SSX2IP is required for centrosome stability in human somatic cells. (A) Localization of endogenous SSX2IP in human retinal pigment epithelium (RPE) cells in interphase and metaphase as indicated. Green, α-tubulin; blue, chromatin; red, SSX2IP; magnified merges do not display chromatin. Magnified panels (magn.) show enlarged views of the boxed regions. (B) Colocalization of SSX2IP (red), PCM-1 (green), and γ-tubulin (blue) in RPE-1 cells in interphase and mitosis; merges display colocalization between SSX2IP and PCM-1, or SSX2IP and γ-tubulin as indicated. Merges do not display chromatin. Magnified panels (magn.) show enlarged views of the boxed regions. (C) Immunoblots to document siRNA-mediated down-regulation of SSX2IP with two different siRNA oligos (Ol. 1 and 2). α-Tubulin served as a loading control. (D) Mitotic figures after knockdown of SSX2IP in RPE-1 cells. Green, α-tubulin; red, γ-tubulin; blue, DAPI/chromatin. Arrowheads indicate fragmentation of the γ-tubulin signals. (E–G) Analysis of mitotic figures in HEK293T wt cells and cells constitutively expressing low levels (“low”) of an siRNA-resistant, FLAG-tagged version of human SSX2IP and high levels after addition of doxycycline (“high”). (E) Green, γ-tubulin; red, SSX2IP; blue, DAPI/chromatin. (F) Quantification of mitotic fragmentation of γ-tubulin. The graph shows mean ± SD from five (wt) or three (stable expression of FLAG-SSX2IP) independent experiments; the significance was calculated by a Student’s t test (two-tailed) and scored as *, P < 0.05; **, P < 0.01. (G) SSX2IP (left) and γ-tubulin (right) levels at mitotic centrosomes after knockdown of SSX2IP in wt cells or cell lines stably expressing SSX2IP. The data distribution from one representative experiment out of four repetitions is shown; n > 35. Bars, 10 µm.