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Research Article
Gli2 modulates cell cycle re-entry through autophagy-mediated regulation of the length of primary cilia
Ching-Ju Hsiao, Chia-Hsiang Chang, Ridwan Babatunde Ibrahim, I-Hsuan Lin, Chun-Hung Wang, Won-Jing Wang, Jin-Wu Tsai
Journal of Cell Science 2018 131: jcs221218 doi: 10.1242/jcs.221218 Published 17 December 2018
Ching-Ju Hsiao
1Institute of Brain Science, School of Medicine, National Yang-Ming University, Taipei 112, Taiwan
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Chia-Hsiang Chang
1Institute of Brain Science, School of Medicine, National Yang-Ming University, Taipei 112, Taiwan
2Taiwan International Graduate Program (TIGP) in Molecular Medicine, National Yang-Ming University and Academia Sinica, Taipei 112, Taiwan
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Ridwan Babatunde Ibrahim
1Institute of Brain Science, School of Medicine, National Yang-Ming University, Taipei 112, Taiwan
3Taiwan International Graduate Program (TIGP) in Interdisciplinary Neuroscience, National Yang-Ming University and Academia Sinica, Taipei 112, Taiwan
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I-Hsuan Lin
2Taiwan International Graduate Program (TIGP) in Molecular Medicine, National Yang-Ming University and Academia Sinica, Taipei 112, Taiwan
4Institute of Biochemistry and Molecular Biology, College of Life Sciences, National Yang-Ming University, Taipei 112, Taiwan
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Chun-Hung Wang
1Institute of Brain Science, School of Medicine, National Yang-Ming University, Taipei 112, Taiwan
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Won-Jing Wang
4Institute of Biochemistry and Molecular Biology, College of Life Sciences, National Yang-Ming University, Taipei 112, Taiwan
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Jin-Wu Tsai
1Institute of Brain Science, School of Medicine, National Yang-Ming University, Taipei 112, Taiwan
5Brain Research Center (BRC), and Biophotonics and Molecular Imaging Research Center (BMIRC), National Yang-Ming University, Taipei 112, Taiwan
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  • ORCID record for Jin-Wu Tsai
  • For correspondence: tsaijw@ym.edu.tw
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  • Fig. 1.
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    Fig. 1.

    Generation of Gli2-knockout cells with CRISPR/Cas9 technology. (A) Gli2 sequences of genomic DNA from NIH3T3WT and NIH3T3Gli2−/− cells at the sgRNA-targeted region. Eleven and five nucleotides in the exon 2 regions of the two Gli2 alleles were deleted. Blue box: sgRNA-targeted regions; red letters, sgRNA-targeting sequence; green letters, PAM sequence. Arrows indicate the breaking points. (B) Western blot of Gli2 protein in NIH3T3Gli2−/− and NIH3T3WT cells. Gli2 in NIH3T3Gli2−/− cells was barely detected. Gli2 (full-length, FL) can be restored by transfecting a pUS2-Gli2 construct. GAPDH was immunoblotted as the loading control. (C) Luciferase assay indicating that the binding activity of Gli was significantly decreased in NIH3T3Gli2−/− cells. The Gli-binding activity can be restored after Gli2 overexpression (pUS2-Gli2). The value of Gli–luciferase is normalized to that for Renilla luciferase, and further normalized to mutant Gli–luciferase. Bar graph along with individual data points represents the relative value of luciferase activity in each group (n=3 trials). ***P<0.001 (post-hoc Mann–Whitney U-test).

  • Fig. 2.
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    Fig. 2.

    Longer primary cilia in NIH3T3Gli2−/− cells after serum starvation. (A) Immunofluorescence staining of primary cilia (red, Arl13b), the basal body [magenta, γ-tubulin (γ-tub)] and nucleus (DAPI) in NIH3T3WT and NIH3T3Gli2−/− cells after serum starvation for 24 h. The scatter plot shows that NIH3T3Gli2−/− cells (n=60) possessed longer primary cilia compared to NIH3T3WT cells (n=63), while Gli2 overexpression (OX) (green, GFP-tagged Gli2; n=68) restored the ciliary length in NIH3T3Gli2−/− cells. The bars represent mean±s.d. ***P<0.001 (one-way ANOVA with post-hoc Bonferroni test). (B) Immunofluorescence staining of primary cilia (red, acetylated α-tubulin) and the nucleus (blue, DAPI) in NIH3T3WT and NIH3T3Gli2−/− cells after 24 h of serum starvation. NIH3T3Gli2−/− cells (n=76) possessed longer primary cilia compared to NIH3T3WT (n=110) cells. Scale bars: 2 μm. The bars represent mean±s.d. ***P<0.001 (Student's t-test). (C) Time course of ciliary length in NIH3T3 cells after serum starvation. Immunostaining the primary cilia (red, Arl13b), centriolar satellites (gray, Pcm1) and the nucleus (DAPI) in NIH3T3WT and NIH3T3Gli2−/− cells after serum starvation for 0, 4, 8, 12, 24 and 48 h. The ciliary lengths of NIH3T3Gli2−/− cells were significantly longer than NIH3T3WT after serum starvation for 4, 8, 12, 24 and 48 h. Scale bars: 2 μm. NIH3T3WT at 0 h, n=74; 4 h, n=102; 8 h, n=105; 12 h, n=65; 24 h, n=135; 48 h, n=63; NIH3T3Gli2−/− at 0 h, n=65; 4 h, n=156, 8 h, n=152; 12 h, n=97; 24 h, n=115; 48 h, n=65. ***P<0.001 (Student's t-test). Scatter plot displays individual cilia in NIH3T3WT cells (white circles) and NIH3T3Gli2−/− cells (black dots). Red lines mark the mean.

  • Fig. 3.
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    Fig. 3.

    Increase in autophagic activities in NIH3T3Gli2−/− cells. (A) Protein lysates were collected from NIH3T3WT and NIH3T3Gli2−/− cells after serum starvation (SS) for 0, 4, 8, 12, 24, and 48 h, and immunoblotted for LC3, p62 and Ofd1. β-actin was used as the loading control. Line charts show the relative protein levels of LC3-II (left), p62 (middle) and Ofd1 (right) in NIH3T3Gli2−/− cells (white circles) compared to WT cells (black circles). Results are mean±s.e.m.; n=3, *P<0.05; **P<0.01 (Student's t-test). (B) Autophagosomes and autolysosomes labeled with RFP–GFP–LC3 transfected into NIH3T3WT and NIH3T3Gli2−/− cells. Autophagosomes (white arrows) appear as yellow whereas autolysosomes (black arrows) appear red due to GFP fluorescence being quenched in the acidic environment. Scale bar: 5 μm. (C) Bar graph showing that the relative density of both autophagosome and autolysosome puncta are higher in NIH3T3Gli2−/− cells (n=59 cells) than those in NIH3T3WT cells (n=21 cells). ***P<0.001 (Student's t-test). (D) Bar graph showing that there is no difference in the percentage of autolysosome versus total LC3 puncta between NIH3T3WT and NIH3T3Gli2−/− cells. NS, not significant (Student's t-test). Results in C and D are mean±s.e.m.

  • Fig. 4.
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    Fig. 4.

    Inhibition of autophagy restores the ciliary length in NIH3T3Gli2−/− cells. (A) Western blot of LC3 in NIH3T3 cells treated with an autophagy inhibitor, 3-methyladenine (3-MA). α-tubulin (α-tub) was immunoblotted as the loading control. Bar graph along with individual data points shows that 3-MA reduced LC3-II expression both in NIH3T3WT and NIH3T3Gli2−/− cells (n=3 trials). **P<0.01, ***P<0.001 (one-way ANOVA with a post-hoc Bonferroni test). (B) Immunofluorescence staining of primary cilia (red, Arl13b), basal body [magenta, γ-tubulin (γ-tub)] and the nucleus (blue, DAPI) in NIH3T3WT and NIH3T3Gli2−/− cells treated with DMSO or 3-MA at the first 4 h of the 24 h serum starvation period. The inset shows the boxed region in each panel. Scale bar: 2 μm. Scatter plots show that primary cilia are longer in NIH3T3Gli2−/− cells (n=60) than NIH3T3WT cells (n=63), while 3-MA significantly reduced the length of primary cilia in both cells (n=61 and 62 in NIH3T3Gli2−/− cells and NIH3T3WT cells, respectively). ***P<0.001 (one-way AVOVA with post-hoc Bonferroni test). The red line indicates the mean. (C) Immunoblots of LC3 in NIH3T3 cells transfected with sh-Ctrl-GFP or sh-Atg3-GFP and selected by puromycin treatment. α-tubulin (α-tub) serves as the loading control. Bar graph along with individual data points shows that cells transfected with sh-Atg3-GFP exhibited lower LC3-II protein compared to control group upon serum starvation. Results are mean±s.e.m.; n=3 trials. *P<0.05 (Student's t-test). (D) Immunostaining for primary cilia (Arl13b, red), centriolar satellites (Pcm1, magenta) and the nucleus (DAPI, blue) in NIH3T3WT and NIH3T3Gli2−/− cells transfected with sh-Ctrl-GFP or sh-Atg3-GFP. Insets show the primary cilia in the boxed regions of each panel. Scale bar: 2 μm. Scatter plots show that primary cilia were longer in NIH3T3Gli2−/− cells (n=105) than in NIH3T3WT cells (n=100), while sh-Atg3-GFP reduced the length of primary cilia in both cells (n=133 and 131 in NIH3T3Gli2−/− cells and NIH3T3WT cells, respectively). Red lines mark the mean. ***P<0.001 (one-way AVOVA with post-hoc Bonferroni test).

  • Fig. 5.
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    Fig. 5.

    Delay in ciliary resorption in NIH3T3Gli2−/− cells. (A) Time course of ciliary length in NIH3T3 cells after serum re-stimulation. The primary cilium (red, Arl13b), centrioles (gray, γ-tubulin) and nuclei (DAPI) in NIH3T3WT and NIH3T3Gli2−/− cells were stained after serum re-supplementation for 0, 8, 16, and 24 h following 24 h of serum starvation. The scatter plots display individual cilia in NIH3T3WT cells (white circles) and NIH3T3Gli2−/− cells (black dots). Red lines mark the mean. Insets represent the boxed region in each panel. Scale bars: 2 μm. NIH3T3WT at 0 h, n=269; 8 h, n=64; 16 h, n=95; 24 h, n=64; NIH3T3Gli2−/− at 0 h, n=196; 8 h, n=113; 16 h, n=113; 24 h, n=261. ***P<0.001 (Mann–Whitney U-test). (B) Bar graph along with individual data points representing the percentage of centrioles possessing an Arl13b-positive signal (indicating that the primary cilium is present on the centrioles) after serum starvation for 24 h (ss24), followed by serum re-stimulation for indicated conditions (+8h, +16h, +24h). n=3 trials. P-values are shown (Student's t-test).

  • Fig. 6.
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    Fig. 6.

    Ablation of the primary cilia by Kif3a knockdown rescues the delay of cell cycle re-entry in NIH3T3Gli2−/− cells. (A) Immunostaining of the primary cilia (Arl13b, red), centrioles (γ-tubulin, magenta) and nucleus (DAPI, blue) in NIH3T3WT and NIH3T3Gli2−/− cells transfected with sh-Ctrl-GFP or sh-Kif3a-GFP (green). The boxed regions are magnified in right of each panel, and show the primary cilia in non-transfected cells (GFP−; top) and transfected cells (GFP+; bottom). Kif3a shRNA effectively eliminates the growth of primary cilia in both NIH3T3WT and NIH3T3Gli2−/− cells. Scale bar: 2 μm. (B) Cell cycle analysis by flow cytometry in NIH3T3WT and NIH3T3Gli2−/− cells and cells transfected with sh-Ctrl-GFP or sh-Kif3a-GFP. Cells were synchronized via serum starvation for 24 h and then collected for 0 h, 8 h, 16 h, and 24 h after serum re-stimulation. (C) The line chart shows the time course of cell cycle re-entry after 0 h, 8 h, 16 h, and 24 h of serum add-back in NIH3T3WT cells and NIH3T3Gli2−/− cells transfected with either sh-Ctrl-GFP or sh-Kif3a-GFP. n=3 trials. While NIH3T3Gli2−/− cells showed a delay in cell cycle re-entry, Kif3a shRNA rescued this effect. **P<0.01, ***P<0.001 (one-way ANOVA with post-hoc Bonferroni test).

  • Fig. 7.
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    Fig. 7.

    Proposed model for the relationship between Gli2 and primary cilia-dependent cell cycle re-entry. In NIH3T3WT cells, the presence of Gli2 represses the progression of autophagy, which reduces the autophagy-dependent Ofd1 removal during serum starvation. In NIH3T3Gli2−/− cells, loss of Gli2 releases the inhibition of autophagy, leading to more Ofd1 removal upon serum starvation. This process contributes to the elongation of ciliary length, resulting in the delay of cell cycle re-entry after serum re-stimulation.

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Keywords

  • Gli2
  • CRISPR/Cas9 technology
  • Primary cilium
  • Cell cycle
  • Autophagy
  • Ofd1

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Research Article
Gli2 modulates cell cycle re-entry through autophagy-mediated regulation of the length of primary cilia
Ching-Ju Hsiao, Chia-Hsiang Chang, Ridwan Babatunde Ibrahim, I-Hsuan Lin, Chun-Hung Wang, Won-Jing Wang, Jin-Wu Tsai
Journal of Cell Science 2018 131: jcs221218 doi: 10.1242/jcs.221218 Published 17 December 2018
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Research Article
Gli2 modulates cell cycle re-entry through autophagy-mediated regulation of the length of primary cilia
Ching-Ju Hsiao, Chia-Hsiang Chang, Ridwan Babatunde Ibrahim, I-Hsuan Lin, Chun-Hung Wang, Won-Jing Wang, Jin-Wu Tsai
Journal of Cell Science 2018 131: jcs221218 doi: 10.1242/jcs.221218 Published 17 December 2018

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