The Nup133-anchored network tethers centrosomes to the NPCs specifically in prophase. (A) GFP_3x-mNup133 or GFP-hNup133_CTD cells treated for 3 d with scramble or hNup133 siRNAs (a), HeLa cells treated for 2 d with scramble, CENP-F, or NudE/EL siRNA duplexes (b), or HeLa cells transfected with a GFP-p50/dynamitin or a DsRec-p150cc1 construct (c) were processed for immunofluorescence using anti-pericentrin and anti–phospho-H3 antibodies. In b, cells were incubated before fixation with 40 µM BrdU for 3 h and anti-BrdU antibodies were further used. Bars, 10 µm. (B) Distances between centrosomes and the NE were measured on cells processed as in A. Prophase cells were identified by phospho-H3 staining and S/early G2 cells as BrdU-positive cells after a 3-h pulse with BrdU (G1 cells were not analyzed because centrioles were reported to be very mobile at that stage of the cell cycle; Piel et al., 2000). Distances are represented as box-plots using KaleidaGraph (see Materials and methods). The black and red bars indicate the median and mean values, respectively. The total number of cells quantified is indicated (n). ***, P < 10⁻⁵; **, P < 10⁻³; ns, P > 0.1 obtained using the Student’s t test.