(A) Indicated RPE1 cell lines were infected with lentiviral vectors carrying tet-inducible PLK4 expression construct and induced to overexpress PLK4. Doxycycline was applied to cells for 2 days before examination. FOP (green) marks centrosomes. Wild type RPE1 cells have centrosomes cluster in a small area. In contrast, centrosomes are well separated in the CEP128−/−; C-Nap1−/− double KO cells.
(B) EM images of two clustered cilia that grew from two centrosomes in the same ciliary pit. PLK4 was expressed in wild type RPE1 cells to induce formation of extra centrosomes before serum starvation and preparation for TEM.
(C) Multiple cilia in CEP128−/−; C-Nap1−/− double knockout cells were distantly separated with undiluted Arl13b. Multiple cilia formation in the same cell was induced by overexpression of the PLK4 kinase, followed by 48 hours of serum starvation. Cells were stained with indicated antibodies.
(D) Quantification of Arl13b intensity. Mean value of Arl13b intensity in regions surrounding cilia was taken. Plot displays Arl13b intensity per cilia in bi-ciliated or triciliated cells. Error bars represent standard deviations.
(See also Figure S3)