Figure 4.

Subcellular Localization of KIZ in Monocilia from Human Fibroblasts

Primary fibroblasts derived from human skin biopsies were cultured via standard conditions. To induce formation of monocilia in human fibroblasts, we deprived cell culture from fetal bovine serum for 24 hr. Serum-starved fibroblasts were fixed 5 min with methanol at −20°C, and immunofluorescence staining was performed as previously described.³¹ Human fibroblasts were immunolabeled with a KIZ antibody (PLK1S1 rabbit-polyclonal antibody,1/250, Sigma Aldrich SAB2700541; A, C, D, and F), an acetyl α-tubulin mouse-monoclonal antibody (1/1,000, Sigma-Aldrich; B and C), and a γ-tubulin mouse-monoclonal antibody (1/1,000, Sigma-Aldrich; E and F), visualized with standard secondary antibodies, and counterstained with nuclear DAPI (blue, 1/1,000, Euromedex, Souffelweyersheim). The overlay view in (C) shows KIZ localization (A, red) at the bottom of the cilia (B, green) and colocalization (F) of KIZ (D, red) to the basal body of the cilium-associated centriole (E, green). Scale bars represent 20 μm.