Nuclear localization of Greatwall is required for its function. (A–C) Nuclear localization of Gwl is required for its mitotic function in cells. Stable cell lines allowing the inducible expression of RNAi-resistant forms of Gwl-GFP (wt or NLSmut) were generated. Simultaneously with induction, D-Mel cells were transfected with dsRNA to deplete endogenous Gwl (G), or with a dsRNA against the bacterial KAN gene (K) as a control. After 4 d, cells were analyzed by immunofluorescence (A) and Western blotting for Gwl (B). Bar, 10 µm. (C) Quantification of cells with bipolar spindle that have scattered chromosomes (A, arrows). Results shown are averages of three independent experiments ± SEM. Between 30 and 50 cells were examined for each condition in each experiment. Asterisks: P < 0.001 after Student’s t test. ns: nonsignificant. (D) The regulated localization of Gwl is essential for its functions in vivo. UASp-Myc-Gwl transgenes were expressed in gwl mutant flies as indicated and adult viability (left) and embryo hatch rates (right) were scored. Error bars: SEM For each construction, results from two independent transgenes were combined. (E) Western blot of ovaries expressing the indicated forms of UASp-Myc-Gwl driven by Maternal α-Tubulin-Gal4-VP16 from flies used in D. Note that expression levels are similar for all transgenes and was approximately threefold higher than endogenous Gwl (Archambault et al., 2007).