( 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 .

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(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 - 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 .

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

O O O B-EXP_ASSAY O O B-CELL_LINE O 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 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 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 .

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

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(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 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 .

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

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(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 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 .

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(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 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 .

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(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 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 .

O O O B-EXP_ASSAY O O B-CELL_LINE O 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 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 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 .

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(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 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 .

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(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 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 .

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

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(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 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 .

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(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 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 .

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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.


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 .

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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 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 .

O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O B-CELL_LINE O O 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 O B-SUBCELLULAR O O 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 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 .

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(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 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 .

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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 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 .

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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.


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 .

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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 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 .

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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 staining 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 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 .

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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 . 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 ) .

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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 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 ) .

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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 protein levels of Oct4 , Sox2 and Nanog in miRNA mimic transfected Dgcr8 - / - ESCs and 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 and Nestin immunofluorescence staining of mimic transfected Dgcr8 - / - ( H ) and 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 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 .

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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.


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 .

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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.


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 .

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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 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 .

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

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


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 .

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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 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 .

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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 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 .

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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 and - 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 the 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 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 .

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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 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 .

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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.


Morphology 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 and exogenous Oct4 , Sox2 , Klf4 and Nanog expression in iPSCs , ESCs and 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 staining of Oct4 , Nanog and SSEA1 of the iPSC colonies .

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


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 ) .

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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 and eosin staining of teratoma derived from the iPSC colonies . Neuronal rosette ( Ectoderm ) , Cartilage ( Mesoderm ) and 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 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 .

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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 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 ) .

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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 staining of wild - type and 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 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 ) .

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

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


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 ) .

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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 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 .

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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 analysis of several cardiac markers expression during cardiac differentiation of wild - type and 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 and Troponin Iimmunofluorescence staining of differentiation cultures derived from wild - type and 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.


Weight of teratomas derived from wild - type and 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 staining of teratomas derived from wild - type and 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 and eosin staining of teratomas derived from wild - type and knockout ESCs . Ectoderm , Mesoderm and 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 ) 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 .

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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 the interaction of MBD9 with HTA9 , HTA11 , PIE1 , CHR11 , and 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 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 .

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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 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 .

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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 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 .

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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 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 .

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(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 the WT , pie1 , swc2 , and rin2 mutant plants . Rosette leaves were cut from bolting plants .

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


( 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 .

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(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 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 .

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(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 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 .

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(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 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 .

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(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 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 .

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(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 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 .

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(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 ) 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 .

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(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 . The 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 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 the WT , pie1 , chr11 / 17 , and mbd9 - 1 mutants over the PIE1

O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD O 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 the WT , pie1 , chr11 / 17 , and mbd9 - 1 mutants over the

O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD 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 ) .

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

(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 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 .

O O O O O O B-GENEPROD O B-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 O B-GENEPROD O B-GENEPROD O B-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 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 .

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

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

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 O O B-SUBCELLULAR O O O O B-SMALL_MOLECULE O O B-EXP_ASSAY O O O O O O B-EXP_ASSAY O O B-EXP_ASSAY I-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 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 .

O O O B-EXP_ASSAY O B-SUBCELLULAR O O O O O O O O O O O O B-GENEPROD O O O B-GENEPROD O O B-GENEPROD O 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 and ATGL protein expression and quantification ( n = 5 - 6 / group ) . Data are represented as mean ± s . e . m . E , Unpaired Student ' s t - test .

O O O O B-EXP_ASSAY O O 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 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 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 .

O O O B-EXP_ASSAY O O B-EXP_ASSAY I-EXP_ASSAY O B-TISSUE O 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 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 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 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 .

O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O 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 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 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 .

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

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

O O O B-TISSUE O 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 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 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 .

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

O O O O O B-GENEPROD O B-CELL_TYPE I-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 I-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 O B-SUBCELLULAR O 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 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 ) 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 .

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 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 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 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

(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 ) 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 .

O O O O O O O O O B-TISSUE O B-SMALL_MOLECULE O 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 line represents non - inflamed controls . Data are represented as mean ± s . e . m . Unpaired Student ' s t - test .

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 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 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 .

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

(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 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 .

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

(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 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 .

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

O O O B-DISEASE O O O B-ORGANISM O O O O B-TISSUE I-TISSUE O O O O O O O O B-TISSUE O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O B-TISSUE O 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 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 ) 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 .

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


( C - G ) ssDNA repair efficiency of the indicated TS mutants as determined from the maximal and final percentages of cells with Rad52 foci during the time course ( right panels ) . Cells were synchronized in G1 and released into S phase in the presence of 0 . 033 % MMS for 1 hour ( 60 + ) , treated with 2 . 5 % sodium thiosulfate to inactivate the MMS , washed and released into fresh medium for different times . They were also released into medium without MMS for 1 hour to control the formation of spontaneous Rad52 - YFP foci ( 60 - ) . The percentage of cells with foci at each point was normalized to the highest value of the wild type , taken as 100 ( left panels ) . Cell cycle progression was determined by cell sorting . The mean and SEM of 3 ( wild type , rad18 ∆ , mms2 ∆ , rad51 ∆ , rad57 ∆ , rad54 ∆ ) , 4 ( pol30 - K164R ) and 5 ( rad5 ∆ ) independent experiments are shown . Statistically significant differences according to an unpaired two - tailed Student ' s t - test are shown , where three asterisks represent p - values < 0 . 001 .

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

(C-G) ssDNA repair efficiency of the indicated TS mutants as determined from the maximal and final percentages of cells with Rad52 foci during the time course (right panels). Cells were synchronized in G1 and released into S phase in the presence of 0.033% MMS for 1 hour (60+), treated with 2.5% sodium thiosulfate to inactivate the MMS, washed and released into fresh medium for different times. They were also released into medium without MMS for 1 hour to control the formation of spontaneous Rad52-YFP foci (60-). The percentage of cells with foci at each point was normalized to the highest value of the wild type, taken as 100 (left panels). Cell cycle progression was determined by cell sorting. The mean and SEM of 3 (wild type, rad18∆, mms2∆, rad51∆, rad57∆, rad54∆), 4 (pol30-K164R) and 5 (rad5∆) independent experiments are shown. Statistically significant differences according to an unpaired two-tailed Student's t-test are shown, where three asterisks represent p-values <0.001.


( C - E ) ssDNA repair efficiency of the indicated TLS mutants as determined from the maximal and final percentages of cells with Rad52 foci during the time course ( D and E , right panel ) . Cells were synchronized in G1 and released into S phase in the presence of 0 . 033 % MMS for 1 hour , treated with 2 . 5 % sodium thiosulfate to inactivate the MMS , washed and released into fresh medium for different times . The percentage of cells with foci at each point was normalized to the highest value of the wild type , taken as 100 ( C and E , left panel ) . Data information : The mean and SEM of and 3 - 12 12 ( wild type ) , 6 ( rev1 ∆ , rev3 ∆ , rad30 ∆ , rev1 ∆ rad30 ∆ , rev3 ∆ rad30 ∆ ) , and 3 ( rev1 ∆ rev3 ∆ and rev1 ∆ rev3 ∆ rad30 ∆ ) independent experiments are shown . Statistically significant differences according to an unpaired two - tailed Student ' s t - test are shown , where one , two and three asterisks represent p - values < 0 . 05 , < 0 . 01 and < 0 . 001 , respectively .

O O O O O 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 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 B-SMALL_MOLECULE O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE 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 O O 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 O B-GENEPROD O O B-GENEPROD O O B-GENEPROD O B-GENEPROD O O B-GENEPROD O B-GENEPROD O O O O O O B-GENEPROD O B-GENEPROD 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 O O O O O 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-E) ssDNA repair efficiency of the indicated TLS mutants as determined from the maximal and final percentages of cells with Rad52 foci during the time course (D and E, right panel). Cells were synchronized in G1 and released into S phase in the presence of 0.033% MMS for 1 hour, treated with 2.5% sodium thiosulfate to inactivate the MMS, washed and released into fresh medium for different times. The percentage of cells with foci at each point was normalized to the highest value of the wild type, taken as 100 (C and E, left panel). Data information: The mean and SEM of and 3-12 12 (wild type), 6 (rev1∆, rev3∆, rad30∆, rev1∆ rad30∆, rev3∆ rad30∆), and 3 (rev1∆ rev3∆ and rev1∆ rev3∆ rad30∆) independent experiments are shown. Statistically significant differences according to an unpaired two-tailed Student's t-test are shown, where one, two and three asterisks represent p-values <0.05, <0.01 and <0.001, respectively.


( C - D ) ssDNA repair efficiency of the rad5 ∆ and rev1 ∆ mutants as determined from the maximal and final percentages of cells with Rad54 foci during the time course ( right panels ) . Cells were synchronized in G1 and released into S phase in the presence of 0 . 033 % MMS for 1 hour , treated with 2 . 5 % sodium thiosulfate to inactivate the MMS , washed and released into fresh medium for different times . The percentage of cells with foci at each point was normalized to the highest value of the wild type , taken as 100 ( left panels ) . Data information : The mean and SEM of independent experiments are shown . Statistically significant differences according to an unpaired two - tailed Student ' s t - test are shown , where one , two and three asterisks represent p - values < 0 . 05 , < 0 . 01 and < 0 . 001 , respectively .

O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD O O B-GENEPROD 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 B-SMALL_MOLECULE O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-D) ssDNA repair efficiency of the rad5∆ and rev1∆ mutants as determined from the maximal and final percentages of cells with Rad54 foci during the time course (right panels). Cells were synchronized in G1 and released into S phase in the presence of 0.033% MMS for 1 hour, treated with 2.5% sodium thiosulfate to inactivate the MMS, washed and released into fresh medium for different times. The percentage of cells with foci at each point was normalized to the highest value of the wild type, taken as 100 (left panels). Data information: The mean and SEM of independent experiments are shown. Statistically significant differences according to an unpaired two-tailed Student's t-test are shown, where one, two and three asterisks represent p-values <0.05, <0.01 and <0.001, respectively.


fluorescence signal of RPA foci per cell ( E ) at times 1 or 2 ( peak ) and 7 hours from the time course shown in ( B ) . Data information : The mean and SEM of 3 independent experiments are shown . Asterisks indicate statistically significant differences according to an unpaired two - tailed Student ' s t - test . One , two and three asterisks represent p - values < 0 . 05 , < 0 . 01 and < 0 . 001 , respectively .

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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

fluorescence signal of RPA foci per cell (E) at times 1 or 2 (peak) and 7 hours from the time course shown in (B). Data information: The mean and SEM of 3 independent experiments are shown. Asterisks indicate statistically significant differences according to an unpaired two-tailed Student's t-test. One, two and three asterisks represent p-values <0.05, <0.01 and <0.001, respectively.


( F ) ssDNA repair efficiency of the indicated mutants as determined by ChIP against Rfa1 - YFP at the ARS305 replication origin . Cells were synchronized in G1 and released into S phase in the presence of 0 . 033 % MMS for 1 hour , treated with 2 . 5 % sodium thiosulfate to inactivate the MMS , washed and released into fresh medium . The amount of Rfa1 at ssDNA was determined at 1 and 7 hours after MMS release . RPA enrichment was calculated as the ratio between immunoprecipitate and input values . All values were normalized to the wild type at 7 hours , taken as 1 . Data information : The mean and SEM of 3 independent experiments are shown . Asterisks indicate statistically significant differences according to an unpaired two - tailed Student ' s t - test . One , two and three asterisks represent p - values < 0 . 05 , < 0 . 01 and < 0 . 001 , respectively . In ( F ) all values were significant relative to the untagged controls ( not shown for clarity ) .

O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O B-EXP_ASSAY O B-GENEPROD 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 B-SMALL_MOLECULE O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O B-SMALL_MOLECULE O O O O O O O O O O O B-GENEPROD O B-SMALL_MOLECULE O O O O O O O O B-SMALL_MOLECULE O O B-GENEPROD 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) ssDNA repair efficiency of the indicated mutants as determined by ChIP against Rfa1-YFP at the ARS305 replication origin. Cells were synchronized in G1 and released into S phase in the presence of 0.033% MMS for 1 hour, treated with 2.5% sodium thiosulfate to inactivate the MMS, washed and released into fresh medium. The amount of Rfa1 at ssDNA was determined at 1 and 7 hours after MMS release. RPA enrichment was calculated as the ratio between immunoprecipitate and input values. All values were normalized to the wild type at 7 hours, taken as 1. Data information: The mean and SEM of 3 independent experiments are shown. Asterisks indicate statistically significant differences according to an unpaired two-tailed Student's t-test. One, two and three asterisks represent p-values <0.05, <0.01 and <0.001, respectively. In (F) all values were significant relative to the untagged controls (not shown for clarity).


Effect of the lack of HR factors in spontaneous and MMS - induced mutagenesis . The frequency of spontaneous and MMS - induced forward mutagenesis at the CAN1 locus was determined in mid - log phase cell cultures before ( spontaneous mutagenesis ) and after treatment with 0 . 01 % MMS for 4 hours ( DNA damage - induced ) . Thus , the frequency of DNA damage - induced mutagenesis includes the mutants that arose spontaneously before MMS addition . The frequency of mutagenesis was determined at 0 . 01 % MMS because at higher MMS concentrations the loss of viability of the rad mutants did not allow us to select for canavanine - resistant cells . Survival ( % ) after 4 hours in MMS relative to the wild type ( taken as 100 ) is shown below the genotypes . The mean and SEM of 6 ( wild type ) , 9 ( rad54 ∆ ) and 3 ( rest ) independent fluctuation tests are shown . Asterisks indicate statistically significant differences according to an unpaired two - tailed Student ' s t - test ( one , two and three asterisks represent p - values < 0 . 05 , < 0 . 01 and < 0 . 001 , respectively ) . Only the statistical analysis of the effect of rad52 ∆ , rad57 ∆ and rad51 ∆ on rad54 ∆ - mediated mutagenesis is included for clarity . All mutants displayed statistically significant differences with the wild type and not with each other in the absence of MMS .

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

Effect of the lack of HR factors in spontaneous and MMS-induced mutagenesis. The frequency of spontaneous and MMS-induced forward mutagenesis at the CAN1 locus was determined in mid-log phase cell cultures before (spontaneous mutagenesis) and after treatment with 0.01% MMS for 4 hours (DNA damage-induced). Thus, the frequency of DNA damage-induced mutagenesis includes the mutants that arose spontaneously before MMS addition. The frequency of mutagenesis was determined at 0.01% MMS because at higher MMS concentrations the loss of viability of the rad mutants did not allow us to select for canavanine-resistant cells. Survival (%) after 4 hours in MMS relative to the wild type (taken as 100) is shown below the genotypes. The mean and SEM of 6 (wild type), 9 (rad54∆) and 3 (rest) independent fluctuation tests are shown. Asterisks indicate statistically significant differences according to an unpaired two-tailed Student's t-test (one, two and three asterisks represent p-values <0.05, <0.01 and <0.001, respectively). Only the statistical analysis of the effect of rad52∆, rad57∆ and rad51∆ on rad54∆-mediated mutagenesis is included for clarity. All mutants displayed statistically significant differences with the wild type and not with each other in the absence of MMS.


( A ) Effect of the rad54 ∆ and rad52 ∆ mutations in spontaneous and UV - induced uSCE . The frequency of spontaneous and UV - induced recombinants was determined by irradiating or not cells plated onto solid medium after the corresponding dilutions ( SMM and SMM without histidine to calculate total and recombinant cells , respectively ) . The mean and SEM of 3 - 5 independent fluctuation tests are shown . Asterisks indicate statistically significant differences according to an unpaired two - tailed Student ' s t - test . Two and three asterisks represent p - values < 0 . 01 and < 0 . 001 , respectively .

O O O O O O B-GENEPROD O O B-GENEPROD O 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 B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O B-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 O O O O O O O O O O O O O O O O O O O O O O

(A) Effect of the rad54∆ and rad52∆ mutations in spontaneous and UV-induced uSCE. The frequency of spontaneous and UV-induced recombinants was determined by irradiating or not cells plated onto solid medium after the corresponding dilutions (SMM and SMM without histidine to calculate total and recombinant cells, respectively). The mean and SEM of 3-5 independent fluctuation tests are shown. Asterisks indicate statistically significant differences according to an unpaired two-tailed Student's t-test. Two and three asterisks represent p-values <0.01 and <0.001, respectively.


( B ) Rad6 binds to DNA in the absence and presence of MMS . ChEC analysis of G1 - synchronyzed cells released in the absence or the presence of 0 . 033 % MMS for the indicated times . Data information : DNA gel electrophoresis , DNA content ( cell sorting ) and quantification of DNA digestion profiles are shown . The experiments were repeated twice with similar results .

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

(B) Rad6 binds to DNA in the absence and presence of MMS. ChEC analysis of G1-synchronyzed cells released in the absence or the presence of 0.033% MMS for the indicated times. Data information: DNA gel electrophoresis, DNA content (cell sorting) and quantification of DNA digestion profiles are shown. The experiments were repeated twice with similar results.


( C - F ) Rad6 binding to DNA depends on Rad18 , Rad52 ( C ) , Rad51 ( D ) , Rad57 ( E ) but not Rad54 ( F ) as determined by ChEC analyses of asynchronous cell cultures without or with 0 . 05 % MMS for 2 hours . Data information : DNA gel electrophoresis , DNA content ( cell sorting ) and quantification of DNA digestion profiles are shown . The experiments were repeated twice with similar results .

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

(C-F) Rad6 binding to DNA depends on Rad18, Rad52 (C), Rad51 (D), Rad57 (E) but not Rad54 (F) as determined by ChEC analyses of asynchronous cell cultures without or with 0.05% MMS for 2 hours. Data information: DNA gel electrophoresis, DNA content (cell sorting) and quantification of DNA digestion profiles are shown. The experiments were repeated twice with similar results.


( A - D ) Effect of rad52 ∆ , rad51 ∆ ( A ) , rad57 ∆ ( B ) and rad54 ∆ ( C ) on PCNA ubiquitylation in cells synchronized in G1 and released into fresh medium in the presence of 0 . 025 % MMS . Cell cycle progression was followed by cell sorting analysis and western blot against Clb2 . Pgk1 was used as loading control . The amounts of mono - and polyubiquitylated PCNA were normalized to the total amount of PCNA . For each time point , rad mutant values were relativized to those of the wild type , taken as 1 . The mean and SEM of 3 independent experiments are shown ( D ) . An asterisk indicates a mean significantly different than 1 , according to a One - sample t - test .

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

(A-D) Effect of rad52∆, rad51∆ (A), rad57∆ (B) and rad54∆ (C) on PCNA ubiquitylation in cells synchronized in G1 and released into fresh medium in the presence of 0.025% MMS. Cell cycle progression was followed by cell sorting analysis and western blot against Clb2. Pgk1 was used as loading control. The amounts of mono- and polyubiquitylated PCNA were normalized to the total amount of PCNA. For each time point, rad mutant values were relativized to those of the wild type, taken as 1. The mean and SEM of 3 independent experiments are shown (D). An asterisk indicates a mean significantly different than 1, according to a One-sample t-test.


( A ) Immunofluorescence staining against Iba - 1 in cortex ( Cx ) , hippocampus ( Hip ) and cerebellum ( Cb ) of NPA affected and control 3 year - old children . Scale bars , 50 μm . ( B ) Magnified image showing ramified ( left ) versus amoeboid ( right ) morphology in control and NPA microglia . Scale bars , 10 μm . ( C ) Mean ± SEM area of Iba - 1 positive cells in different brain regions from a NPA affected and a control child ( n = 20 cells , Student ´ s t - test ) .

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

(A) Immunofluorescence staining against Iba-1 in cortex (Cx), hippocampus (Hip) and cerebellum (Cb) of NPA affected and control 3 year-old children. Scale bars, 50 μm. (B) Magnified image showing ramified (left) versus amoeboid (right) morphology in control and NPA microglia. Scale bars, 10 μm. (C) Mean ± SEM area of Iba-1 positive cells in different brain regions from a NPA affected and a control child (n = 20 cells, Student´s t-test).


( D ) Immunofluorescence staining against Iba - 1 in Cx , Hip and Cb of 3 month - old ASMko and wt mice . DAPI staining shows cell nuclei . Scale bars , 50 μm .

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

(D) Immunofluorescence staining against Iba-1 in Cx, Hip and Cb of 3 month-old ASMko and wt mice. DAPI staining shows cell nuclei. Scale bars, 50 μm.


( E ) Mean ± SEM number of Iba - 1 positive cells in different brain regions from ASMko and wt mice ( n = 5 mice per group , Student ´ s t - test ) .

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

(E) Mean ± SEM number of Iba-1 positive cells in different brain regions from ASMko and wt mice (n = 5 mice per group, Student´s t-test).


( F ) 3D rendered images obtained with Imaris Surface software from high magnification images of immunofluorescence staining against Iba - 1 showing representative microglial morphology in Cx , Hip and Cb of ASMko and wt mice . Scale bars , 10 μm .

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

(F) 3D rendered images obtained with Imaris Surface software from high magnification images of immunofluorescence staining against Iba-1 showing representative microglial morphology in Cx, Hip and Cb of ASMko and wt mice. Scale bars, 10 μm.


( G ) Mean ± SEM area of Iba - 1 positive cells in the different brain regions from ASMko and wt mice ( n = 5 mice per group , > 10 cells per mouse , Student ´ s t - test ) . Scale bars , 15 μm .

O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O B-TISSUE O O B-GENEPROD O O B-ORGANISM O O O O B-ORGANISM O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O

(G) Mean ± SEM area of Iba-1 positive cells in the different brain regions from ASMko and wt mice (n = 5 mice per group, >10 cells per mouse, Student´s t-test). Scale bars, 15 μm.


( H ) Example of projection detection by the Filament Imaris software . ( I ) Mean ± SEM branch point number per process and process length in iba1 + cells obtained with the Imaris Filament software ( Student ´ s t - test ) .

O O O O 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 O O O O O O O O O O O O O O O O

(H) Example of projection detection by the Filament Imaris software. (I) Mean ± SEM branch point number per process and process length in iba1+ cells obtained with the Imaris Filament software (Student´s t-test).


( A ) Immunofluorescence staining against Iba - 1 in Cb of ASMko and wt mice treated or not with PLX for 2 months . DAPI staining shows cell nuclei . Scale bar , 100 μm . ( B ) Magnified images ( from A ) showing ramified ( left ) versus amoeboid ( right ) morphology in wt and ASMko microglia treated or not with PLX . Scale bars , 30 μm . ( C ) Mean ± SEM number of Iba - 1 positive cells in the Cb of the different mouse groups ( n = 7 mice per group , Two - way Anova , Bonferroni post hoc ) .

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

(A) Immunofluorescence staining against Iba-1 in Cb of ASMko and wt mice treated or not with PLX for 2 months. DAPI staining shows cell nuclei. Scale bar, 100 μm. (B) Magnified images (from A) showing ramified (left) versus amoeboid (right) morphology in wt and ASMko microglia treated or not with PLX. Scale bars, 30 μm. (C) Mean ± SEM number of Iba-1 positive cells in the Cb of the different mouse groups (n = 7 mice per group, Two-way Anova, Bonferroni post hoc).


( D , E ) Performance in the Rotarod test of ASMko and wt mice treated or not with PLX for 2 months expressed as the mean ± SEM time spent in the rotating rod on each of four trials ( D ) or the mean ± SEM time of the four trials ( E ) ( n = 7 mice per group , Two - way Anova , Games - Howell ( D ) and Bonferroni ( E ) post hoc ) .

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

(D, E) Performance in the Rotarod test of ASMko and wt mice treated or not with PLX for 2 months expressed as the mean ± SEM time spent in the rotating rod on each of four trials (D) or the mean ± SEM time of the four trials (E) (n = 7 mice per group, Two-way Anova, Games-Howell (D) and Bonferroni (E) post hoc).


( F ) Mean ± SEM body weight of ASMko and wt mice treated or not with PLX at the indicated time during long - term treatment ( n = 7 mice per group , Two - way Anova , Bonferroni post hoc ) .

O O O O O O O O O B-GENEPROD O O B-ORGANISM O O O O B-SMALL_MOLECULE O O 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

(F) Mean ± SEM body weight of ASMko and wt mice treated or not with PLX at the indicated time during long-term treatment (n= 7 mice per group, Two-way Anova, Bonferroni post hoc).


( G ) Survival curve of ASMko and wt mice with or without long - term PLX treatment ( n = 7 mice per group ) .

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(G) Survival curve of ASMko and wt mice with or without long-term PLX treatment (n = 7 mice per group).


( H ) Immunofluorescence staining against the PC marker Calbindin in mid and posterior lobules of Cb in ASMko and wt mice treated or not with PLX for 2 months . DAPI staining shows cell nuclei . Scale bar , 500 μm . ( I ) Mean ± SEM number of Calbindin positive cells in the anterior ( lobules I - V ) , middle ( lobules VI - VIII ) and posterior zone ( lobules IX - X ) of the Cb from the different mouse groups ( n = 7 mouse per group , Two - way Anova , Bonferroni post hoc ) .

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(H) Immunofluorescence staining against the PC marker Calbindin in mid and posterior lobules of Cb in ASMko and wt mice treated or not with PLX for 2 months. DAPI staining shows cell nuclei. Scale bar, 500 μm. (I) Mean ± SEM number of Calbindin positive cells in the anterior (lobules I-V), middle (lobules VI-VIII) and posterior zone (lobules IX-X) of the Cb from the different mouse groups (n = 7 mouse per group, Two-way Anova, Bonferroni post hoc).


( A ) Immunofluorescence staining against CD16 / CD32 or Arg - 1 in Cb of ASMko mice ( red ) . Microglia were identified by F4 / 80 or Iba - 1 staining ( green ) . DAPI staining shows cell nuclei . Scale bar , 50 μm . ( B ) Mean ± SEM number of Arg - 1 and CD16 / CD32 positive microglia per area in Cb from ASMko and wt mice ( n = 6 mice per group ) .

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(A) Immunofluorescence staining against CD16/CD32 or Arg-1 in Cb of ASMko mice (red). Microglia were identified by F4/80 or Iba-1 staining (green). DAPI staining shows cell nuclei. Scale bar, 50 μm. (B) Mean ± SEM number of Arg-1 and CD16/CD32 positive microglia per area in Cb from ASMko and wt mice (n = 6 mice per group).


( C ) Mean ± SEM levels of TNFa protein in the Cb from ASMko and wt mice measured by ELISA ( n = 7 mice per group , Student ´ s t - test ) .

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(C) Mean ± SEM levels of TNFa protein in the Cb from ASMko and wt mice measured by ELISA (n = 7 mice per group, Student´s t-test).


( D ) Mean ± SEM levels of TNFa mRNA in Cb of ASMko and wt mice determined by qRT - PCR ( n = 7 mice per group , Student ´ s t - test ) .

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(D) Mean ± SEM levels of TNFa mRNA in Cb of ASMko and wt mice determined by qRT-PCR (n = 7 mice per group, Student´s t-test).


( E ) Survival after LPS challenge in ASMko and wt mice treated or not with dexamethasone ( n = 8 mice per group ) .

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(E) Survival after LPS challenge in ASMko and wt mice treated or not with dexamethasone (n = 8 mice per group).


( F ) Immunofluorescence staining against MBP in Cb of ASMko and wt mice treated or not with PLX for 2 months . Scale bar , 50 μm .

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(F) Immunofluorescence staining against MBP in Cb of ASMko and wt mice treated or not with PLX for 2 months. Scale bar, 50 μm.


( G ) Mean ± SEM MBP intensity in the different mouse groups ( n = 7 mice per group , Two - way Anova , Bonferroni post hoc ) .

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(G) Mean ± SEM MBP intensity in the different mouse groups (n = 7 mice per group, Two-way Anova, Bonferroni post hoc).