( E ) IFN Module Score of viral - - negative ( grey ; 715 958 in SARS - CoV SARS - CoV - 2 exposed cultures , respectively ) , SARS - CoV - - Positive ( blue ; 56 ) SARS - CoV - 2 - - Positive ( magenta ; 173 ) monocytes . Statistical significance was tested using a Wilcoxon rank sum with continuity correction . P - values < 0 . 05 were considered significant ( * ) , < 0 . 01 very significant ( * * ) or ≥ 0 . 05 not significant Data information : Data shown in this figure are on of two donors .

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(E) IFN Module Score of viral-RNA-negative (grey; 715 and 958 cells in SARS-CoV and SARS-CoV-2 exposed cultures, respectively), SARS-CoV-RNA-Positive (blue; 56 cells) and SARS-CoV-2-RNA-Positive (magenta; 173 cells) monocytes. Statistical significance was tested using a Wilcoxon rank sum test with continuity correction. P-values < 0.05 were considered significant (*), < 0.01 very significant (**) or ≥ 0.05 not significant Data information: Data shown in this figure are based on the analysis of two donors.


Results of ELISAs used to determine IFN - β ( A ) secretion in PMA - differentiated THP - 1 transfected with siCtrl , siRNF34 - 1 , siRNF34 - 2 or siRNF34 - 3 oligos infected with VSV for indicated times . ELISA data are presented as means ± . Two - Student ' s t - was used for statistical analyses : * , P < 0 . 05 ; * * , P < 0 . 01 ; * * * , P < 0 . 001 .

O O B-EXP_ASSAY O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-EXP_ASSAY O B-SMALL_MOLECULE O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O O O O O O O O O O O O O O B-ORGANISM O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Results of the ELISAs used to determine IFN-β (A) secretion in PMA-differentiated THP-1 cells transfected with siCtrl, siRNF34-1, siRNF34-2 or siRNF34-3 oligos and infected with VSV for the indicated times. ELISA data are presented as means ± SEM. Two-tailed Student's t-test was used for statistical analyses: *, P < 0.05; **, P < 0.01; and ***, P < 0.001.


Results of ELISAs used to determine IL6 ( B ) secretion in PMA - differentiated THP - 1 transfected with siCtrl , siRNF34 - 1 , siRNF34 - 2 or siRNF34 - 3 oligos infected with VSV for indicated times . ELISA data are presented as means ± . Two - Student ' s t - was used for statistical analyses : * , P < 0 . 05 ; * * , P < 0 . 01 ; * * * , P < 0 . 001 .

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Results of the ELISAs used to determine IL6 (B) secretion in PMA-differentiated THP-1 cells transfected with siCtrl, siRNF34-1, siRNF34-2 or siRNF34-3 oligos and infected with VSV for the indicated times. ELISA data are presented as means ± SEM. Two-tailed Student's t-test was used for statistical analyses: *, P < 0.05; **, P < 0.01; and ***, P < 0.001.


Luciferase activity driven by IFN - β ( C ) promoter in HEK293T transfected with Flag - RNF34 or its E3 ligase - dead mutant H342A followed by VSV infection for indicated times . Luciferase assays were performed 24 hrs after transfection , luciferase activity was reported as fold induction . luciferase reporter data are presented as means ± . Two - Student ' s t - was used for statistical analyses : * , P < 0 . 05 ; * * , P < 0 . 01 ; * * * , P < 0 . 001 .

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Luciferase activity driven by the IFN-β (C) promoter in HEK293T cells transfected with Flag-RNF34 or its E3 ligase-dead mutant H342A followed by VSV infection for the indicated times. Luciferase assays were performed 24 hrs after transfection, and luciferase activity was reported as the fold induction. The luciferase reporter data are presented as means ± SEM. Two-tailed Student's t-test was used for statistical analyses: *, P < 0.05; **, P < 0.01; and ***, P < 0.001.


Luciferase activity driven by NF - κB ( D ) promoter in HEK293T transfected with Flag - RNF34 or its E3 ligase - dead mutant H342A followed by VSV infection for indicated times . Luciferase assays were performed 24 hrs after transfection , luciferase activity was reported as fold induction . luciferase reporter data are presented as means ± . Two - Student ' s t - was used for statistical analyses : * , P < 0 . 05 ; * * , P < 0 . 01 ; * * * , P < 0 . 001 .

Luciferase activity driven by the NF-κB (D) promoter in HEK293T cells transfected with Flag-RNF34 or its E3 ligase-dead mutant H342A followed by VSV infection for the indicated times. Luciferase assays were performed 24 hrs after transfection, and luciferase activity was reported as the fold induction. The luciferase reporter data are presented as means ± SEM. Two-tailed Student's t-test was used for statistical analyses: *, P < 0.05; **, P < 0.01; and ***, P < 0.001.


( E ) Plaque of VSV loads in supernatants from transfected with shCtrl , shRNF34 - 1 , shRNF34 - 2 or shRNF34 - 3 subsequently infected with VSV . - studies were performed independently at least three times with comparable results .

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(E) Plaque assay of VSV loads in supernatants from cells transfected with shCtrl, shRNF34-1, shRNF34-2 or shRNF34-3 and subsequently infected with VSV. Cell-based studies were performed independently at least three times with comparable results.


( F ) Microscopy of shCtrl - , shRNF34 - 1 - , shRNF34 - 2 - or shRNF34 - 3 infected with NDV - GFP ( MOI = 1 . 0 ) for indicated times . Gray represents ; green represents NDV . Scale bar , 300 μm .

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(F) Microscopy images of shCtrl-, shRNF34-1-, shRNF34-2- or shRNF34-3 cells infected with NDV-GFP (MOI = 1.0) for the indicated times. Gray represents cells; green represents NDV. Scale bar, 300 μm.


( G ) Immunoblot showing of phosphorylated ( p ) total IRF3 , TBK1 , VSV - G in PMA - differentiated THP - 1 transfected with siCtrl or siRNF34 - 1 oligos infected with VSV for indicated times .

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(G) Immunoblot showing the levels of the phosphorylated (p) and total IRF3, TBK1, and VSV-G proteins in PMA-differentiated THP-1 cells transfected with siCtrl or siRNF34-1 oligos and infected with VSV for the indicated times.


RT - PCR of expression of IFN - β ( H ) mRNAs in PBMCs transfected with siCtrl or siRNF34 - 1 oligos infected with VSV for indicated times . results were normalized to values obtained prior to VSV infection .

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RT-PCR analysis of the expression of the IFN-β (H) mRNAs in PBMCs transfected with siCtrl or siRNF34-1 oligos and infected with VSV for the indicated times. The results were normalized to the values obtained prior to VSV infection.


( I ) Results of ELISA used to determine IFN - β secretion in PBMCs transfected with siCtrl or siRNF34 - 1 oligos infected with VSV for indicated times . ELISA data are presented as means ± . Two - Student ' s t - was used for statistical analyses : * , P < 0 . 05 ; * * , P < 0 . 01 ; * * * , P < 0 . 001 .

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(I) Results of the ELISA used to determine IFN-β secretion in PBMCs transfected with siCtrl or siRNF34-1 oligos and infected with VSV for the indicated times. ELISA data are presented as means ± SEM. Two-tailed Student's t-test was used for statistical analyses: *, P < 0.05; **, P < 0.01; and ***, P < 0.001.


RT - PCR of expression of IL8 , ISG54 ISG56 ( J ) mRNAs in PBMCs transfected with siCtrl or siRNF34 - 1 oligos infected with VSV for indicated times . results were normalized to values obtained prior to VSV infection .

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RT-PCR analysis of the expression of the IL8, ISG54 and ISG56 (J) mRNAs in PBMCs transfected with siCtrl or siRNF34-1 oligos and infected with VSV for the indicated times. The results were normalized to the values obtained prior to VSV infection.


( A ) Luciferase activity driven by ISRE promoter in HEK293T transfected with Myc - RNF34 Flag - V , Flag - N - RIG - I , Flag - MAVS , Flag - STING or Flag - TBK1 . Luciferase assays were performed 24 hrs after transfection . luciferase reporter data are presented as means ± . Two - Student ' s t - were used for statistical analyses : * , P < 0 . 05 ; * * , P < 0 . 01 ; * * * , P < 0 . 001 .

O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O B-CELL_LINE O O B-GENEPROD O B-GENEPROD I-GENEPROD O O O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(A) Luciferase activity driven by the ISRE promoter in HEK293T cells transfected with Myc-RNF34 and Flag-V, Flag-N-RIG-I, Flag-MAVS, Flag-STING or Flag-TBK1. Luciferase assays were performed 24 hrs after transfection. The luciferase reporter data are presented as means ± SEM. Two-tailed Student's t-tests were used for statistical analyses: *, P < 0.05; **, P < 0.01; and ***, P < 0.001.


( B ) Y2H in AH109 yeast co - transformed with indicated plasmids . A positive RNF34 - MAVS interaction resulted in colony formation on synthetic medium lacking tryptophan , leucine , adenine , histidine containing X - gal . pGBKT7 - TP53 + pGADT7 - T pGBKT7 - lam + pGADT7 - T were used as positive negative controls , respectively . AH109 co - transfected with pGBKT7 - RNF34 + pACT - 2 was used to exclude self - activation of RNF34 .

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(B) Y2H analysis in the AH109 yeast strain co-transformed with the indicated plasmids. A positive RNF34-MAVS interaction resulted in colony formation on synthetic medium lacking tryptophan, leucine, adenine, and histidine containing X-gal. pGBKT7-TP53+pGADT7-T and pGBKT7-lam+pGADT7-T were used as positive and negative controls, respectively. AH109 co-transfected with pGBKT7-RNF34+pACT-2 was used to exclude the self-activation of RNF34.


( C ) Immunoprecipitation of HEK293T transfected with Myc - RNF34 Flag - MAVS or Flag - V . Anti - Flag or IgG agarose immunoprecipitates were analyzed using immunoblotting with an anti - Myc or anti - Flag antibody .

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(C) Immunoprecipitation analysis of HEK293T cells transfected with Myc-RNF34 and Flag-MAVS or Flag-V. Anti-Flag or IgG agarose immunoprecipitates were analyzed using immunoblotting with an anti-Myc or anti-Flag antibody.


( D ) GST - tagged RNF34 was subjected to a pull - down with HEK293T lysates . Immunoblot with an anti - MAVS antibody are shown in top panel . Loading of GST assessed using Coomassie blue is shown in bottom panel . GST was used as a negative control .

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(D) GST-tagged RNF34 was subjected to a pull-down assay with HEK293T cell lysates. Immunoblot with an anti-MAVS antibody are shown in the top panel. Loading of the GST proteins assessed using Coomassie blue staining is shown in the bottom panel. GST was used as a negative control.


( E ) Anti - Flag or IgG immunoprecipitates prepared from transfected with Flag - MAVS or Flag - vector - expressing plasmids were subjected to SDS - PAGE blotted onto a nitrocellulose . nitrocellulose was incubated with soluble GST - RNF34 ( upper panel ) or GST ( middle panel ) for 2 hrs then analyzed with anti - Flag antibody .

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(E) Anti-Flag or IgG immunoprecipitates prepared from cells transfected with Flag-MAVS or Flag-vector-expressing plasmids were subjected to SDS-PAGE and blotted onto a nitrocellulose membrane. The nitrocellulose membrane was incubated with soluble GST-RNF34 (upper panel) or GST (middle panel) for 2 hrs and then analyzed with anti-Flag antibody.


( F ) Representative confocal of immunofluorescence for Flag - RNF34 colocalization with endogenous MAVS in THP - 1 infected with VSV for 12 hrs . Scale bar , 10 μm . - studies were performed independently at least three times with comparable results .

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(F) Representative confocal images of immunofluorescence staining for Flag-RNF34 colocalization with endogenous MAVS in THP-1 cells infected with VSV for 12 hrs. Scale bar, 10 μm. Cell-based studies were performed independently at least three times with comparable results.


( G ) Immunoblot showing of RNF34 in THP - 1 infected with VSV ( MOI = 1 . 0 ) for indicated times . α - Tubulin was used as a loading control .

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(G) Immunoblot showing the levels of the RNF34 protein in THP-1 cells infected with VSV (MOI = 1.0) for the indicated times. α-Tubulin was used as a loading control.


( H ) In situ PLA of RNF34 - MAVS in HEK293T infected with VSV ( MOI = 1 . 0 ) for indicated times using an anti - RNF34 or anti - MAVS antibody . RNF34 - MAVS , red ; nuclei , blue . Scale bar , 5 μm . - studies were performed independently at least three times with comparable results .

O O O O O B-EXP_ASSAY O B-GENEPROD O B-GENEPROD O B-CELL_LINE O O B-ORGANISM O O O O O O O O O O O O O O B-GENEPROD O O O B-GENEPROD O O B-GENEPROD O B-GENEPROD O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O

(H) In situ PLA assay of the RNF34-MAVS complex in HEK293T cells infected with VSV (MOI =1.0) for the indicated times using an anti-RNF34 or anti-MAVS antibody. RNF34-MAVS complex, red; nuclei, blue. Scale bar, 5 μm. Cell-based studies were performed independently at least three times with comparable results.


( I ) One hundred were counted , of PLA signals per is shown . - studies were performed independently at least three times with comparable results .

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(I) One hundred cells were counted, and the quantification of PLA signals per cell is shown. Cell-based studies were performed independently at least three times with comparable results.


( J ) Immunoprecipitation of HEK293T transfected with Myc - RNF34 Flag - MAVS or Flag - mMAVS . Anti - Flag immunoprecipitates were analyzed using immunoblotting with anti - Myc or anti - Flag antibody .

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(J) Immunoprecipitation analysis of HEK293T cells transfected with Myc-RNF34 and Flag-MAVS or Flag-mMAVS. Anti-Flag immunoprecipitates were analyzed using immunoblotting with anti-Myc or anti-Flag antibody.


( A ) Immunoprecipitation in HEK293T expressing Flag - MAVS Myc - Ub together with HA - RNF34 or HA - H342A . Anti - Flag immunoprecipitates were analyzed using immunoblotting with an anti - Myc or anti - Flag antibody . of transfected were analyzed using immunoblotting with an anti - HA , anti - Myc or anti - Flag antibody . - studies were performed independently at least three times with comparable results .

O O O B-EXP_ASSAY O B-CELL_LINE O B-GENEPROD O B-GENEPROD I-GENEPROD O B-GENEPROD O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O B-GENEPROD B-EXP_ASSAY O O O B-EXP_ASSAY O O O O B-GENEPROD O O O B-GENEPROD O O O O O O O B-EXP_ASSAY O O O O B-GENEPROD O O O B-GENEPROD O O O B-GENEPROD O O O O O O O O O O O O O O O

(A) Immunoprecipitation analysis in HEK293T cells expressing Flag-MAVS and Myc-Ub together with HA-RNF34 or HA-H342A. Anti-Flag immunoprecipitates were analyzed using immunoblotting with an anti-Myc or anti-Flag antibody. The levels of the transfected proteins were analyzed using immunoblotting with an anti-HA, anti-Myc or anti-Flag antibody. Cell-based studies were performed independently at least three times with comparable results.


( B ) Immunoprecipitation of HEK293T expressing Flag - MAVS HA - RNF34 together with Myc - Ub ( K6 , K11 , K27 , K29 , K33 , K48 , or K63 only ) as indicated . Anti - Flag immunoprecipitates were analyzed using immunoblotting with an anti - Myc or anti - Flag antibody . of transfected were analyzed using immunoblotting with an anti - HA , anti - Myc or anti - Flag antibody . - studies were performed independently at least three times with comparable results .

O O O B-EXP_ASSAY O B-CELL_LINE O B-GENEPROD O B-GENEPROD I-GENEPROD O B-GENEPROD O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD B-EXP_ASSAY O O O B-EXP_ASSAY O O O O B-GENEPROD O O O B-GENEPROD O O O O O O O B-EXP_ASSAY O O O O B-GENEPROD O O O B-GENEPROD O O O B-GENEPROD O O O O O O O O O O O O O O O

(B) Immunoprecipitation analysis of HEK293T cells expressing Flag-MAVS and HA-RNF34 together with Myc-Ub (K6, K11, K27, K29, K33, K48, or K63 only) as indicated. Anti-Flag immunoprecipitates were analyzed using immunoblotting with an anti-Myc or anti-Flag antibody. Levels of the transfected proteins were analyzed using immunoblotting with an anti-HA, anti-Myc or anti-Flag antibody. Cell-based studies were performed independently at least three times with comparable results.


( C ) Immunoprecipitation of HEK293T expressing Flag - MAVS HA - RNF34 together with Myc - Ub ( wild - type , WT ; Lys - to - Arg mutants of Ub lysines , KO ; K27 / 29R ) . Anti - Flag immunoprecipitates were analyzed using immunoblotting with an anti - Myc or anti - Flag antibody . of transfected were analyzed using immunoblotting with an anti - HA , anti - Myc or anti - Flag antibody . - studies were performed independently at least three times with comparable results .

O O O B-EXP_ASSAY O B-CELL_LINE O B-GENEPROD O B-GENEPROD I-GENEPROD O B-GENEPROD O O B-GENEPROD O B-GENEPROD O O O O O O O B-SMALL_MOLECULE O O O B-SMALL_MOLECULE O O B-GENEPROD B-SMALL_MOLECULE O O O O O O O O O O B-GENEPROD B-EXP_ASSAY O O O B-EXP_ASSAY O O O O B-GENEPROD O O O B-GENEPROD O O O O O O O B-EXP_ASSAY O O O O B-GENEPROD O O O B-GENEPROD O O O B-GENEPROD O O O O O O O O O O O O O O O

(C) Immunoprecipitation analysis of HEK293T cells expressing Flag-MAVS and HA-RNF34 together with Myc-Ub (wild-type, WT; Lys-to-Arg mutants of all Ub lysines, KO; K27/29R). Anti-Flag immunoprecipitates were analyzed using immunoblotting with an anti-Myc or anti-Flag antibody. Levels of the transfected proteins were analyzed using immunoblotting with an anti-HA, anti-Myc or anti-Flag antibody. Cell-based studies were performed independently at least three times with comparable results.


( D ) Immunoprecipitation of K27 ubiquitination of MAVS in HEK293T infected with VSV for indicated times . Anti - MAVS immunoprecipitates were analyzed using immunoblotting with an anti - MAVS or anti - Ub - K27 antibody . WCL was immunoblotted with an anti - MAVS or anti - Ub - K27 antibody . - studies were performed independently at least three times with comparable results .

O O O B-EXP_ASSAY O O B-EXP_ASSAY O B-GENEPROD O B-CELL_LINE O O B-ORGANISM O O O O O O B-GENEPROD B-EXP_ASSAY O O O B-EXP_ASSAY O O O O B-GENEPROD O O O B-SMALL_MOLECULE O O O O O O B-EXP_ASSAY O O O O B-GENEPROD O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O

(D) Immunoprecipitation analysis of K27 ubiquitination of MAVS in HEK293T cells infected with VSV for the indicated times. Anti-MAVS immunoprecipitates were analyzed using immunoblotting with an anti-MAVS or anti-Ub-K27 antibody. The WCL was immunoblotted with an anti-MAVS or anti-Ub-K27 antibody. Cell-based studies were performed independently at least three times with comparable results.


( E ) Immunoprecipitation of siCtrl - or siRNF34 - 1 - transfected infected with VSV for indicated times . Anti - MAVS immunoprecipitates were analyzed using immunoblotting with an anti - MAVS or anti - Ub - K27 antibody . WCL was immunoblotted with an anti - MAVS or anti - Ub - K27 antibody . - studies were performed independently at least three times with comparable results .

O O O B-EXP_ASSAY O O O O O O O O O O O B-ORGANISM O O O O O O B-GENEPROD B-EXP_ASSAY O O O B-EXP_ASSAY O O O O B-GENEPROD O O O B-GENEPROD O O O O O O B-EXP_ASSAY O O O O B-GENEPROD O O O B-GENEPROD O O O O O O O O O O O O O O O O O

(E) Immunoprecipitation analysis of siCtrl- or siRNF34-1-transfected cells infected with VSV for the indicated times. Anti-MAVS immunoprecipitates were analyzed using immunoblotting with an anti-MAVS or anti-Ub-K27 antibody. The WCL was immunoblotted with an anti-MAVS or anti-Ub-K27 antibody. Cell-based studies were performed independently at least three times with comparable results.


Immunoprecipitation of HEK293T expressing Flag - MAVS or Lys to Arg mutants together with Myc - Ub HA - RNF34 . Anti - Flag immunoprecipitates were analyzed using immunoblotting with an anti - Myc or anti - Flag antibody . of transfected were analyzed using immunoblotting with an anti - HA , anti - Myc or anti - Flag antibody . - studies were performed independently at least three times with comparable results .

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Immunoprecipitation analysis of HEK293T cells expressing Flag-MAVS or Lys to Arg mutants together with Myc-Ub and HA-RNF34. Anti-Flag immunoprecipitates were analyzed using immunoblotting with an anti-Myc or anti-Flag antibody. Levels of the transfected proteins were analyzed using immunoblotting with an anti-HA, anti-Myc or anti-Flag antibody. Cell-based studies were performed independently at least three times with comparable results.


Immunoprecipitation of HEK293T expressing Flag - MAVS or 4KR mutant together with Myc - Ub HA - RNF34 . Anti - Flag immunoprecipitates were analyzed using immunoblotting with an anti - Myc or anti - Ub - K27 antibody . of transfected were analyzed by performing immunoblotting with an anti - Flag antibody . - studies were performed independently at least three times with comparable results .

B-EXP_ASSAY O B-CELL_LINE O B-GENEPROD O B-GENEPROD O O O O O B-GENEPROD O B-GENEPROD I-GENEPROD O B-GENEPROD O O O B-GENEPROD B-EXP_ASSAY O O O B-EXP_ASSAY O O O O B-GENEPROD O O O B-GENEPROD O O O O O O O O O O B-EXP_ASSAY O O O O B-GENEPROD O O O O O O O O O O O O O O O

Immunoprecipitation analysis of HEK293T cells expressing Flag-MAVS or 4KR mutant together with Myc-Ub and HA-RNF34. Anti-Flag immunoprecipitates were analyzed using immunoblotting with an anti-Myc or anti-Ub-K27 antibody. Levels of the transfected proteins were analyzed by performing immunoblotting with an anti-Flag antibody. Cell-based studies were performed independently at least three times with comparable results.


( C ) Immunoprecipitation of HEK293T expressing Flag - MAVS or K311R mutant together with HA - RNF34 / or Myc - TRIM31 following a 3MA ( 0 . 2 mM ) treatment . Anti - Flag immunoprecipitates were analyzed using immunoblotting with an anti - Flag , anti - Ub - K63 or anti - Ub - K27 antibody . of transfected were analyzed using immunoblotting with an anti - HA or anti - Flag antibody . ell - studies were performed independently at least three times with comparable results .

O O O B-EXP_ASSAY O B-CELL_LINE O B-GENEPROD O B-GENEPROD O O O O O B-GENEPROD O B-GENEPROD O O B-GENEPROD O B-GENEPROD O O B-SMALL_MOLECULE O O O O O O O O O O B-GENEPROD B-EXP_ASSAY O O O B-EXP_ASSAY O O O O B-GENEPROD O O O B-GENEPROD O O O O O B-GENEPROD O O O O O O O O O B-EXP_ASSAY O O O O B-GENEPROD O O O B-GENEPROD O O O O O O O O O O O O O O O O

(C) Immunoprecipitation analysis of HEK293T cells expressing Flag-MAVS or K311R mutant together with HA-RNF34 and/or Myc-TRIM31 following a 3MA (0.2 mM) treatment. Anti-Flag immunoprecipitates were analyzed using immunoblotting with an anti-Flag, anti-Ub-K63 or anti-Ub-K27 antibody. Levels of the transfected proteins were analyzed using immunoblotting with an anti-HA or anti-Flag antibody. ell-based studies were performed independently at least three times with comparable results.


( D ) Co - immunoprecipitation of ubiquitination of endogenous MAVS in THP - 1 transfected with siCtrl , siRNF34 , or siTRIM31 oligos infected with VSV for indicated times . - studies were performed independently at least three times with comparable results .

O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY O O B-GENEPROD O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O

(D) Co-immunoprecipitation analysis of the ubiquitination of endogenous MAVS in THP-1 cells transfected with siCtrl, siRNF34, or siTRIM31 oligos and infected with VSV for the indicated times. Cell-based studies were performed independently at least three times with comparable results.


( E ) Co - immunoprecipitation of endogenous interaction of MAVS - TRIM31 MAVS - RNF34 in PBMCs infected with VSV for indicated times . - studies were performed independently at least three times with comparable results .

O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-EXP_ASSAY O B-GENEPROD O B-GENEPROD I-GENEPROD O B-GENEPROD O B-CELL_TYPE O O B-ORGANISM O O O O O O O O O O O O O O O O O

(E) Co-immunoprecipitation analysis of the endogenous interaction of MAVS-TRIM31 and MAVS-RNF34 in PBMCs infected with VSV for the indicated times. Cell-based studies were performed independently at least three times with comparable results.


( A ) Immunoblot showing degradation of MAVS in HEK293T subjected to VSV infection ( MOI = 1 . 0 ) for indicated times . α - Tubulin was used as a loading control . - studies were performed independently at least three times with comparable results .

O O O B-EXP_ASSAY O B-EXP_ASSAY O B-GENEPROD O B-CELL_LINE O O B-ORGANISM O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O

(A) Immunoblot showing the degradation of MAVS in HEK293T cells subjected to VSV infection (MOI = 1.0) for the indicated times. α-Tubulin was used as a loading control. Cell-based studies were performed independently at least three times with comparable results.


( B ) Immunoblot showing degradation of MAVS in HEK293T infected with VSV ( MOI = 1 . 0 ) for indicated times in presence or absence of NH4Cl ( 3 mM ) . α - Tubulin was used as a loading control . - studies were performed independently at least three times with comparable results .

O O O B-EXP_ASSAY O B-EXP_ASSAY O B-GENEPROD O B-CELL_LINE O O B-ORGANISM O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O

(B) Immunoblot showing the degradation of MAVS in HEK293T cells infected with VSV (MOI = 1.0) for the indicated times in the presence or absence of NH4Cl (3 mM). α-Tubulin was used as a loading control. Cell-based studies were performed independently at least three times with comparable results.


( C ) Immunoblot showing of MAVS in HEK293T expressing Flag - MAVS or Flag - mMAVS together with Myc - RNF34 . α - Tubulin was used as a loading control . - studies were performed independently at least three times with comparable results .

O O O B-EXP_ASSAY O O B-GENEPROD O B-CELL_LINE O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O B-GENEPROD O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O

(C) Immunoblot showing the levels of the MAVS protein in HEK293T cells expressing Flag-MAVS or Flag-mMAVS together with Myc-RNF34. α-Tubulin was used as a loading control. Cell-based studies were performed independently at least three times with comparable results.


( D ) Immunoblot showing of MAVS in HEK293T expressing Flag - MAVS or its 4KR mutant together with increasing amounts of Myc - RNF34 . α - Tubulin was used as a loading control . - studies were performed independently at least three times with comparable results .

O O O B-EXP_ASSAY O O B-GENEPROD O B-CELL_LINE O B-GENEPROD O B-GENEPROD O O O O O O O B-EXP_ASSAY O B-GENEPROD O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O

(D) Immunoblot showing the levels of the MAVS protein in HEK293T cells expressing Flag-MAVS or its 4KR mutant together with increasing amounts of Myc-RNF34. α-Tubulin was used as a loading control. Cell-based studies were performed independently at least three times with comparable results.


( E ) Immunoblot showing of MAVS in HEK293T expressing Flag - MAVS with increasing amounts of HA - RNF34 or its H342A mutant . α - Tubulin was used as a loading control . - studies were performed independently at least three times with comparable results .

O O O B-EXP_ASSAY O O B-GENEPROD O B-CELL_LINE O B-GENEPROD O B-GENEPROD O O B-EXP_ASSAY O B-GENEPROD O B-GENEPROD O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O

(E) Immunoblot showing the levels of the MAVS protein in HEK293T cells expressing Flag-MAVS with increasing amounts of HA-RNF34 or its H342A mutant. α-Tubulin was used as a loading control. Cell-based studies were performed independently at least three times with comparable results.


( F ) SDD - of MAVS in HEK293T expressing Flag - MAVS together with WT Myc - RNF34 or its ligase - dead mutant cultured in presence or absence of MG132 ( 10 μM ) , 3 - MA ( 1 mM ) or NH4Cl ( 3 mM ) . SDS - PAGE immunoblotting was used as a loading control . - studies were performed independently at least three times with comparable results .

O O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O B-CELL_LINE O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O B-SUBCELLULAR O O O O O O O O O B-SMALL_MOLECULE O O O O O O O B-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O

(F) SDD-AGE analysis of MAVS aggregates in HEK293T cells expressing Flag-MAVS together with WT Myc-RNF34 or its ligase-dead mutant and cultured in the presence or absence of MG132 (10 μM), 3-MA (1 mM) or NH4Cl (3 mM). SDS-PAGE immunoblotting was used as a loading control. Cell-based studies were performed independently at least three times with comparable results.


( G ) SDD - of MAVS in siCtrl - or siRNF34 - 1 - transfected using an anti - MAVS antibody . SDS - PAGE immunoblotting was used as a loading control . - studies were performed independently at least three times with comparable results .

O O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O O O O O O O O O O O O B-GENEPROD O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O

(G) SDD-AGE analysis of MAVS aggregates in siCtrl- or siRNF34-1-transfected cells using an anti-MAVS antibody. SDS-PAGE immunoblotting was used as a loading control. Cell-based studies were performed independently at least three times with comparable results.


( H ) Immunoblot showing of MAVS in siCtrl - or siRNF34 - 1 - transfected PBMCs . α - Tubulin was used as a loading control . - studies were performed independently at least three times with comparable results .

O O O B-EXP_ASSAY O O B-GENEPROD O O O O O O O O O B-CELL_TYPE O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O

(H) Immunoblot showing the levels of the MAVS protein in siCtrl- or siRNF34-1-transfected PBMCs. α-Tubulin was used as a loading control. Cell-based studies were performed independently at least three times with comparable results.


( I ) SDD - of MAVS in THP - 1 expressing Flag - MAVS or K311R mutant together with Myc - RNF34 or Myc - TRIM31 . SDS - PAGE immunoblotting was used as a loading control . - studies were performed independently at least three times with comparable results .

O O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O B-CELL_LINE I-CELL_LINE I-CELL_LINE O B-GENEPROD O B-GENEPROD O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O

(I) SDD-AGE analysis of MAVS aggregates in THP-1 cells expressing Flag-MAVS or the K311R mutant together with Myc-RNF34 or Myc-TRIM31. SDS-PAGE immunoblotting was used as a loading control. Cell-based studies were performed independently at least three times with comparable results.


( A ) Immunoprecipitation of HEK293T transfected with Flag - SQSTM1 or its I431A mutant together with HA - MAVS . Anti - Flag or IgG agarose immunoprecipitates were analyzed using immunoblotting with an anti - HA or anti - Flag antibody . - studies were performed independently at least three times with comparable results .

O O O B-EXP_ASSAY O B-CELL_LINE O O B-GENEPROD O B-GENEPROD O O O O O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O O B-SMALL_MOLECULE B-EXP_ASSAY O O O B-EXP_ASSAY O O O O B-GENEPROD O O O B-GENEPROD O O O O O O O O O O O O O O O

(A) Immunoprecipitation analysis of HEK293T cells transfected with Flag-SQSTM1 or its I431A mutant together with HA-MAVS. Anti-Flag or IgG agarose immunoprecipitates were analyzed using immunoblotting with an anti-HA or anti-Flag antibody. Cell-based studies were performed independently at least three times with comparable results.


( B ) Luciferase activity driven by IFN - β promoter in HEK293T transfected with HA - MAVS or its 4KR mutant together with Flag - SQSTM1 or its I431A mutant . Luciferase assays were performed 24 hrs after transfection . luciferase reporter data are presented as means ± . Two - Student ' s t - were used for statistical analyses : * , P < 0 . 05 ; * * , P < 0 . 01 ; * * * , P < 0 . 001 .

O O O B-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD I-GENEPROD I-GENEPROD O O B-CELL_LINE O O B-GENEPROD O B-GENEPROD O O O O O O B-GENEPROD O B-GENEPROD O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(B) Luciferase activity driven by the IFN-β promoter in HEK293T cells transfected with HA-MAVS or its 4KR mutant together with Flag-SQSTM1 or its I431A mutant. Luciferase assays were performed 24 hrs after transfection. The luciferase reporter data are presented as means ± SEM. Two-tailed Student's t-tests were used for statistical analyses: *, P < 0.05; **, P < 0.01; and ***, P < 0.001.


( C ) Immunoblot showing of MAVS in HEK293T expressing HA - MAVS or its 4KR mutant together with Flag - SQSTM1 . α - Tubulin was used as a loading control . - studies were performed independently at least three times with comparable results .

O O O B-EXP_ASSAY O O B-GENEPROD O B-CELL_LINE O B-GENEPROD O B-GENEPROD O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O

(C) Immunoblot showing the levels of the MAVS protein in HEK293T cells expressing HA-MAVS or its 4KR mutant together with Flag-SQSTM1. α-Tubulin was used as a loading control. Cell-based studies were performed independently at least three times with comparable results.


( D ) Luciferase activity driven by IFN - β promoter in HEK293T transfected with HA - MAVS or its 4KR mutant together with Flag - NDP52 or its D439R / C443K mutant . Luciferase assays were performed 24 hrs after transfection . luciferase reporter data are presented as means ± . Two - Student ' s t - were used for statistical analyses : * , P < 0 . 05 ; * * , P < 0 . 01 ; * * * , P < 0 . 001 .

O O O B-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD I-GENEPROD I-GENEPROD O O B-CELL_LINE O O B-GENEPROD O B-GENEPROD O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(D) Luciferase activity driven by the IFN-β promoter in HEK293T cells transfected with HA-MAVS or its 4KR mutant together with Flag-NDP52 or its D439R/C443K mutant. Luciferase assays were performed 24 hrs after transfection. The luciferase reporter data are presented as means ± SEM. Two-tailed Student's t-tests were used for statistical analyses: *, P < 0.05; **, P < 0.01; and ***, P < 0.001.


( E ) Immunoprecipitation of HEK293T transfected with Flag - MAVS or its 4KR mutant together with Myc - NDP52 . Anti - Flag immunoprecipitates were analyzed using immunoblotting with an anti - Myc or anti - Flag antibody . - studies were performed independently at least three times with comparable results .

O O O B-EXP_ASSAY O B-CELL_LINE O O B-GENEPROD O B-GENEPROD O O O O O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD B-EXP_ASSAY O O O B-EXP_ASSAY O O O O B-GENEPROD O O O B-GENEPROD O O O O O O O O O O O O O O O

(E) Immunoprecipitation analysis of HEK293T cells transfected with Flag-MAVS or its 4KR mutant together with Myc-NDP52. Anti-Flag immunoprecipitates were analyzed using immunoblotting with an anti-Myc or anti-Flag antibody. Cell-based studies were performed independently at least three times with comparable results.


( F ) Immunoprecipitation of DNP52 - RNF34 interaction in infected with VSV . Anti - DNP52 immunoprecipitates were analyzed using immunoblotting with an anti - DNP52 or anti - RNF34 antibody . WCL was immunoblotted with an anti - MAVS or anti - RNF34 antibody . - studies were performed independently at least three times with comparable results .

O O O B-EXP_ASSAY O B-GENEPROD O B-GENEPROD B-EXP_ASSAY O O O B-ORGANISM O O O B-GENEPROD B-EXP_ASSAY O O O B-EXP_ASSAY O O O O B-GENEPROD O O O B-GENEPROD O O O O B-EXP_ASSAY O O O O B-GENEPROD O O O B-GENEPROD O O O O O O O O O O O O O O O

(F) Immunoprecipitation analysis of the DNP52-RNF34 interaction in cells infected with VSV. Anti-DNP52 immunoprecipitates were analyzed using immunoblotting with an anti-DNP52 or anti-RNF34 antibody. The WCL was immunoblotted with an anti-MAVS or anti- RNF34 antibody. Cell-based studies were performed independently at least three times with comparable results.


( G ) Immunoprecipitation of interaction between Flag - NDP52 HA - RNF34 in WT or MAVS - / - . Anti - Flag immunoprecipitates were analyzed using immunoblotting with an anti - HA antibody . - studies were performed independently at least three times with comparable results .

O O O B-EXP_ASSAY O B-EXP_ASSAY O B-GENEPROD O B-GENEPROD I-GENEPROD O B-GENEPROD O O O B-GENEPROD O O O O O O B-GENEPROD B-EXP_ASSAY O O O B-EXP_ASSAY O O O O B-GENEPROD O O O O O O O O O O O O O O O

(G) Immunoprecipitation analysis of the interaction between Flag-NDP52 and HA-RNF34 in WT or MAVS-/- cells. Anti-Flag immunoprecipitates were analyzed using immunoblotting with an anti-HA antibody. Cell-based studies were performed independently at least three times with comparable results.


( H ) Immunoprecipitation of HEK293T transfected with Flag - NDP52 or its D439R / C443K mutant together with HA - MAVS followed by VSV infection . Anti - Flag immunoprecipitates were analyzed using immunoblotting with an anti - HA or anti - Flag antibody . - studies were performed independently at least three times with comparable results .

O O O B-EXP_ASSAY O B-CELL_LINE O O B-GENEPROD O B-GENEPROD O O O O O O O O B-GENEPROD O B-GENEPROD O O B-ORGANISM O O O O B-GENEPROD B-EXP_ASSAY O O O B-EXP_ASSAY O O O O B-GENEPROD O O O B-GENEPROD O O O O O O O O O O O O O O O

(H) Immunoprecipitation analysis of HEK293T cells transfected with Flag-NDP52 or its D439R/C443K mutant together with HA-MAVS followed by VSV infection. Anti-Flag immunoprecipitates were analyzed using immunoblotting with an anti-HA or anti-Flag antibody. Cell-based studies were performed independently at least three times with comparable results.


( I ) Immunoblot showing of MAVS in siCtrl - or siNDP52 - transfected expressing Flag - RNF34 or Flag - V . α - Tubulin was used as a loading control . - studies were performed independently at least three times with comparable results .

O O O B-EXP_ASSAY O O B-GENEPROD O O O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O

(I) Immunoblot showing the levels of the MAVS protein in siCtrl- or siNDP52-transfected cells expressing Flag-RNF34 or Flag-V. α-Tubulin was used as a loading control. Cell-based studies were performed independently at least three times with comparable results.


( J ) Immunoblot showing of MAVS in siCtrl - or siNDP52 - transfected infected with VSV ( MOI = 1 , 12 hrs ) . α - Tubulin was used as a loading control . - studies were performed independently at least three times with comparable results .

O O O B-EXP_ASSAY O O B-GENEPROD O O O O O O O O O B-ORGANISM O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O

(J) Immunoblot showing the levels of the MAVS protein in siCtrl- or siNDP52-transfected cells infected with VSV (MOI = 1, 12 hrs). α-Tubulin was used as a loading control. Cell-based studies were performed independently at least three times with comparable results.


( K ) Immunoprecipitation of MAVS - LC3 interaction in siCtrl - or siNDP52 - transfected infected with VSV . Anti - MAVS immunoprecipitates were analyzed using immunoblotting with an anti - MAVS or anti - LC3 antibody . NDP52 were analyzed by performing immunoblotting with an anti - NDP52 antibody . α - Tubulin was used as a loading control . - studies were performed independently at least three times with comparable results .

O O O B-EXP_ASSAY O B-GENEPROD O B-GENEPROD B-EXP_ASSAY O O O O O O O O O B-ORGANISM O O O B-GENEPROD B-EXP_ASSAY O O O B-EXP_ASSAY O O O O B-GENEPROD O O O B-GENEPROD O O B-GENEPROD O O O O B-EXP_ASSAY O O O O B-GENEPROD O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O

(K) Immunoprecipitation analysis of the MAVS-LC3 interaction in siCtrl- or siNDP52-transfected cells infected with VSV. Anti-MAVS immunoprecipitates were analyzed using immunoblotting with an anti-MAVS or anti-LC3 antibody. NDP52 levels were analyzed by performing immunoblotting with an anti-NDP52 antibody. α-Tubulin was used as a loading control. Cell-based studies were performed independently at least three times with comparable results.


( A ) Immunoblot showing MAVS , IRF3 - p , LC3 , TOM20 HSP60 in siCtrl - or siRNF34 - 1 infected with VSV cultured in presence of Z - VAD ( 50 μM ) for indicated times . α - Tubulin was used as a loading control . - studies were performed independently at least three times with comparable results .

O O O B-EXP_ASSAY O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O B-GENEPROD I-GENEPROD O O O O O O O O O B-ORGANISM O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O

(A) Immunoblot showing MAVS, IRF3-p, LC3, TOM20 and HSP60 levels in siCtrl- or siRNF34-1 cells infected with VSV and cultured in the presence of Z-VAD (50 μM) for the indicated times. α-Tubulin was used as a loading control. Cell-based studies were performed independently at least three times with comparable results.


( B ) Immunoblot showing MAVS , LC3 , TOM20 HSP60 in transfected with Flag - RNF34 or its H342A mutant subsequently infected with VSV for indicated times . - studies were performed independently at least three times with comparable results .

O O O B-EXP_ASSAY O B-GENEPROD O B-GENEPROD O B-GENEPROD I-GENEPROD O O O B-GENEPROD O B-GENEPROD O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O

(B) Immunoblot showing MAVS, LC3, TOM20 and HSP60 levels in cells transfected with Flag-RNF34 or its H342A mutant and subsequently infected with VSV for the indicated times. Cell-based studies were performed independently at least three times with comparable results.


Representative confocal ( C ) of immunofluorescence for NDP52 colocalization with MAVS in shCtrl - or shRNF34 - 1 infected with VSV for indicated times . Red represents MAVS green represents NDP52 . Scale bar , 10 μm . - studies were performed independently at least three times with comparable results .

O B-EXP_ASSAY O O O O B-EXP_ASSAY O B-GENEPROD B-EXP_ASSAY O B-GENEPROD O O O O O O O O O B-ORGANISM O O O O O O B-GENEPROD O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O

Representative confocal images (C) of immunofluorescence staining for NDP52 colocalization with MAVS in shCtrl- or shRNF34-1 cells infected with VSV for the indicated times. Red represents MAVS and green represents NDP52. Scale bar, 10 μm. Cell-based studies were performed independently at least three times with comparable results.


( D ) of immunofluorescence for NDP52 colocalization with MAVS in shCtrl - or shRNF34 - 1 infected with VSV for indicated times . Scale bar , 10 μm . - studies were performed independently at least three times with comparable results .

O O O O B-EXP_ASSAY O B-GENEPROD B-EXP_ASSAY O B-GENEPROD O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O

quantification (D) of immunofluorescence staining for NDP52 colocalization with MAVS in shCtrl- or shRNF34-1 cells infected with VSV for the indicated times. Scale bar, 10 μm. Cell-based studies were performed independently at least three times with comparable results.


( E ) Transmission electron microscopy of shCtrl - shRNF34 - 1 HEK293T cultured in presence or absence of VSV . Arrows indicate mitochondria engulfed in autophagosomes . Double arrows indicate swollen mitochondria with disrupted cristae . M , mitochondria N , nucleus . 40000 × magnification . - studies were performed independently at least three times with comparable results .

O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O B-CELL_LINE O O O O O O B-ORGANISM O O O B-SUBCELLULAR O O B-SUBCELLULAR O O O O O B-SUBCELLULAR O O B-SUBCELLULAR O O O B-SUBCELLULAR O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O

(E) Transmission electron microscopy images of shCtrl- and shRNF34-1 HEK293T cells cultured in the presence or absence of VSV. Arrows indicate mitochondria engulfed in autophagosomes. Double arrows indicate swollen mitochondria with disrupted cristae. M, mitochondria and N, nucleus. 40000× magnification. Cell-based studies were performed independently at least three times with comparable results.


( F ) Representative confocal of shift in emission of Keima from 458 nm to 543 nm . Scale bar , 10 μm . - studies were performed independently at least three times with comparable results .

O O O O B-EXP_ASSAY O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O

(F) Representative confocal images of the shift in the fluorescence emission of Keima from 458 nm to 543 nm. Scale bar, 10 μm. Cell-based studies were performed independently at least three times with comparable results.


Colony formation of miRNA mimic transfected - Dgcr8 - / - ESCs . There were 17 miRNAs that decreased colony forming ability , 15 miRNAs that did not affect colony formation 8 miRNAs enhanced colony formation of ESCs . Representative pictures are shown . Full data are shown in Supplementary Fig S1 .

B-EXP_ASSAY I-EXP_ASSAY O O O O O B-GENEPROD O O O B-CELL_TYPE O O O O O O O O O O O O O O O O O O O O O O O O O B-CELL_TYPE O O O O O O O O O O O O O O O

Colony formation assay of miRNA mimic transfected-Dgcr8-/- ESCs. There were 17 miRNAs that decreased the colony forming ability, 15 miRNAs that did not affect colony formation and 8 miRNAs enhanced colony formation of ESCs. Representative pictures are shown. Full data are shown in Supplementary Fig S1.


Alkaline phosphatase of ESCs after miRNA mimic transfection . There were 21 miRNAs that decreased AP activity , 11 miRNAs that mildly affected AP activity 8 miRNAs boosted AP activity of ESCs . Representative pictures are shown . Full data are shown in Supplementary Fig S2 .

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Alkaline phosphatase staining of ESCs after miRNA mimic transfection. There were 21 miRNAs that decreased the AP activity, 11 miRNAs that mildly affected the AP activity and 8 miRNAs boosted the AP activity of ESCs. Representative pictures are shown. Full data are shown in Supplementary Fig S2.


cycle distribution in representative miRNAs evoking G1 phase accumulation in miRNA mimic transfected Dgcr8 - / - ESCs . Error bars indicate s . d . ( n = 3 ) . Full data are shown in Supplementary Fig S3A , B .

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Cell cycle distribution in representative miRNAs evoking G1 phase accumulation in miRNA mimic transfected Dgcr8-/- ESCs. Error bars indicate s. d. (n=3). Full data are shown in Supplementary Fig S3A, B.


Immunofluorescence of Oct4 in miRNA mimic transfected ESCs . 20 miRNAs decreased Oct4 expression produced small grossly differentiated colonies , 12 miRNAs mildly affected Oct4 expression , 8 miRNAs improved Oct4 expression yielded compact undifferentiated colonies . Representative pictures are shown . Full data are shown in Supplementary Fig S3C .

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Immunofluorescence staining of Oct4 in miRNA mimic transfected ESCs. 20 miRNAs decreased Oct4 expression and produced small and grossly differentiated cell colonies, 12 miRNAs mildly affected Oct4 expression, and 8 miRNAs improved Oct4 expression and yielded compact and undifferentiated cell colonies. Representative pictures are shown. Full data are shown in Supplementary Fig S3C.


AP of V6 . 5 ESCs after mimic transfection ( Full data are shown in Supplementary Fig S4B ) .

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AP staining of V6.5 ESCs after mimic transfection (Full data are shown in Supplementary Fig S4B).


B , C . Northern blot of expression of miR - 23a , miR - 27a miR - 24 during EB formation ( B ) RA induced differentiation of V6 . 5 ESCs ( C ) . was representive of results of quantitative .

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B,C. Northern blot analysis of the expression of miR-23a, miR-27a and miR-24 during EB formation (B) and RA induced differentiation of V6.5 ESCs (C). The heatmap was representive of the results of quantitative analysis.


D , E . expression of miR - 23a ~ 27a ~ 24 - 2 transcript during EB formation ( D ) RA induced differentiation ( E ) . Error bars indicate s . d . ( n = 3 ) .

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D,E. The expression of primary miR-23a~27a~24-2 transcript during EB formation (D) and RA induced differentiation (E). Error bars indicate s.d. (n=3).


F . Q - PCR of miR - 23a , miR - 27a miR - 24 expression in ESCs ( JM8A3 ) a variety of C57BL / 6J adult tissues . Data are shown as mean + s . d . ( n = 3 ) .

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F. Q-PCR analysis of miR-23a, miR-27a and miR-24 expression in ESCs (JM8A3) and a variety of C57BL/6J adult tissues. Data are shown as mean + s.d. (n=3).


G . Relative of Oct4 , Sox2 Nanog in miRNA mimic transfected Dgcr8 - / - ESCs wild - type ESCs .

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G. Relative protein levels of Oct4, Sox2 and Nanog in miRNA mimic transfected Dgcr8-/- ESCs and wild- type ESCs.


H , I . Oct4 , Nanog , Foxa2 , Gata2 Nestin immunofluorescence of mimic transfected Dgcr8 - / - ( H ) wild - type ESCs ( I ) .

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H,I. Oct4, Nanog, Foxa2, Gata2 and Nestin immunofluorescence staining of mimic transfected Dgcr8-/- (H) and wild- type ESCs(I).


Western blot of expression of target genes in miR - 27a or miR - 24 over - expressed V6 . 5 ESCs . Candidate target genes whose expression was decreased were framed .

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Western blot analysis of the expression of target genes in miR-27a or miR-24 over-expressed V6.5 ESCs. Candidate target genes whose expression was decreased were framed.


relative luciferase activity of reporter constructs co - transfected with scramble or miR - 27a miR - 24 mimics . Error bars indicate s . d . ( n = 3 ) ; * indicates P - value < 0 . 05 . Candidate target genes whose luciferase activity was decreased were framed . PC group was positive reporter control which harbored reverse complementary of miR - 27a or miR - 24 .

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The relative luciferase activity of the reporter constructs co-transfected with scramble or miR-27a and miR-24 mimics. Error bars indicate s.d. (n=3); * indicates P-value < 0.05. Candidate target genes whose luciferase activity was decreased were framed. PC group was positive reporter control which harbored reverse complementary sequence of miR-27a or miR-24.


relative luciferase activity of wild - type 3 ′ UTR or mutated 3 ′ UTR reporter co - transfected with scramble or miR - 27a miR - 24 mimics . Data are shown as mean + s . d . ( n = 3 ) ; * indicates P - value < 0 . 05 .

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The relative luciferase activity of the wild-type 3′ UTR or mutated 3′ UTR reporter co-transfected with scramble or miR-27a and miR-24 mimics. Data are shown as mean + s.d. (n=3); * indicates P-value < 0.05.


F , G RT - PCR qRT - PCR of in vivo binding of miRNAs with their targets . Error bars indicate s . d . ( n = 3 ) ; * indicates P - value < 0 . 05 ; * * indicates P - value < 0 . 01 ; * * * indicates P - value < 0 . 001 .

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F,G RT-PCR and qRT-PCR analysis of in vivo binding of the miRNAs with their targets. Error bars indicate s.d. (n=3); * indicates P-value < 0.05; ** indicates P-value < 0.01; *** indicates P-value < 0.001.


H . Western blot of c - Myc expression in siRNA treated V6 . 5 ESCs .

H. Western blot analysis of c-Myc expression in siRNA treated V6.5 ESCs.


I . Q - PCR of - 23a ~ 27a ~ 24 - 2 , miR - 23a , miR - 27a miR - 24 expression in c - Myc knocked down ESCs . Error bars indicate s . d . ( n = 3 ) ; * indicates P - value < 0 . 05 ; * * indicates P - value < 0 . 01 ; * * * indicates P - value < 0 . 001 .

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I. Q-PCR analysis of primary-23a~27a~24-2, miR-23a, miR-27a and miR-24 expression in c-Myc knocked down ESCs. Error bars indicate s.d. (n=3); * indicates P-value < 0.05; ** indicates P-value < 0.01; *** indicates P-value < 0.001.


K . ChIP - PCR ChIP - q - PCR of c - Myc binding to putative sites in promoter . Error bars indicate s . d . ( n = 3 ) ; * * * indicates P - value < 0 . 001 .

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K. ChIP-PCR and ChIP-q-PCR analysis of c-Myc binding to the putative sites in the promoter. Error bars indicate s.d. (n=3); *** indicates P-value < 0.001.


L . Relative luciferase activity of wild - type - 15 binding site mutated promoter in NIH3T3 co - transfected with c - Myc or blank vector ( NC ) . Error bars indicate s . d . ( n = 3 ) ; * indicates P - value < 0 . 05 .

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L. Relative luciferase activity of the wild-type and -15 binding site mutated promoter in NIH3T3 cells co-transfected with c-Myc or the blank vector (NC). Error bars indicate s.d. (n=3); * indicates P-value < 0.05.


M . Relative luciferase activity of wild - type - 15 binding site mutated promoter in V6 . 5 ESCs . Error bars indicate s . d . ( n = 3 ) ; * indicates P - value < 0 . 05 .

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M. Relative luciferase activity of wild-type and -15 binding site mutated promoter in V6.5 ESCs. Error bars indicate s.d. (n=3); * indicates P-value < 0.05.


Validation of effect of miRNA inhibition in MEFs by q - PCR . Error bars indicate s . d . ( n = 3 ) .

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Validation of the effect of miRNA inhibition in MEFs by q-PCR. Error bars indicate s.d. (n=3).


AP of iPSC colonies in reprogramming with OSK combined with scramble control , inhibitors of miR - 23a , miR - 27a , miR - 24 let - 7c respectively .

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AP staining of the iPSC colonies in reprogramming with OSK combined with scramble control, inhibitors of miR-23a, miR-27a, miR-24 and let-7c respectively.


Fold increase in Oct4 - GFP - positive colonies in above . Data are shown as mean + s . d . ( n = 5 ) ; * * indicates P - value < 0 . 01 ; * * * indicates P - value < 0 . 001 .

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Fold increase in Oct4-GFP-positive cell colonies in the above cells. Data are shown as mean + s.d. (n=5); ** indicates P-value < 0.01; *** indicates P-value < 0.001.


of typical Oct4 - GFP - positive iPSC colonies that were reprogrammed by OSK combined with miRNA inhibitors .

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Morphology of typical Oct4-GFP-positive iPSC colonies that were reprogrammed by OSK combined with miRNA inhibitors.


Detection of endogenous exogenous Oct4 , Sox2 , Klf4 Nanog expression in iPSCs , ESCs MEFs by RT - PCR .

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Detection of endogenous and exogenous Oct4, Sox2, Klf4 and Nanog expression in iPSCs, ESCs and MEFs by RT-PCR.


Immunofluorescence of Oct4 , Nanog SSEA1 of iPSC colonies .

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Immunofluorescence staining of Oct4, Nanog and SSEA1 of the iPSC colonies.


Q - PCR of expression of self - renewal ( Oct4 , Sox2 , Nanog ) differentiation ( Cdx2 , Foxa2 , Brachyury , Hand1 , Nestin , FGF5 ) relative markers in EBs derived from iPSCs . Error bars indicate s . d . ( n = 3 ) .

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Q-PCR analysis of the expression of self-renewal (Oct4, Sox2, Nanog) and differentiation (Cdx2, Foxa2, Brachyury, Hand1, Nestin, FGF5) relative markers in EBs derived from the iPSCs. Error bars indicate s.d. (n=3).


Hematoxylin eosin of teratoma derived from iPSC colonies . Neuronal rosette ( Ectoderm ) , Cartilage ( Mesoderm ) respiratory epithelium ( Endoderm ) are marked with black arrows .

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Hematoxylin and eosin staining of teratoma derived from the iPSC colonies. Neuronal rosette (Ectoderm), Cartilage (Mesoderm) and respiratory epithelium (Endoderm) are marked with black arrows.


Genomic PCR of specific fragments spanning miR - 23 ~ 27 ~ 24 clusters . WT indicate wild - type ESCs ; DKO indicate miR - 23a ~ 27a ~ 24 - 2 - / - , miR - 23b ~ 27b ~ 24 - 1 - / - ESCs ; KO indicate miR - 23a ~ 27a ~ 24 - 2 - / - ESCs .

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Genomic PCR of the specific fragments spanning miR-23~27~24 clusters. WT indicate wild-type ESCs; DKO indicate miR-23a~27a~24-2-/-, miR-23b~27b~24-1-/- ESCs; KO indicate miR-23a~27a~24-2-/- ESCs.


Real - time PCR of mature miR - 23a / b , miR - 27a / b miR - 24 expression in wild - type , DKO KO ESCs . Data are shown as mean + s . d . ( n = 3 ) .

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Real-time PCR analysis of mature miR-23a/b, miR-27a/b and miR-24 expression in wild-type, DKO and KO ESCs. Data are shown as mean + s.d. (n=3).


AP of wild - type miR - 23 ~ 27 ~ 24 cluster knockout ESCs .

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AP staining of wild-type and miR-23~27~24 cluster knockout ESCs.


Real - time PCR of Oct4 , Sox2 Nanog expression in wild - type miR - 23 ~ 27 ~ 24 cluster knockout ESCs . Data are shown as mean + s . d . ( n = 3 ) .

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Real-time PCR analysis of Oct4, Sox2 and Nanog expression in wild-type and miR-23~27~24 cluster knockout ESCs. Data are shown as mean + s.d. (n=3).


Oct4 , Sox2 Nanog immunofluorescence of wild - type knockout ESCs .

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Oct4, Sox2 and Nanog immunofluorescence staining of wild-type and the knockout ESCs.


Real - time PCR of Oct4 several lineage differentiation markers expression during EB formation of wild - type knockout ESCs . Data are shown as mean ± s . d . ( n = 3 ) .

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Real-time PCR analysis of Oct4 and several lineage differentiation markers expression during EB formation of wild-type and knockout ESCs. Data are shown as mean ± s.d. (n=3).


Beating EB incidence during cardiac differentiation of wild - type knockout ESCs . Data are shown as mean + s . d . ( n = 3 ) . * indicates P - value < 0 . 05 ; * * indicates P - value < 0 . 01 ; * * * indicates P - value < 0 . 001 .

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Beating EB incidence during cardiac differentiation of wild-type and knockout ESCs. Data are shown as mean + s.d. (n=3). * indicates P-value < 0.05; ** indicates P-value < 0.01; *** indicates P-value < 0.001.


Real - time PCR of several cardiac markers expression during cardiac differentiation of wild - type knockout ESCs . Data are shown as mean ± s . d . ( n = 3 ) .

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Real-time PCR analysis of several cardiac markers expression during cardiac differentiation of wild-type and knockout ESCs. Data are shown as mean ± s.d. (n=3).


Actinin , ANP Troponin Iimmunofluorescence of differentiation cultures derived from wild - type knockout ESCs .

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Actinin, ANP and Troponin Iimmunofluorescence staining of differentiation cultures derived from wild-type and knockout ESCs.


Volumes of teratomas at different days after injection . Data are shown as mean + s . d . ( n = 4 ) . * indicates P - value < 0 . 05 ; * * indicates P - value < 0 . 01 ; * * * indicates P - value < 0 . 001 .

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Volumes of teratomas at different days after injection. Data are shown as mean + s.d. (n=4). * indicates P-value < 0.05; ** indicates P-value < 0.01; *** indicates P-value < 0.001.


of teratomas derived from wild - type knockout ESCs . Data are shown as mean ± s . d . * * indicates P - value < 0 . 01 .

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Weight of teratomas derived from wild-type and knockout ESCs. Data are shown as mean ± s.d. ** indicates P-value < 0.01.


Oct4 immunohistochemistry of teratomas derived from wild - type knockout ESCs . Representative Oct4 positive regions are shown .

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Oct4 immunohistochemistry staining of teratomas derived from wild-type and knockout ESCs. Representative Oct4 positive regions are shown.


Hematoxylin eosin of teratomas derived from wild - type knockout ESCs . Ectoderm , Mesoderm Endoderm are marked with different color arrows .

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Hematoxylin and eosin staining of teratomas derived from wild-type and knockout ESCs. Ectoderm, Mesoderm and Endoderm are marked with different color arrows.


( A ) list of co - precipitated as determined by immunoprecipitation followed by mass spectrometry ( IP - MS ) . SWR1 subunits not shared by ISWI complexes are shown in blue ; DDT domain - containing ISWI specific subunits are shown in orange ; CHR11 CHR17 are shared subunits of SWR1 ISWI complexes are shown in green .

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(A) The list of co-precipitated proteins as determined by immunoprecipitation followed by mass spectrometry (IP-MS). The SWR1 subunits not shared by the ISWI complexes are shown in blue; The DDT domain-containing ISWI specific subunits are shown in orange; CHR11 and CHR17 are shared subunits of the SWR1 and ISWI complexes and are shown in green.


( C ) Determination of interaction of MBD9 with HTA9 , HTA11 , PIE1 , CHR11 , CHR17 by co - immunoprecipitation .

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(C) Determination of the interaction of MBD9 with HTA9, HTA11, PIE1, CHR11, and CHR17 by co-immunoprecipitation.


Determination of interaction of PIE1 - Flag , MBD9 - Myc , CHR11 - HA by in vitro pull down assays . PIE1 - Flag , MBD9 - Myc , CHR11 - HA were expressed from yeast subjected to pull down assays . Anti - Flag agaroses were independently used in pull down assays .

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Determination of the interaction of PIE1-Flag, MBD9-Myc, and CHR11-HA by in vitro pull down assays. PIE1-Flag, MBD9-Myc, and CHR11-HA were expressed from yeast and subjected to the pull down assays. Anti-Flag agaroses were independently used in the pull down assays.


C ) Determination of interaction of MBD9 - Myc , CHR11 - HA by in vitro pull down assays . MBD9 - Myc , CHR11 - HA were expressed from yeast subjected to pull down assays . anti - HA ( C ) agaroses were independently used in pull down assays .

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C) Determination of the interaction of MBD9-Myc, and CHR11-HA by in vitro pull down assays. MBD9-Myc, and CHR11-HA were expressed from yeast and subjected to the pull down assays. anti-HA (C) agaroses were independently used in the pull down assays.


D ) he effect of pie1 mbd9 - 1 mutations on formation of SWR1 as determined by IP - MS . Transgenic expressing Flag - tagged MBD9 , PIE1 , CHR11 in WT mutant backgrounds were subjected to IP - MS experiment with anti - Flag agarose .

O O O O O B-GENEPROD I-GENEPROD O O O O O O B-SUBCELLULAR O O O B-EXP_ASSAY O B-EXP_ASSAY O O O B-GENEPROD O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O B-EXP_ASSAY O B-EXP_ASSAY O O O O B-GENEPROD B-SMALL_MOLECULE O

D) he effect of the pie1 and mbd9-1 mutations on the formation of the SWR1 complex as determined by IP-MS. Transgenic plants expressing Flag-tagged MBD9, PIE1, and CHR11 in the WT and mutant backgrounds were subjected to the IP-MS experiment with anti-Flag agarose.


( A ) Morphological of WT , chr11 / 17 , pie1 , swc2 , rin2 mutant . Shown are 24 - day - old . top panel shows of in soil ; bottom panel shows of on black cloth .

O O O B-EXP_ASSAY O O O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O

(A) Morphological phenotypes of the WT, chr11/17, pie1, swc2, and rin2 mutant plants. Shown are 24-day-old plants. The top panel shows the phenotypes of plants in the soil; the bottom panel shows the phenotypes of plants on the black cloth.


( B ) Shown are rosette leaves of WT , pie1 , swc2 , rin2 mutant . Rosette leaves were cut from bolting .

O O O O O O B-TISSUE O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O B-TISSUE O O O O O

(B) Shown are rosette leaves of the WT, pie1, swc2, and rin2 mutant plants. Rosette leaves were cut from bolting plants.


( C ) early - flowering phenotype of mbd9 - 1 , mbd9 - 3 , mbd9 - 4 hta9 - 1 / 11 - 1 mutants compared to WT . 24 - day - old in soil are shown .

O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O O B-GENEPROD O O O B-GENEPROD O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O

(C) The early-flowering phenotype of the mbd9-1, mbd9-3, mbd9-4 and hta9-1/11-1 mutants compared to the WT. 24-day-old plants in soil are shown.


( D ) Numbers of rosette leaves of WT mutant shortly after bolting . Rosette leaves from at least 20 were counted for each genotype . Values are mean ± SD . * * P < 0 . 01 , student ' s t .

O O O B-EXP_ASSAY O O B-TISSUE O O O O O O O O B-TISSUE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(D) Numbers of rosette leaves of the WT and mutant plants shortly after bolting. Rosette leaves from at least 20 plants were counted for each genotype. Values are mean ± SD. ** P<0.01, student's t test.


( E ) Determination of effect of arp4 - 1 , arp6 swc6 - 1 mutations on flowering time . top panel shows number of rosette leaves in WT , arp4 - 1 , arp6 , swc6 - 1 mutant ; bottom panel shows a photograph of . Rosette leaves from at least 20 were counted for each genotype . Values are mean ± SD . * * P < 0 . 01 , student ' s t .

O O O O O O O B-GENEPROD O O O B-GENEPROD I-GENEPROD O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O B-EXP_ASSAY O O B-TISSUE O O O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O B-TISSUE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(E) Determination of the effect of the arp4-1, arp6 and swc6-1 mutations on flowering time. The top panel shows the number of rosette leaves in the WT, arp4-1, arp6, and swc6-1 mutant plants; the bottom panel shows a photograph of the plants. Rosette leaves from at least 20 plants were counted for each genotype. Values are mean ± SD. ** P<0.01, student's t test.


( F ) Determination of effect of tra1a tra1b mutations on flowering time . top panel shows numbers of rosette leaves of WT , tra1a - 1 , tra1a - 2 , tra1b mutant ; bottom panel shows a photograph of . Rosette leaves from at least 20 null were counted for each genotype . Values are mean ± SD . * * P < 0 . 01 , student ' s t .

O O O O O O O B-GENEPROD I-GENEPROD O O B-EXP_ASSAY I-EXP_ASSAY O O O O B-EXP_ASSAY O O B-TISSUE O O O B-GENEPROD O O O B-GENEPROD O O O B-GENEPROD O O O O O O O O O O B-TISSUE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(F) Determination of the effect of the tra1a and tra1b mutations on flowering time. The top panel shows numbers of rosette leaves of the WT, tra1a-1, tra1a-2, and tra1b mutant plants; the bottom panel shows a photograph of the plants. Rosette leaves from at least 20 null were counted for each genotype. Values are mean ± SD. ** P<0.01, student's t test.


( B ) Heat maps showing effect of indicated mutations on expression of differentially expressed genes identified in mbd9 - 1 mutant . color bar shows log2 ( fold change ) . Up - down - regulation are shown by red blue , respectively .

O O O O O O O O O O O B-EXP_ASSAY O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O

(B) Heat maps showing the effect of indicated mutations on the expression of differentially expressed genes identified in the mbd9-1 mutant. The color bar shows log2(fold change). Up- and down-regulation are shown by red and blue, respectively.


( C ) Scatter plots showing that expression pattern in hta9 - 1 / 11 - 1 mutant is correlated with that in chr11 / 17 , mbd9 - 1 , pie1 mutants . differentially expressed genes identified in hta9 - 1 / 11 - 1 mutant were subjected to .

O O O O O O O B-EXP_ASSAY O O B-GENEPROD O O O O O O O O O O O O B-GENEPROD O O O B-GENEPROD O O O B-GENEPROD O O O O O O O B-GENEPROD O O O O O O O O O O O

(C) Scatter plots showing that the expression pattern in the hta9-1/11-1 mutant is correlated with that in the chr11/17, mbd9-1, and pie1 mutants. The differentially expressed genes identified in the hta9-1/11-1 mutant were subjected to the analysis.


( A ) effect of indicated mutations on H2A . Z deposition as determined by immunoblotting . - associated were extracted subjected to immunoblotting by anti - H2A . Z antibody . histone H4 level was detected by anti - H4 antibody indicated as a loading control .

O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-EXP_ASSAY O O O O O O O B-EXP_ASSAY O O O B-GENEPROD I-GENEPROD I-GENEPROD O O B-GENEPROD I-GENEPROD O O O O O O B-GENEPROD O O O O O O O

(A) The effect of indicated mutations on H2A.Z deposition as determined by immunoblotting. Chromatin-associated proteins were extracted and subjected to immunoblotting by the anti-H2A.Z antibody. The histone H4 level was detected by the anti-H4 antibody and indicated as a loading control.


( B ) Meta plots showing H2A . Z occupancy on H2A . Z target genes . TSS , transcription start site ; TTS , transcription termination site . RPKM , reads per kilobase per million . H2A . Z ChIP - seq results were generated from two independent biological replicates .

O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O

(B) Meta plots showing H2A.Z occupancy on H2A.Z target genes. TSS, transcription start site; TTS, transcription termination site. RPKM, reads per kilobase per million. The H2A.Z ChIP-seq results were generated from two independent biological replicates.


( D ) Heat maps showing H2A . Z enrichment in WT , pie1 , chr11 / 17 , mbd9 - 1 mutants over PIE1

O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O O O O B-GENEPROD

(D) Heat maps showing H2A.Z enrichment in the WT, pie1, chr11/17, and mbd9-1 mutants over the PIE1


( D ) Heat maps showing H2A . Z enrichment in WT , pie1 , chr11 / 17 , mbd9 - 1 mutants over

O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O O O O


( E ) Shown are H2A . Z changes in pie1 , chr11 / 17 , mbd9 - 1 mutants relative to WT . PIE1 - dependent H2A . Z target genes were subjected to sorted by H2A . Z density in WT . color bar shows log2 ( fold change ) .

O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O O O O O O O B-GENEPROD O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O

(E) Shown are H2A.Z changes in the pie1, chr11/17, and mbd9-1 mutants relative to the WT. PIE1-dependent H2A.Z target genes were subjected to the analysis and sorted by H2A.Z density in the WT. The color bar shows log2(fold change).


( F ) Meta plots showing CHR11 PIE1 occupancy on PIE1 - dependent H2A . Z target genes . TSS , transcription start site ; TTS , transcription termination site . CHR11 - Flag PIE1 - Flag ChIP - seq data are from two independent biological replicates .

O O O O O O B-GENEPROD I-GENEPROD O O B-GENEPROD O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD I-GENEPROD O B-GENEPROD B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O

(F) Meta plots showing CHR11 and PIE1 occupancy on PIE1-dependent H2A.Z target genes. TSS, transcription start site; TTS, transcription termination site. The CHR11-Flag and PIE1-Flag ChIP-seq data are from two independent biological replicates.


( G ) Heat maps showing enrichment of PIE1 - Flag CHR11 - Flag over PIE1 - dependent H2A . Z target genes . peaks were sorted by H2A . Z density in WT .

O O O O O O O O B-GENEPROD O B-GENEPROD I-GENEPROD O B-GENEPROD O B-GENEPROD O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O

(G) Heat maps showing the enrichment of PIE1-Flag and CHR11-Flag over the PIE1-dependent H2A.Z target genes. The peaks were sorted by H2A.Z density in the WT.


( H ) Box plot showing enrichment of CHR11 PIE1 over H2A . Z target genes non - H2A . Z target genes . log2 ( fold change ) values of RPKM between CHR11 - Flag or PIE1 - Flag signals WT signals are shown . Center lines box edges represent medians interquartile range ( IQR ) , respectively . Whiskers extend to minimum maximum values within 1 . 5 times of IQR . data shown are from two independent biological replicates . difference between CHR11 - Flag PIE1 - Flag ChIP - seq was determined by student ' s t . * * P < 0 . 01 .

O O O O O O O O B-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O B-EXP_ASSAY O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD I-GENEPROD O B-GENEPROD B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O

(H) Box plot showing the enrichment of CHR11 and PIE1 over H2A.Z target genes and non-H2A.Z target genes. The log2(fold change) values of RPKM between the CHR11-Flag or PIE1-Flag signals and the WT signals are shown. Center lines and box edges represent medians and the interquartile range (IQR), respectively. Whiskers extend to minimum and maximum values within 1.5 times of the IQR. The data shown are from two independent biological replicates. The difference between the CHR11-Flag and PIE1-Flag ChIP-seq levels was determined by student's t test. ** P<0.01.


( A ) Increasing amounts of CHR11 ( left panel ) or PIE1 ( right panel ) were incubated with end - positioned ( lane 1 - 4 9 - 12 ) or center - positioned ( lane 5 - 8 13 - 16 ) mono - nucleosomes in presence of ATP . sliding results were then visualized on native PAGE gels by EB .

O O O O O O B-GENEPROD O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-SUBCELLULAR O O O B-SMALL_MOLECULE O B-EXP_ASSAY O O O O O O B-EXP_ASSAY O O B-EXP_ASSAY O

(A) Increasing amounts of CHR11 (left panel) or PIE1 (right panel) were incubated with end-positioned (lane 1-4 and 9-12) or center-positioned (lane 5-8 and 13-16) mono-nucleosomes in the presence of ATP. The sliding results were then visualized on native PAGE gels by EB staining.


( B ) Distribution of nucleosomes over TSS of - coding genes in WT , chr11 / 17 , pie1 , mbd9 - 1 mutants . MNase - seq results were generated from two independent biological replicates .

O O O B-EXP_ASSAY O B-SUBCELLULAR O O O O O O O O O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD O B-EXP_ASSAY O O O O O O O O O

(B) Distribution of nucleosomes over TSS of all protein-coding genes in the WT, chr11/17, pie1, and mbd9-1 mutants. The MNase-seq results were generated from two independent biological replicates.


( E ) Representative immunoblot for key lipolytic enzymes HSL ATGL expression ( n = 5 - 6 / group ) . Data are represented as mean ± s . e . m . E , Unpaired Student ' s t - .

O O O O B-EXP_ASSAY O O O O B-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(E) Representative immunoblot for key lipolytic enzymes HSL and ATGL protein expression and quantification (n = 5-6/group). Data are represented as mean ± s.e.m. E, Unpaired Student's t-test.


( F ) Immunoblot of autophagic flux in mWAT ( upper panel ) gWAT ( lower panel ) adipose tissue stimulated ex vivo with lysosomal inhibitors 100nM Bafilomycin A1 20mM NH4Cl for 4 hours or DMSO ( Vehicle ) ( n = 3 - 4 / group ) . Data are represented as mean ± s . e . m . Unpaired Student ' s t - .

O O O B-EXP_ASSAY O B-EXP_ASSAY I-EXP_ASSAY O B-TISSUE O O O O B-TISSUE O O O O B-TISSUE I-TISSUE O O O O B-SUBCELLULAR O O B-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(F) Immunoblot analysis of autophagic flux in mWAT (upper panel) and gWAT (lower panel) adipose tissue stimulated ex vivo with lysosomal inhibitors 100nM Bafilomycin A1 and 20mM NH4Cl for 4 hours or DMSO (Vehicle) (n = 3-4/group). Data are represented as mean ± s.e.m. Unpaired Student's t-test.


( G ) Representative transmission electron microscopy from mesenteric adipose tissue 7 days post DSS - induced colitis induction . Lower panel is showing magnification of selected . arrows show autophagosomal structures .

O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-TISSUE I-TISSUE I-TISSUE O O O B-SMALL_MOLECULE O O B-DISEASE O O O O O O O O O O O O B-SUBCELLULAR O O

(G) Representative transmission electron microscopy images from mesenteric adipose tissue 7 days post DSS-induced colitis induction. Lower panel is showing magnification of selected area. White arrows show autophagosomal structures.


( J ) Representative immunoblot for LC3 - I / - II ACTIN expression of autophagic flux in creeping fat tissues ( CrF ) adjacent mesenteric adipose tissues ( Ad . MAT ) of Crohn ' s disease ( n = 3 / group ) . Additionally , autophagic flux was determined in mesenteric adipose tissue ( MAT ) of a colorectal cancer as control ( dotted ) . Data are represented as mean ± s . e . m . ( J ) Paired Student ' s t - .

O O O O B-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-GENEPROD O O B-EXP_ASSAY I-EXP_ASSAY O B-TISSUE I-TISSUE I-TISSUE O O O B-TISSUE I-TISSUE I-TISSUE I-TISSUE O O O O O O B-DISEASE I-DISEASE I-DISEASE I-DISEASE O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O B-TISSUE I-TISSUE I-TISSUE O O O O O B-DISEASE I-DISEASE O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(J) Representative immunoblot for LC3-I/-II and ACTIN protein expression and quantification of autophagic flux in creeping fat tissues (CrF) and adjacent mesenteric adipose tissues (Ad. MAT) of Crohn's disease patients (n = 3/group). Additionally, autophagic flux was determined in the mesenteric adipose tissue (MAT) of a colorectal cancer patient as control (dotted line). Data are represented as mean ± s.e.m. (J) Paired Student's t-test.


( C ) Representative immunoblot for LC3 - I LC3 - II expression of LC3 conversion ( LC3 - II / LC3 - I ) ( n = 3 / group ) . Data are represented as mean ± s . e . m . C , Unpaired Student ' s t - .

O O O O B-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(C) Representative immunoblot for LC3-I and LC3-II protein expression and quantification of LC3 conversion ratio (LC3-II/LC3-I) (n = 3/group). Data are represented as mean ± s.e.m. C, Unpaired Student's t-test.


( F ) Colon after two post - deletion ( steady state ; n = 14 / group ) after DSS at day 7 ( n = 18 - 22 / group ) . Data are represented as mean ± s . e . m . , F , Unpaired Student ' s t - .

O O O B-TISSUE O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(F) Colon length after two weeks post-deletion (steady state; n = 14/group) and after DSS at day 7 (n = 18-22/group). Data are represented as mean ± s.e.m. , F, Unpaired Student's t-test.


( G ) Representative H & E ( 10x magnification ) of colon sections of histological score at steady state ( n = 9 / group ) DSS colitis ( n = 18 - 22 / group ) . Data are represented as mean ± s . e . m . G , Unpaired Student ' s t - .

O O O O O O O O O O O O B-TISSUE O O B-EXP_ASSAY O O O O O O O O O O O B-SMALL_MOLECULE B-DISEASE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(G) Representative H&E staining images (10x magnification) of colon sections and quantification of histological score at steady state (n = 9/group) and DSS colitis (n = 18-22/group). Data are represented as mean ± s.e.m. G, Unpaired Student's t-test.


( I ) Absolute number CD45 + immune from colons at steady state ( n = 13 - 14 / group ) or at 7 days post - DSS induction ( n = 18 - 22 / group ) . ( J ) Frequency of myeloid population in colon at day 7 post - DSS induction ( n = 18 - 25 / group ) . ( K ) Absolute number of Ly6C + monocytes discriminated by absence or presence of MHCII for infiltrating inflammatory monocytes respectively ( n = 18 - 25 / group ) . Data are represented as mean ± s . e . m . I ) Unpaired Student ' s t - . ( J , K ) Two - Way ANOVA .

O O O O O B-GENEPROD O B-CELL_TYPE O B-TISSUE O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O B-CELL_TYPE O O B-TISSUE O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O B-GENEPROD O B-CELL_TYPE O O O O O O B-SUBCELLULAR O O B-CELL_TYPE I-CELL_TYPE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(I) Absolute number CD45+ immune cells from colons at steady state (n = 13-14/group) or at 7 days post-DSS induction (n = 18-22/group). (J) Frequency of myeloid cell population in colon at day 7 post-DSS induction (n = 18-25/group). (K) Absolute number of Ly6C+ monocytes discriminated by the absence or presence of MHCII for infiltrating and inflammatory monocytes respectively (n = 18-25/group). Data are represented as mean ± s.e.m. I) Unpaired Student's t-test. (J, K) Two-Way ANOVA.


( A ) Ex vivo lipolysis measured by released free fatty acid ( left , n = 4 - 5 / group ) glycerol ( right , n = 7 - 8 / group ) in culture supernatant of adipose tissue explants simulated with isoproterenol ( 10µM ) for 1 - 2h . ( B ) Ex vivo lipolysis measured by released free fatty acid ( left , n = 4 / group ) glycerol ( right , n = 7 / group ) adipose tissue explants simulated with TNFα ( 100ng / mL ) for 24h before replacing with fresh medium in absence of TNFα for 3h . Data are represented as mean ± s . e . m . ( A , B , Two - Way ANOVA . ( B , Unpaired Student ' s t - .

O O O O O B-EXP_ASSAY O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O B-TISSUE I-TISSUE O O O B-SMALL_MOLECULE O O O O O O O O O O O O O B-EXP_ASSAY O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O B-TISSUE I-TISSUE O O O B-GENEPROD O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(A) Ex vivo lipolysis measured by released free fatty acid (left, n = 4-5/group) and glycerol (right, n = 7-8/group) in culture supernatant of adipose tissue explants simulated with isoproterenol (10µM) for 1-2h. (B) Ex vivo lipolysis measured by released free fatty acid (left, n = 4/group) and glycerol (right, n = 7/group) adipose tissue explants simulated with TNFα (100ng/mL) for 24h before replacing with fresh medium in the absence of TNFα for 3h. Data are represented as mean ± s.e.m. (A, B, Two-Way ANOVA. (B, Unpaired Student's t-test.


( E ) of FFA species in serum in water - treated DSS - treated mice as measured by FID - GC ( n = 12 - 14 / group ) . Data are represented as mean ± s . e . m . E ) Two - Way ANOVA .

O O O O O O O B-TISSUE O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O O B-ORGANISM O O O B-EXP_ASSAY O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(E) Concentration of individual FFA species in serum in water-treated and DSS-treated mice as measured by FID-GC (n = 12-14/group). Data are represented as mean ± s.e.m. E) Two-Way ANOVA.


( C ) Ex vivo lipolysis assays on Atg7 - deficient adipose tissue explants simulated with isoproterenol ( 10µM ) for 1 - 2h ( n = 5 - 6 / group ) . Data are represented as mean ± s . e . m . ( C , Two - Way ANOVA .

O O O O O B-EXP_ASSAY O O B-GENEPROD O O B-TISSUE I-TISSUE O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(C) Ex vivo lipolysis assays on Atg7-deficient adipose tissue explants simulated with isoproterenol (10µM) for 1-2h (n = 5-6/group). Data are represented as mean ± s.e.m. (C, Two-Way ANOVA.


( H ) Expression of pro - inflammatory cytokines in colon tissues at 7 days post - DSS induction ( n = 8 / group ) . Dotted represents non - inflamed controls . Data are represented as mean ± s . e . m . Unpaired Student ' s t - .

O O O O O O O O O O B-TISSUE O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(H) Expression of pro-inflammatory cytokines in colon tissues at 7 days post-DSS induction (n = 8/group). Dotted line represents non-inflamed controls. Data are represented as mean ± s.e.m. Unpaired Student's t-test.


( K ) Absolute number of Ly6C + monocytes discriminated by absence or presence of MHCII for infiltrating inflammatory monocytes respectively ( n = 8 / group ) . Data are represented as mean ± s . e . m . K ) Unpaired Student ' s t - .

O O O O O O B-GENEPROD O B-CELL_TYPE O O O O O O B-SUBCELLULAR O O B-CELL_TYPE I-CELL_TYPE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(K) Absolute number of Ly6C+ monocytes discriminated by the absence or presence of MHCII for infiltrating and inflammatory monocytes respectively (n = 8/group). Data are represented as mean ± s.e.m. K) Unpaired Student's t-test.


( E ) Fold change expression of NRF2 - target genes in visceral adipocytes at day 7 after DSS induction from normalized of RNAseq dataset ( n = 6 / group ) . Data are represented as mean ± s . e . m . Unpaired Student ' s t - .

O O O O O O O B-GENEPROD O O O O B-CELL_TYPE I-CELL_TYPE O O O O B-SMALL_MOLECULE O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(E) Fold change expression of NRF2-target genes in primary visceral adipocytes at day 7 after DSS induction from normalized counts of RNAseq dataset (n = 6/group). Data are represented as mean ± s.e.m. Unpaired Student's t-test.


( H ) Representative immunoblot of EPHX1 EPHX2 in gonadal adipose tissues at day 7 after DSS induction . Asterix indicating non - specific band ( n = 14 - 18 / group ) . Data are represented as mean ± s . e . m . Unpaired Student ' s t - .

O O O O B-EXP_ASSAY O B-GENEPROD I-GENEPROD O B-TISSUE I-TISSUE I-TISSUE O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(H) Representative immunoblot of EPHX1 and EPHX2 in gonadal adipose tissues at day 7 after DSS induction. Asterix indicating non-specific band (n = 14-18/group). Data are represented as mean ± s.e.m. Unpaired Student's t-test.


( B ) Colitis was induced in mice for 7 days adipose tissues were extracted cultured for 6 hours in serum - starved medium . Secretion of IL - 10 from mesenteric ( left panel ) gonadal adipose tissues ( right panel ) was measured by ELISA . Shapes identify experiments ( n = 5 - 15 / group ) . Data are represented as mean ± s . e . m . ( B ) Two - Way ANOVA with regression for experiment .

O O O B-DISEASE O O O B-ORGANISM O O O B-TISSUE I-TISSUE O O O O O O O B-TISSUE O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-TISSUE O O O O B-TISSUE I-TISSUE I-TISSUE O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(B) Colitis was induced in mice for 7 days and adipose tissues were extracted and cultured for 6 hours in serum-starved medium. Secretion of IL-10 and from mesenteric (left panel) and gonadal adipose tissues (right panel) was measured by ELISA. Shapes identify individual experiments (n = 5-15/group). Data are represented as mean ± s.e.m. (B) Two-Way ANOVA with regression for experiment.


( F ) Serum cytokines upon DSS - induced colitis at day 7 post - induction ( n = 17 - 23 / group ) . Data are represented as mean ± s . e . m . Two - Way ANOVA .

O O O B-TISSUE O O B-SMALL_MOLECULE O O B-DISEASE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(F) Serum cytokines upon DSS-induced colitis at day 7 post-induction (n = 17-23/group). Data are represented as mean ± s.e.m. Two-Way ANOVA.


( A ) of Il10 transcript in RAW264 . 7 upon stimulation to epoxy fatty ( n = 3 / group ) . ( B ) of IL - 10 in supernatant of RAW264 . 7 upon stimulation to epoxy fatty ( n = 3 / group ) . Data are represented as mean ± s . e . m . ( A , B ) One - Way ANOVA .

O O O O B-GENEPROD O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(A) Quantification of Il10 transcript levels in RAW264.7 upon stimulation to epoxy fatty acids (n = 3/group). (B) Quantification of IL-10 protein levels in the supernatant of RAW264.7 upon stimulation to epoxy fatty acids (n = 3/group). Data are represented as mean ± s.e.m. (A,B) One-Way ANOVA.