LIC1-CTD phosphorylation is required for prophase centrosome attachment with the NE. (A and B) Representative merged confocal micrographs of G2/prophase synchronized U2OS cells (10 h after double thymidine block and release) showing the relative centrosome (γ-tubulin, arrows) and NE (inner dashed lines) positions upon the indicated siRNA treatments. Outer dashed lines, cell boundaries. Scale bar = 40 µm. (C) Immunoblots showing cyclin A/B depletion upon the respective siRNA treatments. (D–F) Fraction of G2/prophase U2OS cells showing significant centrosome-NE detachment (D and E) and centrosome fragmentation (F) under various conditions. n = 3 independent experiments, ≥150 cells per experiment. (G) Immunoblots showing affinity precipitates of GST-Pin1/GST from late G2/prophase HeLa cell lysate (8 h after double thymidine block and release), probed for the indicated antigens. (H–J) Quantification of the maximal centrosome-NE distance (H) and the timing from inter-centrosome separation to NEB (I) from time lapse, live-cell videos of double-labeled HeLa cells under the indicated treatments (represented in J, sequential still frames with time stamps shown). n = 2 independent experiments, ≥36 mitotic cells per condition. Scale bar = 10 µm. Arrowheads, centrosomes (green); AP, affinity purification; IB, immunoblot. Error bars = mean ± SEM. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001 (one-way ANOVA).