing Aurora A inhibitor (0.5 µM MLN8237) for 1 h. Cells were then washed into fresh medium and after a further 1 h, fixed, and then stained for tubulin and DNA. (E) The percentage of cells with aligned chromosomes is plotted in the graph (siKIF4A n = 93, KIF4A WT n = 96, FF1154AA n = 96, FF1220AA n = 145, T799A/S801A n = 82, and T799D/S801D n = 60 from two independent experiments). Error bars indicate the SD.

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