Figure 2.

Reduction of cilia assembly in the CEP250 and CROCC KO cells. (A) The CEP250 and CROCC KO RPE1 cells were cultured in serum-deprived medium for 48 h and subjected to coimmunostaining analyses with antibodies specific to CEP250 (cyan), CROCC (cyan), and acetylated tubulin (magenta). (B) The number of cells with cilia was counted. (C) The number of cells with centriole disjunction (>2 μm) was counted after treatment of 20 μM nocodazole for 2 h. (D) The CEP250 KO cells were cultured in serum-deprived medium for 48 h to induce cilia assembly and subjected to coimmunostaining analysis with antibodies specific to CEP250 (cyan) and acetylated tubulin (magenta) with and without daughter centriole association. (E) The number of cells with cilia was counted in CEP250 KO cells with and without daughter centriole association. (F) The number of cells with centrosome/basal body CROCC signals was counted in CEP250 KO cells with and without cilia in two different cell densities. (G) The CEP250 KO cells were cultured in normal and serum-deprived media for 48 h and subjected to coimmunostaining analysis with antibodies specific to CROCC (cyan) and acetylated tubulin (magenta). (H) The number of cells with centrosome/basal body CROCC signals was counted in CEP250 KO cells cultured in two different cell densities. (A, D, and G) Scale bars, 10 μm; inset scale bars, 2 μm. (B, C, E, F, and H) More than 30 cells per group were counted in three independent experiments. Graph values are expressed as mean and SEM. Statistical significance was determined using one-way ANOVA with Tukey’s post hoc test (*, P < 0.05; **, P < 0.01; ***, P < 0.001; n.s., not significant).