Figure 2.
Cep55 deficiency induces cell cycle arrest and decreased Smo enrichment by abnormal cilia. (A)Cep55+/+ and Cep55−/− primary MEFs were stained with Ac-tubulin (green), a centrosome marker (γ-tubulin, red), and DNA (blue) in the presence of serum (+ Serum). Insets show zoomed-in views of the boxed regions. Scale bars, 10 µm (main image) and 1 µm (magnified region). (B) Quantification of the ciliated cells in A (three mice per group). Data are means ± SD. Unpaired two-tailed t test was performed. ***, P < 0.001. n, number of cells. (C)Cep55+/+ and Cep55−/− primary MEFs were stained with Ac-tubulin (green) and DNA (blue) under serum starvation (− Serum). Insets show zoomed-in views of the boxed regions. Scale bars, 10 µm (main image) and 1 µm (magnified region). (D) Graph showing quantitative analysis of the cilium length in C. Each dot represents one cell. Data are means ± SD. Unpaired two-tailed t test was performed. ***, P < 0.001. (E)Cep55+/+ and Cep55−/− primary MEFs were transfected with control and Ift20 siRNA and then stained with EdU (red), Ac-tubulin (green), and DNA (blue). Insets show zoomed-in views of the boxed regions. Scale bars, 20 µm (main image) and 2 µm (magnified region). (F) The percentages of EdU-positive cells and ciliated cells in E were quantified. Data are means ± SD of three independent experiments. A two-way ANOVA test was performed in each group, followed by Dunnett's multiple comparisons. ***, P < 0.001. n, number of cells. (G) Immunoblot of primary MEFs lysates in (E and F) with the indicated antibodies. (H)Cep55+/+ and Cep55−/− primary MEFs were transfected with control and Ift20 siRNA and then stained with propidium iodide for DNA content analysis by FACS. DNA content is represented on the x axis, and the number of cells counted is represented on the y axis. Cell populations in G0/G1, S, and G2/M phases are given as percentage of total cells. (I) Graphical representations of the cell cycle distribution of the samples in H. Data are presented as means ± SD of three independent experiments. One-way ANOVA test was performed to analyze the difference of the percentage of cells in G1 phase in each group followed by Dunnett’s multiple comparisons. ***, P < 0.001. (J) P0.5 Cep55+/+ and Cep55−/− cerebral cortex coronal sections were stained with proliferation marker (Ki67, green) and DNA (blue). Scale bar, 250 µm. (K) Quantification of Ki67-positive (Ki67+) cells in the cerebral cortex in J (three mice per group). Data are means ± SD. An unpaired two-tailed t test was performed. ***, P < 0.001. (L)Cep55+/+ and Cep55−/− primary MEFs treated with SAG in the absence of serum (− Serum) were stained with Smo (green), ARL13B (red), and DNA (blue). Insets show zoomed-in views of the boxed regions. Scale bar, 5 µm (main image) and 1 µm (magnified region). (M) Quantification of the Smo-positive (Smo+) ciliated cell in L. Data are presented as means ± SD of three independent experiments. A two-way ANOVA test was performed followed by Bonferroni’s multiple comparisons. ***, P < 0.001. NT, non-treatment.

Cep55 deficiency induces cell cycle arrest and decreased Smo enrichment by abnormal cilia. (A) Cep55 +/+ and Cep55−/− primary MEFs were stained with Ac-tubulin (green), a centrosome marker (γ-tubulin, red), and DNA (blue) in the presence of serum (+ Serum). Insets show zoomed-in views of the boxed regions. Scale bars, 10 µm (main image) and 1 µm (magnified region). (B) Quantification of the ciliated cells in A (three mice per group). Data are means ± SD. Unpaired two-tailed t test was performed. ***, P < 0.001. n, number of cells. (C)Cep55+/+ and Cep55−/− primary MEFs were stained with Ac-tubulin (green) and DNA (blue) under serum starvation (− Serum). Insets show zoomed-in views of the boxed regions. Scale bars, 10 µm (main image) and 1 µm (magnified region). (D) Graph showing quantitative analysis of the cilium length in C. Each dot represents one cell. Data are means ± SD. Unpaired two-tailed t test was performed. ***, P < 0.001. (E)Cep55+/+ and Cep55−/− primary MEFs were transfected with control and Ift20 siRNA and then stained with EdU (red), Ac-tubulin (green), and DNA (blue). Insets show zoomed-in views of the boxed regions. Scale bars, 20 µm (main image) and 2 µm (magnified region). (F) The percentages of EdU-positive cells and ciliated cells in E were quantified. Data are means ± SD of three independent experiments. A two-way ANOVA test was performed in each group, followed by Dunnett's multiple comparisons. ***, P < 0.001. n, number of cells. (G) Immunoblot of primary MEFs lysates in (E and F) with the indicated antibodies. (H)Cep55+/+ and Cep55−/− primary MEFs were transfected with control and Ift20 siRNA and then stained with propidium iodide for DNA content analysis by FACS. DNA content is represented on the x axis, and the number of cells counted is represented on the y axis. Cell populations in G0/G1, S, and G2/M phases are given as percentage of total cells. (I) Graphical representations of the cell cycle distribution of the samples in H. Data are presented as means ± SD of three independent experiments. One-way ANOVA test was performed to analyze the difference of the percentage of cells in G1 phase in each group followed by Dunnett’s multiple comparisons. ***, P < 0.001. (J) P0.5 Cep55+/+ and Cep55−/− cerebral cortex coronal sections were stained with proliferation marker (Ki67, green) and DNA (blue). Scale bar, 250 µm. (K) Quantification of Ki67-positive (Ki67+) cells in the cerebral cortex in J (three mice per group). Data are means ± SD. An unpaired two-tailed t test was performed. ***, P < 0.001. (L)Cep55+/+ and Cep55−/− primary MEFs treated with SAG in the absence of serum (− Serum) were stained with Smo (green), ARL13B (red), and DNA (blue). Insets show zoomed-in views of the boxed regions. Scale bar, 5 µm (main image) and 1 µm (magnified region). (M) Quantification of the Smo-positive (Smo+) ciliated cell in L. Data are presented as means ± SD of three independent experiments. A two-way ANOVA test was performed followed by Bonferroni’s multiple comparisons. ***, P < 0.001. NT, non-treatment.

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