Figure 6

Aurora A localization and expression in wt and cancer OSE cells. IFM analysis of mitotic (A), interphase (D), and growth-arrested (D, E) wt and cancer OSE cells. Anti-pericentrin (PCNT), anti-EB3, anti-acetylated α-tubulin (Acet.tub), and anti-AURA were applied to detect the pericentriolar material (¤), centrioles (*), mitotic spindle and primary cilia (white arrows), and AURA, respectively. The rightmost column in (D) is a shifted overlay of the Acet.tub, AURA, and EB3 stainings. Cells in A, and rightmost column in E were fixed in mix-fix (see Methods for details), and cells in (D, E) were fixed in PFA + MeOH fix. B, C) AURA mRNA and protein levels were analyzed with RT-PCR (B) and WB (C), respectively, in sub-confluent cells with serum (0 hour) and in confluent cultures serum-depleted for 72 hours. GAPDH (B) and EB1 (C) were used as controls. Anti-phosphorylated retinoblastoma protein (p-RB) was included in WB analysis to verify that starved cells were in growth arrest (C). F) IFM analysis of checkpoint with forkhead-associated and ring finger domains (CHFR) localization in wt OSE cells during mitosis and in growth-arrested, serum-depleted cells. Cells were fixed in mix-fix and stained with antibodies as indicated. Primary cilia are marked with arrows and centrioles/basal bodies are marked with ¤. DNA was stained with DAPI. DAPI, 4′,6-diamidino-2-phenylindole; IFM, immunofluorescence microscopy; OSE, ovarian surface epithelium; WB, western blot; wt, wild type.