(A) Hela cells transfected with siGFP (control), siAstrin, siAurora-A, or both were lysed and subjected to immunoblot analysis. Proteins are indicated on the right. (B), (C), (D), (E) Hela cells seeded on cover slips were transfected twice with indicated siRNAs at a 24-hour interval. Then the cells were fixed for immunofluorescence with Hoechst 33342 and indicated antibodies, respectively.

(A) Hela cells transfected with various siRNAs were collected and fixed for flow cytometry. (B) cells in (A) were stained with anti-phospho-H3 immunofluorescence. (C) Cells in (A) were immunostained with anti-cyclin B1 and -γ-tubulin antibodies. (D) Equal amounts of cell extracts from siGFP (control) and siAstrin treated cells were immunoprecipitated with anti-cyclin B1 antibody. The resulting precipitates were tested both for CDK1 kinase assay using histone H1 as a substrate (upper panel), and by immunoblot with anti-cyclin B1 antibody (lower panel) to ensure equal loading. (E) Hela cells were seeded into 35 mm plates and transfected with siRNAs. At the indicated time (days), cells were collected, cell numbers counted and plotted as growth curves. This is a represent of three repeated experiments.

(A) Transformants of S. cerevisiae pJ64-4a were patched on synthetic complete plates and transferred to Whatman No.1 paper for β-galactosidase assays with X-gal as substrate at 37°C for 1 hour. (B) Hela cell lysates were immunoprecipitated with anti-IgG (negative control), or -astrin, respectively. The immunoprecipitates were analyzed by Western blotting. Proteins are labeled at right. (C) Hela cells were synchronized at S-phase by double thymidine and aphidicolin arrest, and then released into the cell cycle. Cells at various time-points were collected and analyzed by flow cytometry or by immunoblot. “Unsys” indicates the unsynchronized cells. Numbers indicate the hours from the release point. (D) Hela cells grown on cover slips were fixed and stained with Hoechst 33324, and antibodies directly against astrin and Aurora-A.

(A) Hela cells transfected with siGFP (control) or siAstrin were fixed for immunofluorescence with Hoechst 33342 and anti-centrin 2 and γ-tubulin antibodies, respectively. (B) Hela cell lysates from unsynchronized or mitotic cells were immunoprecipitated with anti-IgG (negative control), or -astrin antibodies, then subjected to immunoblot analysis (upper and middle panels). The whole lysates were immunoblotted with anti-tubulin to insure equal loading (lower panel). Proteins are indicated on the right. (C) Hela cells transfected with siGFP (negative control) or siAstrin were treated with DMSO (mock lanes) or monastrol (Monastrol lanes) for 5 hours, or cells released from monastrol inhibition for another 1 hour (Monastrol released lanes). Cells were fixed for anti-γ-tubulin immunofluorescence to assess the centrosome numbers in the cells. (D) siAstrin transfection was done after the first thymidine arrest during the double thymidine and aphidicolin synchronization. Released cells after aphidicolin treatment were collected at the indicated time-points and subjected to anti-centrin-2 and -γ-tubulin immunostaining. Cells carrying multiple (>2) γ-tubulin spots were counted and plotted. More than 300 interphase and 300 mitotic cells were counted.