Centriolar satellites and AURKA exhibit antagonistic effects during cilium assembly and disassembly A.Figure 5A. Representative immunofluorescence images of cilium assembly experiments in control and PCM1-depleted RPE1 cells treated with DMSO or MLN8237. Cells were transfected with control or PCM1 siRNA#1 for 48 h and treated with DMSO (vehicle control) or 0.5 μM MLN8237 in serum starvation medium for 24 h. Cells were fixed and immunostained for the primary cilium with acetylated tubulin antibody (Acet-tub) and the centrosome with gamma-tubulin antibody. DNA was stained with DAPI. Scale bar, 10 μm. B. Quantification of ciliogenesis efficiency for A. n>100 cells per experiment. Data represent mean value from three experiments per condition ± SEM (***p < 0.001; **p < 0.01; ns, non-significant, Unpaired Student's t-test.). C. Quantification of cilium length for A. n>100 cells per experiment. Data represent mean value from three experiments per condition ± SEM (****p < 0.0001; ns, non-significant, Unpaired Student's t-test.). D. Representative immunofluorescence images of cilium assembly experiments in control and PCM1-depleted RPE1 cells treated with DMSO or tubacin. Cells were transfected with control or PCM1 siRNA#1 for 48 h and treated with DMSO or 2 μM tubacin in serum starvation medium for 24 h. Cells were fixed and immunostained for the primary cilium with Arl13b and acetylated tubulin antibody (Acet-tub) and the centrosome with gamma-tubulin antibody. DNA was stained with DAPI. Scale bar, 10 μm E. Quantification of ciliogenesis efficiency for D. n>100 cells per experiment. Data represent mean value from three experiments per condition ± SEM (*p < 0.05; ns, non-significant, Unpaired Student's t-test). F. Representative immunofluorescence images and quantification of IFT88 levels at the centrosome and primary cilium. RPE1 cells were transfected with control or PCM1 siRNA#1 for 48 h, serum starved for 24 h and treated with DMSO or 0.5 μM MLN8237. Cells were fixed and immunostained with antibodies against IFT88, acetylated tubulin and gamma tubulin. DNA was stained with DAPI. Scale bar, 10 μm. G. Quantification of IFT88 centrosomal intensity and ciliary concentration for F. IFT88 fluorescence intensities at the centrosome and axoneme were measured from maximum projections, and average means of the levels in control cells were normalized to 1. Ciliary concentration was calculated by dividing ciliary fluorescence intensity to the cilium length. n>100 cells per experiment. Data represent mean value from three experiments per condition ± SEM (***p < 0.001; ****p < 0.0001, Unpaired Student's t-test) H, I. Quantification of cilium disassembly and centrosomal AURKA levels after serum stimulation. RPE1 cells were transfected with control or PCM1 siRNA#1 for 48 h, serum starved for 24 h and treated with DMSO or 0.5 μM MLN8237 in serum stimulation medium for 2 h and 24 h. Cells were fixed and immunostained with antibodies against acetylated tubulin and gamma tubulin, and percentage of ciliated cells were quantified. x-axis indicates the hours after serum stimulation. (H) n>100 cells per experiment. Data represent mean value from three experiments per condition ± SEM. (I) n>95 cells per experiment. Data represent mean value from three experiments per condition ± SEM (***p < 0.001; ****p < 0.0001, Unpaired Student's t-test).