Characterization of CEP152 Seckel cells. (a) Mitotic morphology of CEP152 Seckel fibroblasts. Immunofluorescence staining of CEP152 Seckel fibroblasts carrying the c.261+1G>C mutation with antibodies against α-tubulin (green), pericentrin (red) and DAPI staining of DNA (blue). Above, from left to right, control fibroblasts showing normal mitotic morphology of interphase, metaphase, anaphase and telophase. Middle, Seckel interphase cells containing three equally sized nuclei and two centrosomes without astral microtubules (inset, tenfold magnification), two unseparated centrosomes per nucleus without asters (inset, threefold magnification), fragmented centrosomes without asters and micronuclei (inset, twofold magnification), and partially depolymerized microtubules together with micronuclei in addition to a main nucleus. Below, abnormal Seckel metaphases showing incorrectly aligned chromosomes on the metaphase plate, a monopolar spindle with a large centrosome and reduced spindle, a tripolar spindle with differently sized and structurally compromised centrosomes, and an abnormal Seckel telophase showing defects in cytokinesis (inset, twofold magnification). Scale bars, 5 μm. (b) Aneuploid metaphase karyotype of a CEP152 Seckel lymphocyte. (c) Centrosomal localization of wildtype CEP152 in HEK293T cells expressing either GFP-tagged wildtype CEP152 (above) or GFP as a control (below). Additional staining was with pericentrin (red) and DAPI (blue). (d) DNA-damage response in wildtype and Seckel fibroblasts. H2AX phosphorylation of wildtype and CEP152 Seckel primary fibroblasts after treatment with hydroxyurea (HU) (left). Protein blot analysis of HU-induced phosphorylation of CHK1 (Ser345) and H2AX (Ser139) (right). Equal protein loading was confirmed by re-probing of the membranes with antibodies against CHK1 or H2AX and actin antibodies.