Figure 5.
CEP55 promotes cilia disassembly by regulating Aurora A stability. (A) Schematic illustration of experimental strategy used for the cilia assembly and disassembly experiments. (B) RPE-1 cells were transfected with control or CEP55 siRNA and serum starved for 48 h. The ciliated RPE-1 cells were then stimulated with serum for the indicated times and stained with Ac-tubulin (green), γ-tubulin (red), and DNA (blue). Insets show zoomed-in views of the boxed regions. Scale bars, 10 µm (main image) and 2 µm (magnified region). (C) Quantification of the percentage of ciliated cells in B with three individual siRNAs against CEP55. Ciliation in siCEP55 groups are compared with their respective time-point controls in siControl group. Data are presented as means ± SD of three independent experiments. A two-way ANOVA test was performed followed by Dunnett’s multiple comparisons. *, P < 0.05; **, P < 0.01; ***, P < 0.001. n, number of cells. (D) Quantification of the percentage of ciliated cells. Cep55+/+ and Cep55−/− primary MEFs were starved for 48 h and then stimulated with serum for the indicated times. Data are presented as means ± SD of three independent experiments. A two-way ANOVA test was performed. *, P < 0.05; **, P < 0.01; ***, P < 0.001. n, number of cells. (E) RPE-1 cells were starved (− Serum) and then stimulated with serum (serum stimulation) for the indicated times. The endogenous expressions of CEP55, Aurora A, HDAC6, and GAPDH were determined by immunoblotting with respective antibodies. (F) RPE-1 cells were transfected with control or CEP55 siRNA and serum starved for 48 h. Then, the cells were stimulated with serum for the indicated times. The treated cells were subjected to immunoblotting with the indicated antibodies. (G) mRNA expression levels of Aurora A by qRT-PCR were determined from RPE-1 cells transfected with control, CEP55, or Aurora A siRNA. Data are means ± SD of three independent experiments. A one-way ANOVA test was performed followed by Dunnett’s multiple comparisons. ***, P < 0.001. (H–J) Ciliation in RPE-1 cells induced by CEP55 knockdown were rescued by expressing GFP-Aurora A. GFP-vector (−) or GF–Aurora A plasmids were transfected in RPE-1 cells after 24 h of siRNA transfection. 24 h later, cell lysates were detected with the indicated antibodies (H) or were stained with Ac-tubulin (red) and DNA (blue; I). Scale bars,10 µm (main image) and 1 µm (magnified region). (J) Quantification of the ciliation in GFP-positive cells in I. 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. n, number of cells.

CEP55 promotes cilia disassembly by regulating Aurora A stability. (A) Schematic illustration of experimental strategy used for the cilia assembly and disassembly experiments. (B) RPE-1 cells were transfected with control or CEP55 siRNA and serum starved for 48 h. The ciliated RPE-1 cells were then stimulated with serum for the indicated times and stained with Ac-tubulin (green), γ-tubulin (red), and DNA (blue). Insets show zoomed-in views of the boxed regions. Scale bars, 10 µm (main image) and 2 µm (magnified region). (C) Quantification of the percentage of ciliated cells in B with three individual siRNAs against CEP55. Ciliation in siCEP55 groups are compared with their respective time-point controls in siControl group. Data are presented as means ± SD of three independent experiments. A two-way ANOVA test was performed followed by Dunnett’s multiple comparisons. *, P < 0.05; **, P < 0.01; ***, P < 0.001. n, number of cells. (D) Quantification of the percentage of ciliated cells. Cep55+/+ and Cep55−/− primary MEFs were starved for 48 h and then stimulated with serum for the indicated times. Data are presented as means ± SD of three independent experiments. A two-way ANOVA test was performed. *, P < 0.05; **, P < 0.01; ***, P < 0.001. n, number of cells. (E) RPE-1 cells were starved (− Serum) and then stimulated with serum (serum stimulation) for the indicated times. The endogenous expressions of CEP55, Aurora A, HDAC6, and GAPDH were determined by immunoblotting with respective antibodies. (F) RPE-1 cells were transfected with control or CEP55 siRNA and serum starved for 48 h. Then, the cells were stimulated with serum for the indicated times. The treated cells were subjected to immunoblotting with the indicated antibodies. (G) mRNA expression levels of Aurora A by qRT-PCR were determined from RPE-1 cells transfected with control, CEP55, or Aurora A siRNA. Data are means ± SD of three independent experiments. A one-way ANOVA test was performed followed by Dunnett’s multiple comparisons. ***, P < 0.001. (H–J) Ciliation in RPE-1 cells induced by CEP55 knockdown were rescued by expressing GFP-Aurora A. GFP-vector (−) or GF–Aurora A plasmids were transfected in RPE-1 cells after 24 h of siRNA transfection. 24 h later, cell lysates were detected with the indicated antibodies (H) or were stained with Ac-tubulin (red) and DNA (blue; I). Scale bars,10 µm (main image) and 1 µm (magnified region). (J) Quantification of the ciliation in GFP-positive cells in I. 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. n, number of cells.

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