Fig 2: Apolo1 is important for sustaining PLK1 activity in mitotic cells(A) Mitotic HeLa cells treated with two independent Apolo1 siRNAs were collected by shake-off, separated by SDS-PAGE, and blotted with the indicated antibodies against Apolo1, pT210-PLK1, PLK1, pT288-Aurora A, Aurora A, Bora, and tubulin.(B) Quantification of pT210-PLK1 intensity (relative to PLK1) in (A).(C) Immunofluorescence staining of pT210-PLK1, PLK1, and ACA in prometaphase HeLa cells transfected with siScramble and siApolo1. Suppression of Apolo1 did not alter the kinetochore distribution of PLK1 but attenuated the pT210-PLK1 labeling (asterisks indicate pT210-PLK1 located at centrosomes). Scale bar, 10 µm.(D) Quantification of the relative pT210-PLK1 level (pT210-PLK1/PLK1) at the kinetochore in (C). Data represent mean ± SEM and were examined with two-sided t test. A total of 150 pairs of kinetochores from at least 10 cells were examined from three independent experiments. Statistical significance was tested by two-sided t test; **p < 0.01, ***p < 0.001.(E) Control or Apolo1-depleted 293T cells expressing the Hec1-targeted PLK1 kinase sensor were imaged at prometaphase. 293T cells were synchronized with 100 ng/mL nocodazole for 2 h after release from single thymidine treatment. BI2536 was added at 100 nM for 0.5 h. Scale bar, 5 µm.(F) Statistical analyses of the FRET/CFP emission ratio as in (E). At least 20 cells of each group were calculated, and data represent mean ± SEM from three independent experiments. Statistical significance was tested by two-sided t test and represented by asterisks corresponding to *p < 0.05 and ***p < 0.001.See also Figures S1 and S2.