Fig. 4

Excessive cholesterol synthesis results in abnormal primary cilium formation. a IC analyses for anti-acetylated tubulin (AT; green) in WT and Insig1/2 conditional KO (cKO) osteoblasts. Nuclei were stained with DAPI (blue). Boxed areas in upper images are enlarged, and arrows indicate primary cilia. Scale bars: 20 µm in the upper images and 5 µm in the lower images. b Percentage of cells with primary cilia in osteoblasts from WT (blue bar) and Insig1/2 cKO (yellow bar) osteoblasts. More than 200 cells were randomly analyzed in three independent experiments. c Quantification of the length of primary cilia in osteoblasts from WT (left) and Insig1/2 cKO (right) mice. More than 200 cells were randomly analyzed in three independent experiments. ***P < 0.001. d IC for RAB8 (red) and AT (green) in WT and Insig1/2 cKO osteoblasts. Nuclei were stained with DAPI (blue). Boxed areas in upper images are enlarged. Scale bars, 5 µm. e IC analyses for γ-tubulin (red) and AT (green) in WT and Insig1/2 cKO osteoblasts. Nuclei were stained with DAPI (blue). Arrows indicate duplicated primary cilia and basal bodies. Primary cilia are enlarged in insets. Scale bars, 5 µm. f ChIP assays of IgG control and SREBP1 or SREBP2 for SRE (BS1 and BS2) of the Plk4 promoter region in WT control (blue bars) and Insig1/2 cKO (yellow bars) osteoblasts. n = 3 per group. ***P < 0.001. g Quantitative RT-PCR for Plk4 in WT (blue bar) and Insig1/2 cKO (yellow bar) osteoblasts. n = 6 per group. **P < 0.01. h Immunoblotting for PLK4 in WT and Insig1/2 cKO osteoblasts. GAPDH was used as loading control.