Vangl2 suppresses NF-κB signaling and ameliorates sepsis by targeting p65 for NDP52-mediated autophagic degradation
- Department of Joint Surgery, the Fifth Affiliated Hospital, Southern Medical University, China
- Department of Immunology, School of Basic Medical Sciences, Southern Medical University, China
- Department of Clinical Laboratory Medicine, Guangdong Provincial People's Hospital (Guangdong Academy of Medical Sciences), Southern Medical University, China
- Department of Orthopaedics, Yuebei People's Hospital Affiliated to Medical College of Shantou University, China
- Department of Pharmacology, School of Medicine, Southern University of Science and Technology, China
- Institute of Biosciences and Technology, College of Medicine, Texas A&M University, United States
- Klarman Cell Observatory, Broad Institute of MIT and Harvard, United States
- Department of Cell Biology, School of Basic Medical Sciences, Southern Medical University, China
- Guangdong Provincial Key Lab of Single Cell Technology and Application, Southern Medical University, China
Figures
Figure 1 with 1 supplement
Van Gogh-like 2 (Vangl2) ablation promotes inflammation during LPS treatment.
(A) Transcription levels of Vangl2 in PBMCs from healthy volunteers (healthy control, HC) and sepsis patients were analyzed by real-time PCR (n = 8). (B) Vangl2 mRNA in different organs from mice …
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Figure 1—source data 1
Uncropped and labelled gels for Figure 1.
- https://cdn.elifesciences.org/articles/87935/elife-87935-fig1-data1-v1.pdf
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Figure 1—source data 2
Raw data for Figure 1.
- https://cdn.elifesciences.org/articles/87935/elife-87935-fig1-data2-v1.xlsx
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Figure 1—source data 3
Raw unedited gels for Figure 1.
- https://cdn.elifesciences.org/articles/87935/elife-87935-fig1-data3-v1.zip
Figure 1—figure supplement 1
Expression of Van Gogh-like 2 (Vangl2) during sepsis and lipopolysaccharide (LPS) treatment.
(A) White blood cell (WBC) count, acute C-reactive protein (CRP), and IL-6 level in the peripheral blood mononuclear cell (PBMC) of healthy volunteers (HC) and sepsis patients (n ≥ 3). And WBC …
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Figure 1—figure supplement 1—source data 1
Uncropped and labelled gels for (Figure 1—figure supplement 1).
- https://cdn.elifesciences.org/articles/87935/elife-87935-fig1-figsupp1-data1-v1.pdf
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Figure 1—figure supplement 1—source data 2
Raw data for (Figure 1—figure supplement 1).
- https://cdn.elifesciences.org/articles/87935/elife-87935-fig1-figsupp1-data2-v1.xlsx
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Figure 1—figure supplement 1—source data 3
Raw unedited gels for (Figure 1—figure supplement 1).
- https://cdn.elifesciences.org/articles/87935/elife-87935-fig1-figsupp1-data3-v1.zip
Figure 2 with 1 supplement
Van Gogh-like 2 (Vangl2) negatively regulates lipopolysaccharide (LPS)-induced NF-κB activation and proinflammatory cytokines.
Wild-type (WT) and Vangl2-deficient (n ≥ 3) pMAC (A, C) or neutrophils (B, D) were stimulated with LPS (100 ng/ml) for the indicated times. Immunoblot analysis of total and phosphorylated p65, …
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Figure 2—source data 1
Uncropped and labelled gels for Figure 2.
- https://cdn.elifesciences.org/articles/87935/elife-87935-fig2-data1-v1.pdf
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Figure 2—source data 2
Raw data for Figure 2.
- https://cdn.elifesciences.org/articles/87935/elife-87935-fig2-data2-v1.xlsx
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Figure 2—source data 3
Raw unedited gels for Figure 2.
- https://cdn.elifesciences.org/articles/87935/elife-87935-fig2-data3-v1.zip
Figure 2—figure supplement 1
Van Gogh-like 2 (Vangl2) defection promotes lipopolysaccharide (LPS)-induced NF-κB activation and production of inflammatory cytokines.
Volcano plot (A), gene ontology (GO) analysis (B), and KEGG analysis (C) of differentially expressed genes in wild-type (WT) and Vangl2-deficient bone marrow-derived macrophages (BMDMs) after LPS …
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Figure 2—figure supplement 1—source data 1
Raw data for (Figure 2—figure supplement 1).
- https://cdn.elifesciences.org/articles/87935/elife-87935-fig2-figsupp1-data1-v1.pdf
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Figure 2—figure supplement 1—source data 2
Raw data for (Figure 2—figure supplement 1).
- https://cdn.elifesciences.org/articles/87935/elife-87935-fig2-figsupp1-data2-v1.xlsx
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Figure 2—figure supplement 1—source data 3
Raw unedited gels for (Figure 2—figure supplement 1).
- https://cdn.elifesciences.org/articles/87935/elife-87935-fig2-figsupp1-data3-v1.zip
Figure 3 with 1 supplement
Van Gogh-like 2 (Vangl2) inhibits NF-κB signaling by interacting with p65.
Cho (A) or HEK293T cells (B, C) were co-transfected with a NF-κB and TK-Renilla reporter along with increasing amounts of Vangl2 for 18 hr, then treated the cells with or without lipopolysaccharide …
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Figure 3—source data 1
Uncropped and labelled gels for Figure 3.
- https://cdn.elifesciences.org/articles/87935/elife-87935-fig3-data1-v1.pdf
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Figure 3—source data 2
Raw data for Figure 3.
- https://cdn.elifesciences.org/articles/87935/elife-87935-fig3-data2-v1.xlsx
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Figure 3—source data 3
Raw unedited gels for Figure 3.
- https://cdn.elifesciences.org/articles/87935/elife-87935-fig3-data3-v1.zip
Figure 3—figure supplement 1
Van Gogh-like 2 (Vangl2) interacts with p65 to inhibit NF-κB activation.
Luciferase activity in HEK293T transfected with plasmids encoding an NF-κB luciferase reporter and TK-Renilla reporter, together with a vector encoding MyD88 (A), IRAK1 (B), TRAF6 (C), IKKα (D), …
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Figure 3—figure supplement 1—source data 1
Uncropped and labelled gels for (Figure 3—figure supplement 1).
- https://cdn.elifesciences.org/articles/87935/elife-87935-fig3-figsupp1-data1-v1.pdf
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Figure 3—figure supplement 1—source data 2
Raw data for (Figure 3—figure supplement 1).
- https://cdn.elifesciences.org/articles/87935/elife-87935-fig3-figsupp1-data2-v1.xlsx
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Figure 3—figure supplement 1—source data 3
Raw unedited gels for (Figure 3—figure supplement 1).
- https://cdn.elifesciences.org/articles/87935/elife-87935-fig3-figsupp1-data3-v1.zip
Figure 4 with 1 supplement
Van Gogh-like 2 (Vangl2) promotes the autophagic degradation of p65.
(A) Immunoblot analysis of HEK293T cells transfected with Flag-p65 and increasing amounts of the vector encoding HA-Vangl2 (0, 250, 500, and 1000 ng) (n ≥ 3). (B) Total RNA from HEK293T cells as in …
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Figure 4—source data 1
Uncropped and labelled gels for Figure 4.
- https://cdn.elifesciences.org/articles/87935/elife-87935-fig4-data1-v1.pdf
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Figure 4—source data 2
Raw data for Figure 4.
- https://cdn.elifesciences.org/articles/87935/elife-87935-fig4-data2-v1.xlsx
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Figure 4—source data 3
Raw unedited gels for Figure 4.
- https://cdn.elifesciences.org/articles/87935/elife-87935-fig4-data3-v1.zip
Figure 4—figure supplement 1
Van Gogh-like 2 (Vangl2) promotes p65 degradation by autophagic pathway.
(A) Immunoblot analysis of HEK293T cells transfected with HA-p65 (wild-type [WT] or S536A) and Flag-Vangl2. (B) Immunoblot analysis of HEK293T cells transfected with HA-p65 and Flag-Vangl2 …
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Figure 4—figure supplement 1—source data 1
Uncropped and labelled gels for (Figure 4—figure supplement 1).
- https://cdn.elifesciences.org/articles/87935/elife-87935-fig4-figsupp1-data1-v1.pdf
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Figure 4—figure supplement 1—source data 2
Raw data for (Figure 4—figure supplement 1).
- https://cdn.elifesciences.org/articles/87935/elife-87935-fig4-figsupp1-data2-v1.xlsx
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Figure 4—figure supplement 1—source dat
a 3. Raw unedited gels for (Figure 4—figure supplement 1).
- https://cdn.elifesciences.org/articles/87935/elife-87935-fig4-figsupp1-data3-v1.zip
Figure 5 with 1 supplement
Van Gogh-like 2 (Vangl2) enhances the recognition of p65 by cargo receptor NDP52.
(A) HEK293T cells transfected with a vector expressing HA-Vangl2 along with the empty vector or vector encoding Flag-p62/NDP52/NBR1/Nix. The cell lysates were subjected to immunoprecipitation with …
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Figure 5—source data 1
Uncropped and labelled gels for Figure 5.
- https://cdn.elifesciences.org/articles/87935/elife-87935-fig5-data1-v1.pdf
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Figure 5—source data 2
Raw data for Figure 5.
- https://cdn.elifesciences.org/articles/87935/elife-87935-fig5-data2-v1.xlsx
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Figure 5—source data 3
Raw unedited gels for Figure 5.
- https://cdn.elifesciences.org/articles/87935/elife-87935-fig5-data3-v1.zip
Figure 5—figure supplement 1
Van Gogh-like 2 (Vangl2) promoted autophagic degradation of p65 is not mediated by cargo receptor p62.
(A) Wild-type (WT) and p62 knockout (KO) HEK293T cells transfected with a vector expressing HA-p65 along with the empty vector or vector encoding Flag-Vangl2. The cell lysates were subjected to …
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Figure 5—figure supplement 1—source data 1
Uncropped and labelled gels for (Figure 5—figure supplement 1).
- https://cdn.elifesciences.org/articles/87935/elife-87935-fig5-figsupp1-data1-v1.pdf
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Figure 5—figure supplement 1—source data 2
Raw data for (Figure 5—figure supplement 1).
- https://cdn.elifesciences.org/articles/87935/elife-87935-fig5-figsupp1-data2-v1.xlsx
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Figure 5—figure supplement 1—source data 3
Raw unedited gels for (Figure 5—figure supplement 1).
- https://cdn.elifesciences.org/articles/87935/elife-87935-fig5-figsupp1-data3-v1.zip
Figure 6
Van Gogh-like 2 (Vangl2) increases the K63-linked ubiquitination of p65.
(A) Wild-type (WT) and Vangl2-deficient bone marrow-derived macrophages (BMDMs) were stimulated with lipopolysaccharide (LPS) (100 ng/ml) for the indicated times. The cell lysates were subjected to …
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Figure 6—source data 1
Uncropped and labelled gels for Figure 6.
- https://cdn.elifesciences.org/articles/87935/elife-87935-fig6-data1-v1.pdf
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Figure 6—source data 2
Raw unedited gels for Figure 6.
- https://cdn.elifesciences.org/articles/87935/elife-87935-fig6-data2-v1.zip
Figure 7 with 1 supplement
Van Gogh-like 2 (Vangl2) recruits PDLIM2 to ubiquitinate p65.
(A) HEK293T cells were transfected with the indicated small interfering RNA (siRNA), NF-κB reporter plasmids together with HA-Vangl2, Flag-p65, or the control vector as indicated for 24 hr, and then …
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Figure 7—source data 1
Uncropped and labelled gels for Figure 7.
- https://cdn.elifesciences.org/articles/87935/elife-87935-fig7-data1-v1.pdf
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Figure 7—source data 2
Raw unedited gels for Figure 7.
- https://cdn.elifesciences.org/articles/87935/elife-87935-fig7-data2-v1.zip
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Figure 7—source data 3
Raw data for Figure 7.
- https://cdn.elifesciences.org/articles/87935/elife-87935-fig7-data3-v1.xlsx
Figure 7—figure supplement 1
Expression of candidate E3 ubiquitin ligases in wild-type (WT) and Van Gogh-like 2 (Vangl2)-deficient bone marrow-derived macrophages (BMDMs) after lipopolysaccharide (LPS) treatment.
(A) Heatmap view of mRNA variations of E3 ubiquitin ligases in WT and Vangl2-deficient BMDMs treated with or without LPS. mRNA levels of Pdlim2 (B), Usp7 (C), and Trim21 (D) in the spleens from …
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Figure 7—figure supplement 1—source code 1
Raw data for Figure 7—figure supplement 1.
- https://cdn.elifesciences.org/articles/87935/elife-87935-fig7-figsupp1-code1-v1.xlsx
Author response image 1
Vangl2 degrades p65 through a selective autophagic pathway, but not by the PCP pathway.
HEK293T cells were transfected with Flag-p65 and HA-Vangl2 plasmids, and treated with DMSO, CQ (50 mM) for 6 h, SP600125 (20 mM) for 1 h or FH535 (30 mM) for 6 h. The cell lysates were analyzed by …
Author response image 2
Vangl2 deficiency promotes NF-kB activation.
(A) The survival rates of WT, Vangl2ΔM/ΔM and Vangl2ΔM/WT mice treated with high-dosage of LPS (30 mg/kg, i.p.) (n≥4). (B) IL-6 and TNF-a secretion by WT and Vangl2-deficient BMDMs treated with LPS …
Additional files
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Supplementary file 1
Reagents and antibodies used in this study.
- https://cdn.elifesciences.org/articles/87935/elife-87935-supp1-v1.docx
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Supplementary file 2
Primers sequences for quantitative real-time reverse transcription polymerase chain reaction (RT-PCR) .
- https://cdn.elifesciences.org/articles/87935/elife-87935-supp2-v1.docx
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Supplementary file 3
Primers sequences for siRNA transfection.
- https://cdn.elifesciences.org/articles/87935/elife-87935-supp3-v1.docx
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Supplementary file 4
The sepsis patients’ information was shown in this study.
- https://cdn.elifesciences.org/articles/87935/elife-87935-supp4-v1.docx
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MDAR checklist
- https://cdn.elifesciences.org/articles/87935/elife-87935-mdarchecklist1-v1.docx
