Figure 3

Characterization of primary cilia formation in OSE cell cultures. Wild type (wt) and cancer OSE cells were grown to confluence followed by serum depletion for 72 hours to induce growth arrest and formation of primary cilia (arrows). Microtubules of the ciliary axoneme were detected with anti-acetylated α-tubulin (Acet.tub) and anti-detyrosinated α-tubulin (Glu.tub) in IFM analysis (A, B). The pericentriolar material of the centrosome and the centrioles were visualized with anti-pericentrin (PCTN) and anti-centrin (CT) antibodies, respectively. C) Cilia frequencies were determined, by IFM analysis with anti-Acet.tub and/or anti-Glu.tub, antibodies, as the number of ciliated cells over the total cell number in sub-confluent cultures in the presence of serum (0 hour) and in confluent cultures serum-depleted for 48 or 72 hours. Error bars represent standard deviations. Data were tested for significance using one-way ANOVA. The level of significance was set at P < 0.001 (***). D) Localization of IFT20 and IFT88 was visualized by IFM in serum-depleted wt OSE cultures. Nuclei were stained with DAPI. E) WB analysis of IFT88 and IFT20 in wt and cancer OSE cells in sub-confluent cultures with serum (0 hour) and confluent cultures depleted for serum for six, 24, or 72 hours. α-Tubulin (α-tub) was applied as loading control. ANOVA, analysis of variance; DAPI, 4′,6-diamidino-2-phenylindole; IFM, immunofluorescence microscopy; OSE, ovarian surface epithelium; WB, western blot; wt, wild type.