Fig. 5: PLK1 Y445F is hyperactive and provides greater resistance to effects on survival and mitosis caused by depletion of endogenous PLK1, EYA4, or EYA inhibition.

A Representative western blots of pT210 and total PLK1 in PLK1-myc WT and PLK1-myc Y445F expressing HeLa cells that have been mitotically synchronized with nocodazole (* represents endogenous pT210 or total PLK1). B Densitometry pertaining to (A) (n = 3 biological replicates, ***p = 0.0006, two-sided Student’s t-test). C Representative western blots of endogenous PLK1 depletion rescue by PLK1-myc WT and PLK1-myc Y445F (* represents endogenous PLK1). D Densitometry of cleaved PARP/PARP pertaining to (A) (n = 4, **p = 0.0013, ***p = 0.0005, ****p ≤ 0.0001, ANOVA with Tukey correction for multiple comparisons). E, F Quantification of mitotic cell death (E) or mitotic duration (F) from live cell microscopy experiments involving endogenous PLK1 depletion and rescue with PLK1-myc WT or PLK1-myc Y445F overexpression (n = 75 mitotic entry events across 3 experiments, pertaining to (E): **p = 0.0011, ***p = 0.0001, ****p ≤ 0.0001, pertaining to (F): *p = 0.0326, **p = 0.0080, ***p = 0.0004, ****p ≤ 0.0001, both panels used ANOVA with Tukey correction for multiple comparisons). G–J Quantification of mitotic cell death (G, I) or mitotic duration (H, J) from live cell microscopy experiments involving depletion of EYA4, and overexpression of PLK1-myc WT or PLK1-myc Y445F overexpression (n = 75 mitotic entry events across 3 experiments, *p = 0.0139, **p = 0.0075). K Alamar blue viability dose-response curves following 72 h treatment with benzarone across a range of concentrations in cells stably overexpressing an EV construct, PLK1-myc WT, or PLK1-myc mutants (n = 4 replicates, mean IC50 values presented, *p ≤ 0.05, ****p ≤ 0.0001, two-sided Student’s t-tests). “AU” stands for arbitrary units. All quantitative data in this figure are presented as mean values +/− SEM. Source data are provided as a Source data file.