Polo T-loop phosphorylation is required for mitotic progression but not for mitotic entry.
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(A) Stable cell lines allowing the copper-inducible expression of PoloWT-GFP or PoloT182A-GFP (or control DMel-2 cells) were treated with CuSO4 for 1 d and transfected with Polo 3′UTR dsRNA the next day in presence of CuSO4. Four days later, cells were analyzed by immunoblotting. (B) The same cells were analyzed by immunofluorescence to measure the mitotic index (± S.E.M.) using anti-phospho-Histone H3 staining. Note that the expression of PoloWT-GFP, but not PoloT182A-GFP, rescued the mitotic index in cells depleted of endogenous Polo. (C) Quantification of the phase distribution of mitotic cells after staining for α-tubulin and DNA (± S.E.M.). Cells expressing PoloT182A-GFP accumulate in prometaphase/metaphase compared with PoloWT-GFP cells. (D) Cells expressing PoloT182A-GFP and depleted of endogenous Polo (D) showed aberrant prometaphase/metaphase figures, with scattered chromosomes, whereas cells expressing PoloWT-GFP progressed into a normal metaphase (E). Scale bar = 10 µm. (F) Quantification of defects in chromosome alignment and bipolar spindle formation in the different experimental conditions, where all cells were depleted of endogenous Polo by 3′UTR dsRNA. Error bar = SEM.