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. Author manuscript; available in PMC: 2015 Mar 31.
Published in final edited form as: Nat Cell Biol. 2014 Apr 28;16(6):538–549. doi: 10.1038/ncb2952

Figure 4.

Figure 4

Endogenous eye Iin B2 regulates aurora-A-mediated centrosome separation. (a-h) Wild-type (WT) MEFs transduced with cycl in B2 shR NA or non-targeting sh RNA negative control lentiviruses and analysed 72 h later. (a) Western blot analysis for eye Iins B1 and B2. (b) lmmunostain ing for cycl in B2 and γ-tubulin. (c) Quantification of cells with unseparated centrosomes (<l μml in G2. (d) G2 cells immunostained for p-Plkl1210 , γ-b.Jbulin and pH3510 . We note that p-Plkl1210 levels at centrosomes of cyclin B2 overexpressing cells were increased irrespective of inter-centrosome distance. (e) Quantification of p-Plkl1210 signa I at centrosomes. (f) Quantification of centrosomal p-aurora A signa Is in prophase. p-aurora A levels at centrosomes of eye Iin B2 over expressing eel Is were increased irrespective of intercentrosome distance. (g) Analysis of spindle geometry. (h) Analysis of chromosome segregation errors by live-cell imaging. The analysis was carried out on three independent MEF lines (15-22 cells were analysed per Iine). Statistical significance was determined by a one-tailed, unpaired t-test. (i) The same as c for mouse epithelial cells. (j,k) Different human cell lines transduced with cyclin B2 shR NA or non-targeting shR NA negative control lentiviruses and analysed 72 h later (HF, human skin fibroblasts). (j) Western blot analysis of human skin fibroblast lysates for cyclins B1 and B2. (k) Quantification of cells with unseparated centrosomes in G2 phase. Colour codes in e-g and i are as in c. DNA in b and d was visualized with Hoechst. Sea le bars in b and d, 10 μm. Quantifications in c,e-g and k were done on three independent cell lines (per line, 20 cells in c,g and k and 10 cells in e and f were analysed). Quantifications in i were done on the same line in triplicate (20 cells for each analysis). Data represent mean± s.e.m. Statistical significance was determined by a two-tailed, unpaired I-test inc,g,i and k and by a one sam pie I-test against a theoretica Imean of unity in eand f. *P < 0.05; **P < 0.01. Statistics source data are provided in Supplementary Table 2. Blots in a and j are representative for three independent experiments. Uncropped images of blots are shown in Supplementary Fig. 9.