GLI1 facilitates collagen-induced arthritis in mice by collaborative regulation of DNA methyltransferases
- Department of Orthopedics, The First Affiliated Hospital of Soochow University, Orthopedic Institute, Medical College, Soochow University, China
- Medical 3D Printing Center, The First Affiliated Hospital, School of Biology & Basic Medical Sciences, Suzhou Medical College, Soochow University, China
- Department of Obstetrics and Gynecology, The First Affiliated Hospital of Soochow University, China
- Department of Orthopedics, The First Affiliated Hospital of USTC, Division of Life Sciences and Medicine, University of Science and Technology of China, China
- Department of Orthopaedics, Suzhou Kowloon Hospital Shanghai Jiao Tong University School of Medicine, China
- Collaborative Innovation Center of Hematology, Soochow University, China
Figures
Figure 1 with 3 supplements
GLI1 is highly expressed in human RA synovium and regulates the pathological process of joint inflammatory bone destruction in CIA mice.
(A) H&E staining of normal and RA human synovium. Scale bars = 200 μm. (B) IHC staining of GLI1 in normal and RA human synovium and (C) quantification of positively stained cells. Scale bars = 50 …
Figure 1—figure supplement 1
GLI1 is highly expressed in synovial tissue of rheumatoid arthritis patients.
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Figure 1—figure supplement 1—source data 1
Uncropped western blot images for Figure 1—figure supplement 1.
- https://cdn.elifesciences.org/articles/92142/elife-92142-fig1-figsupp1-data1-v1.pptx
Figure 1—figure supplement 2
GANT58 shows no obvious toxicity in CIA mice.
Figure 1—figure supplement 3
GANT58 reduces arthritis symptoms and bone destruction in CIA mice.
Figure 2 with 3 supplements
GLI1 plays an active role in M1 macrophage formation and the release of proinflammatory cytokines.
(A, B) RAW264.7 cells were stimulated with LPS/IFN-γ for 24 hr. The total proteins and proteins in the cytoplasm and nucleus were isolated and extracted. GLI1 protein was detected by western blot, …
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Figure 2—source data 1
Uncropped western blot images for Figure 2.
- https://cdn.elifesciences.org/articles/92142/elife-92142-fig2-data1-v1.pptx
Figure 2—figure supplement 1
Cytotoxicity detection of GANT58.
Figure 2—figure supplement 2
GLI1 protein expression after GANT58 and siRNA treatment.
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Figure 2—figure supplement 2—source data 1
Uncropped western blot images for Figure 2—figure supplement 2.
- https://cdn.elifesciences.org/articles/92142/elife-92142-fig2-figsupp2-data1-v1.pptx
Figure 2—figure supplement 3
GANT58 inhibits M1 macrophage polarization.
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Figure 2—figure supplement 3—source data 1
Uncropped western blot images for Figure 2—figure supplement 3.
- https://cdn.elifesciences.org/articles/92142/elife-92142-fig2-figsupp3-data1-v1.pptx
Figure 3 with 1 supplement
GLI1 regulates the formation of osteoclasts in vivo and in vitro.
(A, B) RAW264.7 cells were stimulated by RANKL for 48 hr. Total GLI1 protein and GLI1 in the cytoplasm and nucleus were detected by western blotting during osteoclast induction, and the fold change …
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Figure 3—source data 1
Uncropped western blot images for Figure 3.
- https://cdn.elifesciences.org/articles/92142/elife-92142-fig3-data1-v1.pptx
Figure 3—figure supplement 1
GANT58 at working concentration has no obvious apoptosis-promoting effect on cells.
Figure 4 with 3 supplements
DNA methyltransferases might be a regulatory target downstream of GLI1.
(A) Biological process GO analysis of RNA-seq results for macrophages with or without GANT58 treatment. (B) KEGG rich analysis of RNA-seq results. (C) Heat map of parts of the relevant gene …
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Figure 4—source data 1
Uncropped western blot images for Figure 4.
- https://cdn.elifesciences.org/articles/92142/elife-92142-fig4-data1-v1.pptx
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Figure 4—source data 2
RNA-seq data for Figure 4.
- https://cdn.elifesciences.org/articles/92142/elife-92142-fig4-data2-v1.xlsx
Figure 4—figure supplement 1
Analysis of differentially expressed genes and enrichment pathways of RNA-seq results.
Figure 4—figure supplement 2
DNMT1 and DNMT3a are highly expressed in synovial tissue of patients with rheumatoid arthritis.
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Figure 4—figure supplement 2—source data 1
Uncropped western blot images for Figure 4—figure supplement 2.
- https://cdn.elifesciences.org/articles/92142/elife-92142-fig4-figsupp2-data1-v1.pptx
Figure 4—figure supplement 3
GANT58 reduced the expression of DNMT1 and DNMT3a in macrophages under different induction conditions.
Figure 5 with 4 supplements
GLI1 regulates the expression of DNMT1, which affects macrophage phenotypes and the release of inflammatory cytokines.
(A) IHC staining of DNMT1 and (B) quantification of positive cell numbers in mouse ankle joints. n=5. Scale bars = 100 μm. Statistical analysis was performed using one-way ANOVA. (C) RAW264.7 cells …
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Figure 5—source data 1
Uncropped western blot images for Figure 5.
- https://cdn.elifesciences.org/articles/92142/elife-92142-fig5-data1-v1.pptx
Figure 5—figure supplement 1
GANT58 decreased the expressions of DNMT1 in CIA mice.
Figure 5—figure supplement 2
Cytotoxicity detection of procainamide and TF3.
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Figure 5—figure supplement 2—source data 1
Uncropped western blot images for Figure 5—figure supplement 2.
- https://cdn.elifesciences.org/articles/92142/elife-92142-fig5-figsupp2-data1-v1.pptx
Figure 5—figure supplement 3
Overexpression of DNMT1 rescued the expression of pro-inflammatory cytokines in M1 macrophages treated by GANT58.
Figure 5—figure supplement 4
Effects of overexpression of DNMT1 on induction of M1 macrophages.
Figure 6 with 2 supplements
GLI1 regulates DNMT3a to affect the differentiation of osteoclasts.
(A) RAW264.7 cells were stimulated with RANKL for 3 days. Western blot analysis of DNMT3a and DNMT1 during the process of osteoclast induction and (B) grayscale value ratio to β-actin of western …
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Figure 6—source data 1
Uncropped western blot images for Figure 6.
- https://cdn.elifesciences.org/articles/92142/elife-92142-fig6-data1-v1.pptx
Figure 6—figure supplement 1
GANT58 decreased the expressions of DNMT3a in CIA mice.
Figure 6—figure supplement 2
Effects of overexpression of DNMT3a on induction of osteoclasts.
Author response image 1
(a) M1 macrophages were induced for 24 h with intervention of different concentrations of GANT58.
Immunofluorescence staining of iNOS. (b) Osteoclast were induced for 4 days with intervention of different concentrations of GANT58. TRAP staining images and TRAP positive osteoclast number …
Author response image 2
(a) Immunofluorescence staining of iNOS in RAW264.
7 cells during the stimulation by LPS/IFN-γ with or without 10 μM cyclopamine intervention and mean fluorescence intensity of the iNOS immunofluorescence. (b) TRAP staining of RAW264.7 cells after …
Additional files
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Supplementary file 1
siRNA target sequences.
- https://cdn.elifesciences.org/articles/92142/elife-92142-supp1-v1.docx
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Supplementary file 2
Predictive analysis of protein binding.
- https://cdn.elifesciences.org/articles/92142/elife-92142-supp2-v1.docx
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Supplementary file 3
RT-qPCR primer sequences.
- https://cdn.elifesciences.org/articles/92142/elife-92142-supp3-v1.docx
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MDAR checklist
- https://cdn.elifesciences.org/articles/92142/elife-92142-mdarchecklist1-v1.pdf
