Relationship between the Nup133–CENP-F–NudE/EL and RanBP2–BICD2 pathways in centrosome tethering to the NE in U2OS cells. (A) Distances between centrosomes and nuclear periphery, measured in phospho-H3–positive U2OS cells treated for 3 d with scramble, CENP-F, BICD2, a combination of CENP-F and BICD2, or NudE/EL siRNA duplexes. 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. (B) Extracts from U2OS cells treated with the indicated siRNA duplexes were analyzed by Western blot using anti-BICD2 antibodies. Decreasing amounts of the reference sample (scramble siRNA) were loaded (1-, 0.5-, and 0.25-fold equivalent) and anti–γ-tubulin was used as loading control. (C and D) U2OS cells transfected with the indicated siRNA duplexes were preextracted, fixed, and stained with anti–CENP-F and anti-RanGAP1 (C) or anti-BICD2 (D), along with anti–phospho-H3 antibodies and DAPI. Bars, 10 µm.
