( C ) Immunoprecipitation of Flag - tagged Fbxw7 with anti - Flag affinity gel , followed by Western blotting of Myc to detect Myc - Gata3 in HEK293T cells transfected with Myc - Gata3 and / or Flag - Fbxw7 plasmids .

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

(C) Immunoprecipitation of Flag-tagged Fbxw7 with anti-Flag affinity gel, followed by Western blotting of Myc to detect Myc-Gata3 in HEK293T cells transfected with Myc-Gata3 and/or Flag-Fbxw7 plasmids.


( F ) Western blotting of Gata3 in mouse Th2 cells treated with or without JQ1 ( 500nM ) , and then treated with cycloheximide ( CHX ; 25μg / ml ) for 0h , 0 . 5h , 1h and 2h before harvest to stop protein synthesis .

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

(F) Western blotting of Gata3 in mouse Th2 cells treated with or without JQ1 (500nM), and then treated with cycloheximide (CHX; 25μg/ml) for 0h, 0.5h, 1h and 2h before harvest to stop protein synthesis.


( C ) ChIP - qPCR analysis of histone modifications on the gene loci of Foxp3 and Fbxw7 in mouse Th2 cells treated with or without JQ1 ( 250nM ) .

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

(C) ChIP-qPCR analysis of histone modifications on the gene loci of Foxp3 and Fbxw7 in mouse Th2 cells treated with or without JQ1 (250nM).


( D ) Immunoprecipitation of Brd4 in mouse Th2 cells , followed by Western blotting of Ezh2 , Suz12 , EED and G9a .

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

(D) Immunoprecipitation of Brd4 in mouse Th2 cells, followed by Western blotting of Ezh2, Suz12, EED and G9a.


( F ) ChIP - qPCR analysis of Brd4 , Ezh2 , Suz12 and EED binding on the gene loci of Il4 , Il5 , Foxp3 and Fbxw7 in mouse Th2 cells treat with or without JQ1 ( 250nM ) .

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

(F) ChIP-qPCR analysis of Brd4, Ezh2, Suz12 and EED binding on the gene loci of Il4, Il5, Foxp3 and Fbxw7 in mouse Th2 cells treat with or without JQ1 (250nM).


( B ) HEK293T cells are transfected with Flag - Brd4 and HA - EED , HA - Ezh2 , and Suz12 plasmids . Lysates are immunoprecipitated with Flag - tagged Brd4 , treated in - vitro with JQ1 ( 5μM ) , MS402 ( 5μM ) , and ABBV - 744 ( 5μM ) , followed by Western blotting of HA .

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

(B) HEK293T cells are transfected with Flag-Brd4 and HA-EED, HA-Ezh2, and Suz12 plasmids. Lysates are immunoprecipitated with Flag-tagged Brd4, treated in-vitro with JQ1 (5μM), MS402 (5μM), and ABBV-744 (5μM), followed by Western blotting of HA.


( F ) HEK293T cells are transfected with Flag - Brd4 , HA - EED and HA - EED ( K19R ) plasmids . Lysates are immunoprecipitated with Flag followed by Western blotting of HA , and vice versa .

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

(F) HEK293T cells are transfected with Flag-Brd4, HA-EED and HA-EED(K19R) plasmids. Lysates are immunoprecipitated with Flag followed by Western blotting of HA, and vice versa.


( B ) qPCR analysis of Il4 , Il5 , Gata3 in mouse Th2 cells treated with ABBV - 744 at 500nM and 1μM .

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

(B) qPCR analysis of Il4, Il5, Gata3 in mouse Th2 cells treated with ABBV-744 at 500nM and 1μM.


( D ) Western blotting of Gata3 of mouse Th2 cells treated with ABBV - 744 ( 500nM ) and MG132 ( 20μM ) .

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

(D) Western blotting of Gata3 of mouse Th2 cells treated with ABBV-744 (500nM) and MG132 (20μM).


( E ) qPCR analysis of Foxp3 in mouse Th2 cells treated with ABBV - 744 at 500nM and 1μM . ( F ) qPCR analysis of Fbxw7 in mouse Th2 cells treated with ABBV - 744 at 500nM and 1μM . (

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

(E) qPCR analysis of Foxp3 in mouse Th2 cells treated with ABBV-744 at 500nM and 1μM. (F) qPCR analysis of Fbxw7 in mouse Th2 cells treated with ABBV-744 at 500nM and 1μM. (


( A , B ) Relative change in total abundances between control and ρ0 cells averaged over four independent migration profiles ( Dataset EV1 ) . A , all mitochondrial proteins detected . Grey area indicates proteins that changed less than 2 - fold in both directions . Subunits of selected mitochondrial machineries are highligted as follows : magenta , pyruate dehydrogenase complex ( PDH ) ; blue , mitochondial carriers ( Carrier ) ; brown , translocase of the outer membrane ( TOM ) ; yellow , translocase of the inner membrane ( TIM ) ; green , mitochondrial contact site and cristae organizing system ( MICOS ) ; yellow - green , mitochondrial fusion - fission system ( Dynamics ) ; red , porin or voltage dependent anion channel ( VDAC ) ; B , subunits of OXPHOS complexes ( Complex I - V ) and mitoribosomes ( Ribosome ) . Gene names of the subunit with the highest abundances in ρ0 cells are indicated for each complex . For the full gene name , add NDU ( complex I ) , SDH ( complex II ) , UQC ( R10 ) , COX ( complex IV ) , ATP ( 5L , 5J2 ) , MRP ( Ribosome ) in front of the abbreviated gene names given in the figure .

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

(A, B) Relative change in total abundances between control and ρ0 cells averaged over four independent migration profiles (Dataset EV1). A, all mitochondrial proteins detected. Grey area indicates proteins that changed less than 2-fold in both directions. Subunits of selected mitochondrial machineries are highligted as follows: magenta, pyruate dehydrogenase complex (PDH); blue, mitochondial carriers (Carrier); brown, translocase of the outer membrane (TOM); yellow, translocase of the inner membrane (TIM); green, mitochondrial contact site and cristae organizing system (MICOS); yellow-green, mitochondrial fusion-fission system (Dynamics); red, porin or voltage dependent anion channel (VDAC); B, subunits of OXPHOS complexes (Complex I-V) and mitoribosomes (Ribosome). Gene names of the subunit with the highest abundances in ρ0 cells are indicated for each complex. For the full gene name, add NDU (complex I), SDH (complex II), UQC (R10), COX (complex IV), ATP (5L, 5J2), MRP (Ribosome) in front of the abbreviated gene names given in the figure.


( A , B ) Selected proteins involved in DNA ( A ) and RNA ( B ) maintenance of control ( 143B , solid lines , grey bars ) and ρ0 cells ( dashed lines , colored bars ) . A , for POLG , UNG and TWNK , only abundances > 1 MDa were quantified . Plots and gene names are in corresponding colors . Data are presented as mean ± SD ( n = 4 ) . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 ( Student ' s t - test for individual proteins and two - way ANOVA for changes of multiple proteins in a complex ) .

O O O O O O O O O O O O O O O O O O O O O O B-CELL_LINE 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, B) Selected proteins involved in DNA (A) and RNA (B) maintenance of control (143B, solid lines, grey bars) and ρ0 cells (dashed lines, colored bars). A, for POLG, UNG and TWNK, only abundances >1 MDa were quantified. Plots and gene names are in corresponding colors. Data are presented as mean ± SD (n=4). *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 (Student's t-test for individual proteins and two-way ANOVA for changes of multiple proteins in a complex).


( A - F ) OXPHOS complexes ( A - C ) and selected enzymes involved in the energy metabolism of control ( 143B , solid lines ) and ρ0 cells ( dashed lines ) . A , Complex ( C ) I , III and IV ; B , F1 , FO domain and inhibitor protein IF1 of complex V ; C , subunits SDHA , SDHB - D and assembly factor SDHAF2 of complex II ; D , oxoglutarate ( OGDH ; average of OGDHL , OGDH , DLST ) and pyruvate dehydrogenase complex ( PDH ; average of PDHA1 , PDHB , PDHX , DLAT ) ; E , subunits of GDP forming ( SUCLG2 ) and ADP forming ( SUCLA2 ) succinyl - CoA ligase ; F , urea cycle enzymes and glutaminase ( GLS ) . Plots and gene names are in corresponding colors .

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_LINE O O O O O O O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O O O O O O O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O O O O B-GENEPROD O B-SUBCELLULAR I-SUBCELLULAR O O O O B-GENEPROD O B-GENEPROD O O O O O B-GENEPROD O B-SUBCELLULAR I-SUBCELLULAR O O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O B-SUBCELLULAR I-SUBCELLULAR O O B-SUBCELLULAR O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O B-SMALL_MOLECULE O O B-GENEPROD O O B-SMALL_MOLECULE O O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O

(A-F) OXPHOS complexes (A-C) and selected enzymes involved in the energy metabolism of control (143B, solid lines) and ρ0 cells (dashed lines). A, Complex (C) I, III and IV; B, F1, FO domain and inhibitor protein IF1 of complex V; C, subunits SDHA, SDHB-D and assembly factor SDHAF2 of complex II; D, oxoglutarate (OGDH; average of OGDHL, OGDH, DLST) and pyruvate dehydrogenase complex (PDH; average of PDHA1, PDHB, PDHX, DLAT); E, subunits of GDP forming (SUCLG2) and ADP forming (SUCLA2) succinyl-CoA ligase; F, urea cycle enzymes and glutaminase (GLS). Plots and gene names are in corresponding colors.


( A - D ) Selected metabolite carriers of control ( 143B , solid lines , grey bars ) and ρ0 cells ( dashed lines , colored bars ) . A , isoforms of the nucleotide carrier ( ANT1 - 3 ) and phosphate carrier ( PiC ) ; B , pyruvate carrier ; C , dicarboxylate carriers ; D , mitoferrins ( MFRN1 - 2 ) and glycine transporter ( GlyC ) . Plots and gene names are in corresponding colors . Data are presented as mean ± SD ( n = 4 ) . * p < 0 . 05 , * * p < 0 . 01 ( Student ' s t - test ) .

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

(A-D) Selected metabolite carriers of control (143B, solid lines, grey bars) and ρ0 cells (dashed lines, colored bars). A, isoforms of the nucleotide carrier (ANT1-3) and phosphate carrier (PiC); B, pyruvate carrier; C, dicarboxylate carriers; D, mitoferrins (MFRN1-2) and glycine transporter (GlyC). Plots and gene names are in corresponding colors. Data are presented as mean ± SD (n=4). *p<0.05, **p<0.01 (Student's t-test).


( A - D ) Components of the mitochondrial protein import machinery and selected proteins related to this process of control ( 143B , solid lines , grey bars ) and ρ0 cells ( dashed lines , colored and dark - grey bars ) . A and B , components of the translocase of the outer membrane ( TOM ) machinery ( TOM avg . : average of TOMM40 , TOMM22 , TOMM5 , TOMM6 , TOMM7 ) ; C , components of the trans locase of the inner membrane ( TIM ) machinery ( TIM22 avg . : TIMM22 , AGK , TIMM10B ; TIM23 avg . : TIMM23 , TIMM17A , TIMM17B ) ; D , components of the TIM23 - associated TIMM21 assembly machinery ( left panel ) and its association with prohibitin ( PHB ) and components of the mitochondrial quality control system ( right panel ) . Plots and gene names are in corresponding colors , except for TIMM44 and TIMM50 ( no migration profiles shown ) . In ( A - C ) data are presented as mean ± SD ( n = 4 ) . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 ( Student ' s t - test ) .

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

(A-D) Components of the mitochondrial protein import machinery and selected proteins related to this process of control (143B, solid lines, grey bars) and ρ0 cells (dashed lines, colored and dark-grey bars). A and B, components of the translocase of the outer membrane (TOM) machinery (TOM avg.: average of TOMM40, TOMM22, TOMM5, TOMM6, TOMM7); C, components of the translocase of the inner membrane (TIM) machinery (TIM22 avg.: TIMM22, AGK, TIMM10B; TIM23 avg.: TIMM23, TIMM17A, TIMM17B); D, components of the TIM23-associated TIMM21 assembly machinery (left panel) and its association with prohibitin (PHB) and components of the mitochondrial quality control system (right panel). Plots and gene names are in corresponding colors, except for TIMM44 and TIMM50 (no migration profiles shown). In (A-C) data are presented as mean ± SD (n=4). *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 (Student's t-test).


Selected proteins and complexes involved in controlling mitochondrial morphology and fusion / fission machinery and related processes of control ( 143B , solid lines , grey bars ) and ρ0 cells ( dashed lines , colored bars ) . A , mitochondrial contact site and cristae organizing system ( MICOS : MIC60 , MIC19 , MIC25 ) and SAM complex ( SAM : SAMM50 , MTX2 , MTX3 ) together forming the MIB

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

Selected proteins and complexes involved in controlling mitochondrial morphology and fusion/fission machinery and related processes of control (143B, solid lines, grey bars) and ρ0 cells (dashed lines, colored bars). A, mitochondrial contact site and cristae organizing system (MICOS: MIC60, MIC19, MIC25) and SAM complex (SAM: SAMM50, MTX2, MTX3) together forming the MIB complex


Selected proteins and complexes involved in controlling mitochondrial morphology and fusion / fission machinery and related processes of control ( 143B , solid lines , grey bars ) and ρ0 cells ( dashed lines , colored bars ) . B , proteins involved in controlling mitochondrial morphology ( orange ) and dynamics ( teal ) ;

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

Selected proteins and complexes involved in controlling mitochondrial morphology and fusion/fission machinery and related processes of control (143B, solid lines, grey bars) and ρ0 cells (dashed lines, colored bars). B, proteins involved in controlling mitochondrial morphology (orange) and dynamics (teal);


Selected proteins and complexes involved in controlling mitochondrial morphology and fusion / fission machinery and related processes of control ( 143B , solid lines , grey bars ) and ρ0 cells ( dashed lines , colored bars ) . C , DNM1L / DRP1 and interacting proteins . For DNM1L , MTFR1 and ARL2BP , quantification of the entire migra tion profiles ( Overall ) and the range above 1 , 400 kDa only ( > 1 , 400 kDa ) are shown . Plots and gene names are in corresponding colors , except for ARL2 ( dark - grey ) not shown in the plot . Data are presented as mean ± SD ( n = 4 ) . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 ( Student ' s t - test ) .

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

Selected proteins and complexes involved in controlling mitochondrial morphology and fusion/fission machinery and related processes of control (143B, solid lines, grey bars) and ρ0 cells (dashed lines, colored bars). C, DNM1L/DRP1 and interacting proteins. For DNM1L, MTFR1 and ARL2BP, quantification of the entire migration profiles (Overall) and the range above 1,400 kDa only (>1,400 kDa) are shown. Plots and gene names are in corresponding colors, except for ARL2 (dark-grey) not shown in the plot. Data are presented as mean ± SD (n=4). *p<0.05, **p<0.01, ***p<0.001 (Student's t-test).


B Vero E6 cells were inoculated with SARS - CoV - 2 , fixed with paraformaldehyde after two days , stained with 30 nM of the indicated Alexa Fluor 488 ( AF488 ) - labeled anti - RBD VHH antibodies , and imaged by confocal laser scanning microscopy ( CLSM ) . C Immunofluorescence ( IF ) staining as in B , but with the indicated AF568 - labeled anti - S1ΔRBD VHH antibodies .

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

B Vero E6 cells were inoculated with SARS-CoV-2, fixed with paraformaldehyde after two days, stained with 30 nM of the indicated Alexa Fluor 488 (AF488)-labeled anti-RBD VHH antibodies, and imaged by confocal laser scanning microscopy (CLSM). C Immunofluorescence (IF) staining as in B, but with the indicated AF568-labeled anti-S1ΔRBD VHH antibodies.


BLI sensorgrams of intermediate VHHs A SARS - CoV - 2 RBD - loaded sensors were incubated with nanobodies at the indicated concentrations for 300 s , followed by dipping in assay buffer for 600 s . The response curves ( black ) were fitted using either a 1 : 1 ( for VHH - 72 ) or 2 : 1 ( for Re6B06 and Re9F06 ) binding models to obtain KDs . Fitted curves are shown in red . For Re6B06 and Re9F06 , some secondary binding with weaker affinity at high VHH concentrations was observed ( denoted as KD2 ) .

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

BLI sensorgrams of intermediate VHHs A SARS-CoV-2 RBD-loaded sensors were incubated with nanobodies at the indicated concentrations for 300 s, followed by dipping in assay buffer for 600 s. The response curves (black) were fitted using either a 1:1 (for VHH-72) or 2:1 (for Re6B06 and Re9F06) binding models to obtain KDs. Fitted curves are shown in red. For Re6B06 and Re9F06, some secondary binding with weaker affinity at high VHH concentrations was observed (denoted as KD2).


A Vero E6 cells were infected with SARS - CoV - 2 , pre - incubated with indicated concentrations of the neutralizing VHH - 72 Cells were fixed two days after inoculation , stained with sets of anti - RBD ( green ) and anti - S1ΔRBD ( red ) nanobodies and analyzed by CLSM . We used a cocktail of such fluorophore - labeled VHH antibodies to ensure that negative fluorescence readings truly indicated neutralization and thus absence of viral infection and not just masking of the IF epitope by the tested nanobody .

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

A Vero E6 cells were infected with SARS-CoV-2, pre-incubated with indicated concentrations of the neutralizing VHH-72 Cells were fixed two days after inoculation, stained with sets of anti-RBD (green) and anti-S1ΔRBD (red) nanobodies and analyzed by CLSM. We used a cocktail of such fluorophore-labeled VHH antibodies to ensure that negative fluorescence readings truly indicated neutralization and thus absence of viral infection and not just masking of the IF epitope by the tested nanobody.


B Vero E6 cells were infected as in A . Viral RNA in the culture supernatants was quantified by reverse transcription ( RT ) - qPCR . RNA signals are normalized to the respective minus - VHH controls ( 100 % ) . Note the log10 scale of the y - axis . " Inoculum " ( red dotted line ) marks the RNA level detected at the time of infection .

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B Vero E6 cells were infected as in A. Viral RNA in the culture supernatants was quantified by reverse transcription (RT)-qPCR. RNA signals are normalized to the respective minus-VHH controls (100%). Note the log10 scale of the y-axis. "Inoculum" (red dotted line) marks the RNA level detected at the time of infection.


Neutralization of SARS - CoV - 2 by the indicated VHH antibodies at nanomolar ( A and B ) or picomolar ( C and D ) concentrations . The neutralization experiment was performed ; n " indicates the number of independent biological

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Neutralization of SARS-CoV-2 by the indicated VHH antibodies at nanomolar (A and B) or picomolar (C and D) concentrations. The neutralization experiment was performed ;n" indicates the number of independent biological replicates


A HeLa cells were transiently transfected to express the SARS - CoV - 2 Spike protein . Following fixation , cells were stained for 1 hour with fluorophore - labeled Re10B10 ( 5 nM , green ) and Re7E02 ( 15 nM , red ) in the presence of the indicated unlabeled VHH competitors . Competitor ( 150 nM ) was added 20 min prior to the labeled nanobodies . The weakly binding competitors Re5A08 , Re9F06 , Re6B06 , and , Re6D06 were added as trimers ( see below ) . Cells were imaged by CLSM . For each competitor , the sequence class and the binding site on the RBD ( epitope 1 or 2 ) are indicated .

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A HeLa cells were transiently transfected to express the SARS-CoV-2 Spike protein. Following fixation, cells were stained for 1 hour with fluorophore-labeled Re10B10 (5 nM, green) and Re7E02 (15 nM, red) in the presence of the indicated unlabeled VHH competitors. Competitor (150 nM) was added 20 min prior to the labeled nanobodies. The weakly binding competitors Re5A08, Re9F06, Re6B06, and, Re6D06 were added as trimers (see below). Cells were imaged by CLSM. For each competitor, the sequence class and the binding site on the RBD (epitope 1 or 2) are indicated.


F Neutralization of SARS - CoV - 2 by the thermostable Re5D06 variants R15 and R28 at the indicated concentrations . The neutralization experiment was

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F Neutralization of SARS-CoV-2 by the thermostable Re5D06 variants R15 and R28 at the indicated concentrations. The neutralization experiment was performed


B Re6B06 monomer and trimer variants ( 20 nM ) were analyzed by BLI for RBD - binding , with 900 s association and 1500 s dissociation steps . Note the avidity effect of Re6B06 trimerization .

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B Re6B06 monomer and trimer variants (20 nM) were analyzed by BLI for RBD-binding, with 900 s association and 1500 s dissociation steps. Note the avidity effect of Re6B06 trimerization.


C Neutralization of SARS - CoV - 2 by the indicated VHH antibodies . The neutralization experiment was performed Note the gain in neutralization efficacy when a VHH is trimerized . Re6B06 , for example , is 30 000 - fold more potent when symmetry - matched with the virus Spike .

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C Neutralization of SARS-CoV-2 by the indicated VHH antibodies. The neutralization experiment was performed Note the gain in neutralization efficacy when a VHH is trimerized. Re6B06, for example, is 30 000-fold more potent when symmetry-matched with the virus Spike.


C Binding of nanobodies to the quadruple RBD mutant was monitored by BLI . Immobilized RBD was incubated with 100 nM of nanobodies for 450 s , followed by assay buffer for 900 s .

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C Binding of nanobodies to the quadruple RBD mutant was monitored by BLI. Immobilized RBD was incubated with 100 nM of nanobodies for 450 s, followed by assay buffer for 900 s.


A and B Neutralization of SARS - CoV - 2 B . 1 . 351 / Beta by the indicated VHH antibody constructs . The neutralization experiment was performed as described in Fig 3A , with the difference being that a mutant - optimized anti - RBD / S1 VHH cocktail was used for immunofluorescence staining Two titrations for the Re9F06 trimer are shown , corresponding to the standard ( upper row ) or a five - fold higher virus inoculum ( lower row ) that was used for even more stringent neutralization conditions .

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A and B Neutralization of SARS-CoV-2 B.1.351/Beta by the indicated VHH antibody constructs. The neutralization experiment was performed as described in Fig 3A, with the difference being that a mutant-optimized anti-RBD/S1 VHH cocktail was used for immunofluorescence staining Two titrations for the Re9F06 trimer are shown, corresponding to the standard (upper row) or a five-fold higher virus inoculum (lower row) that was used for even more stringent neutralization conditions.


C ) Time course analysis of triubiquitin ( 2 µM ) hydrolysis using 250 nM SARS2 PLpro , resolved on a Coomassie stained SDS - PAGE gel . Linkage specific cleavage of Lys48 - linked triubiquitin to di - and monoubiquitin resembles SARS PLpro activity ( Békés et al , 2015 ; 2016 ) . See Source Data for uncropped gels , and Appendix Fig S1B - D for gel - based cleavage quantification .

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C) Time course analysis of triubiquitin (2 µM) hydrolysis using 250 nM SARS2 PLpro, resolved on a Coomassie stained SDS-PAGE gel. Linkage specific cleavage of Lys48-linked triubiquitin to di- and monoubiquitin resembles SARS PLpro activity (Békés et al, 2015; 2016). See Source Data for uncropped gels, and Appendix Fig S1B-D for gel-based cleavage quantification.


B ) Fluorescence polarisation assays against ubiquitin - TAMRA and ISG15 - TAMRA using indicated SARS2 PLpro variants performed in technical triplicate and n = 2 for each mutant , and compared to wild - type PLpro as shown in Appendix Fig S1A . Catalytic efficiencies were calculated as described in Methods .

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B) Fluorescence polarisation assays against ubiquitin-TAMRA and ISG15-TAMRA using indicated SARS2 PLpro variants performed in technical triplicate and n=2 for each mutant, and compared to wild-type PLpro as shown in Appendix Fig S1A. Catalytic efficiencies were calculated as described in Methods.


C ) Gel based analysis of PLpro variant activity against Lys48 - triubiquitin and proISG15 . Experiments were performed in duplicate , see Fig EV3 and Source Data for uncropped gels .

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C) Gel based analysis of PLpro variant activity against Lys48-triubiquitin and proISG15. Experiments were performed in duplicate, see Fig EV3 and Source Data for uncropped gels.


C ) Calculated enzymatic efficiencies for K48 - diUb - TAMRA cleavage , followed by fluorescence polarisation with PLpro wild - type ( reproduced from Appendix Fig S1A ) , and PLpro F69S . ( Left ) Using the same concentration range of SARS2 PLpro , the F69S mutant activity could not be fitted ; ( middle ) a higher concentration recovered an activity slightly lower as compared to PLpro cleaving ubiquitin - TAMRA ( 45 vs . 86 M - 1s - 1 , compare Fig 1D , Appendix Fig S1A ) . ( Right ) A ~ 3 - fold lower efficiency for PLpro F69S cleaving ISG15 - TAMRA yields values similar to cleavage of ISG15CTD - TAMRA ( Appendix Fig S1A ) , suggesting that the S2 site contributes the difference in binding for the N - terminal Ubl - fold . Experiments for F69S were performed in technical triplicate and biological duplicate ; wild - type data is reproduced from experiments shown in Fig 1D , Appendix Fig S1A .

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C) Calculated enzymatic efficiencies for K48-diUb-TAMRA cleavage, followed by fluorescence polarisation with PLpro wild-type (reproduced from Appendix Fig S1A), and PLpro F69S. (Left) Using the same concentration range of SARS2 PLpro, the F69S mutant activity could not be fitted; (middle) a higher concentration recovered an activity slightly lower as compared to PLpro cleaving ubiquitin-TAMRA (45 vs. 86 M-1s-1, compare Fig 1D, Appendix Fig S1A). (Right) A ~3-fold lower efficiency for PLpro F69S cleaving ISG15-TAMRA yields values similar to cleavage of ISG15CTD-TAMRA (Appendix Fig S1A), suggesting that the S2 site contributes the difference in binding for the N-terminal Ubl-fold. Experiments for F69S were performed in technical triplicate and biological duplicate; wild-type data is reproduced from experiments shown in Fig 1D, Appendix Fig S1A.


D - F ) Gel based analysis showing hydrolysis time course of triubiquitin ( D ) , proISG15CTD ( E ) and proISG15 ( F ) using wild - type PLpro ( left , wild - type gels reproduced from Appendix Fig S1B - D to enable direct comparison ) or PLpro F69S ( right ) . PLpro F69S has a strong effect on triubiquitin hydrolysis ( D ) , no marked effect on hydrolysis of proISG15CTD ( E ) and reduces proISG15 cleavage to the same levels as proISG15CTD ( compare E , F ) , Experiments were performed in duplicate , see Source Data for uncropped gels .

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D-F) Gel based analysis showing hydrolysis time course of triubiquitin (D), proISG15CTD (E) and proISG15 (F) using wild-type PLpro (left, wild-type gels reproduced from Appendix Fig S1B-D to enable direct comparison) or PLpro F69S (right). PLpro F69S has a strong effect on triubiquitin hydrolysis (D), no marked effect on hydrolysis of proISG15CTD (E) and reduces proISG15 cleavage to the same levels as proISG15CTD (compare E, F), Experiments were performed in duplicate, see Source Data for uncropped gels.


B ) Hit compounds and compound rac5c ( see Fig 5 ) , were further assessed in 10 - point IC50 titrations using the Ub - Rhodamine assay , using a starting concentration of 100 µM serially diluted in 1 : 3 steps . Degree of inhibition is shown as a titration heat - map from dark ( full inhibition ) to light blue ( low / no inhibition ) . The catalytic domain of human USP21 ( Ye et al , 2011 ) was used as a counterscreen . Each PLpro hit compound showed either no activity in the titration analysis , or an identical inhibition profile against PLpro and USP21 , suggesting assay interference . Rac5c was specific for SARS2 PLpro and did not inhibit USP21 even at the highest concentration of 100 µM . IC50 assays were performed in technical triplicate in two independent experiments .

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B) Hit compounds and compound rac5c (see Fig 5), were further assessed in 10-point IC50 titrations using the Ub-Rhodamine assay, using a starting concentration of 100 µM serially diluted in 1:3 steps. Degree of inhibition is shown as a titration heat-map from dark (full inhibition) to light blue (low/no inhibition). The catalytic domain of human USP21 (Ye et al, 2011) was used as a counterscreen. Each PLpro hit compound showed either no activity in the titration analysis, or an identical inhibition profile against PLpro and USP21, suggesting assay interference. Rac5c was specific for SARS2 PLpro and did not inhibit USP21 even at the highest concentration of 100 µM. IC50 assays were performed in technical triplicate in two independent experiments.


C ) In vitro inhibition ( IC50 ) for rac5c inhibiting SARS2 PLpro . Experiments were performed using the HTS assay ( Fig 4 ) , in technical triplicate in three independent experiments . A geometric mean was used to determine IC50 .

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C) In vitro inhibition (IC50) for rac5c inhibiting SARS2 PLpro. Experiments were performed using the HTS assay (Fig 4), in technical triplicate in three independent experiments. A geometric mean was used to determine IC50.


D ) Full - length nsp3 was expressed from a C - terminally GFP - tagged vector in HEK293T cells and treated with increasing concentrations of rac5c for 24 h . GFP is released from the C - terminus , presumably by nsp3 protease activity . Nsp3 can be detected by a SARS / SARS2 PLpro antibody ( see Fig EV5E for antibody validation ) . Lysates were blotted for Lys48 - linked polyubiquitin with a linkage specific antibody ( K48 ) . Experiments were performed in duplicate with similar results . Also see Fig EV5F , G and Source Data for uncropped blots .

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D) Full-length nsp3 was expressed from a C-terminally GFP-tagged vector in HEK293T cells and treated with increasing concentrations of rac5c for 24 h. GFP is released from the C-terminus, presumably by nsp3 protease activity. Nsp3 can be detected by a SARS/SARS2 PLpro antibody (see Fig EV5E for antibody validation). Lysates were blotted for Lys48-linked polyubiquitin with a linkage specific antibody (K48). Experiments were performed in duplicate with similar results. Also see Fig EV5F, G and Source Data for uncropped blots.


B ) Reduction in SARS - CoV - 2 induced cytopathic effect with rac5c , Remdesivir ( RDV ) and hydroxychloroquine ( HCQ ) treatment . DMSO 0 . 3 % ( v / v ) was required to keep 33 µM rac5c in solution ( see Fig EV6A , B ) . Mean ( black line ) is provided for 18 samples in each group , representing 3 independent experiments with 6 biological replicates per experiment across the different concentrations of rac5c . HCQ data is pooled from 2 independent experiments and RDV from 1 experiment using 6 biological repeats . P values were calculated using a one - way ANOVA , with regular Dunnet ' s post - hoc test for multiple comparisons between treatment arms and infected / vehicle treated control using a single pooled variance .

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B) Reduction in SARS-CoV-2 induced cytopathic effect with rac5c, Remdesivir (RDV) and hydroxychloroquine (HCQ) treatment. DMSO 0.3% (v/v) was required to keep 33 µM rac5c in solution (see Fig EV6A, B). Mean (black line) is provided for 18 samples in each group, representing 3 independent experiments with 6 biological replicates per experiment across the different concentrations of rac5c. HCQ data is pooled from 2 independent experiments and RDV from 1 experiment using 6 biological repeats. P values were calculated using a one-way ANOVA, with regular Dunnet's post-hoc test for multiple comparisons between treatment arms and infected/vehicle treated control using a single pooled variance.


C ) TCID50 data , mean and SD , for one representative experiment from B with 6 technical replicates .

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C) TCID50 data, mean and SD, for one representative experiment from B with 6 technical replicates.


Tagged MTA proteins in heterozygously targeted cell lines were immunoprecipitated and subsequent western blots probed with antibodies indicated at right . Solid black triangles indicate the locations of untagged proteins and open triangles show the position of epitope - tagged proteins in the anti - Mta1 , Mta2 or Mta3 panels ; and a grey triangle shows the location of IgG bands in the anti - Mbd3 panel . 10 % of the input was loaded into " Input " lanes . Molecular weights in kDa are shown at left .

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Tagged MTA proteins in heterozygously targeted cell lines were immunoprecipitated and subsequent western blots probed with antibodies indicated at right. Solid black triangles indicate the locations of untagged proteins and open triangles show the position of epitope-tagged proteins in the anti-Mta1, Mta2 or Mta3 panels; and a grey triangle shows the location of IgG bands in the anti-Mbd3 panel. 10% of the input was loaded into "Input" lanes. Molecular weights in kDa are shown at left.


Proteins co - purifying with Mta1 , Mta2 or Mta3 in IP - mass spectrometry experiments in wild type cells ( top ) or Mbd3 - null cells ( bottom ) . Proteins showing significant enrichment with the bait protein are located outside the dotted lines . For all panels the protein being immunoprecipitated is indicated in red , the other MTA proteins in purple , and other NuRD components in blue . Each IP / Mass spec experiment set comprised three independent immunoprecipitations from one nuclear extract preparation .

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Proteins co-purifying with Mta1, Mta2 or Mta3 in IP-mass spectrometry experiments in wild type cells (top) or Mbd3-null cells (bottom). Proteins showing significant enrichment with the bait protein are located outside the dotted lines. For all panels the protein being immunoprecipitated is indicated in red, the other MTA proteins in purple, and other NuRD components in blue. Each IP/Mass spec experiment set comprised three independent immunoprecipitations from one nuclear extract preparation.


Relative enrichment of indicated proteins in MTA pulldowns from wild type ( WT ) or Mbd3 - null ( Mbd3KO ) ES cells , normalised to 2x MTA proteins . NuRD components comprising the remodelling subunit are labelled in blue , those comprising the deacetylase subunit in red . As MTA proteins are the bait in these experiments , they may be isolated more efficiently than their interaction partners . Error bars represent standard deviation from three ( Mta1 and Mta2 ) or six ( Mta3 ) replicate pulldowns .

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Relative enrichment of indicated proteins in MTA pulldowns from wild type (WT) or Mbd3-null (Mbd3KO) ES cells, normalised to 2x MTA proteins. NuRD components comprising the remodelling subunit are labelled in blue, those comprising the deacetylase subunit in red. As MTA proteins are the bait in these experiments, they may be isolated more efficiently than their interaction partners. Error bars represent standard deviation from three (Mta1 and Mta2) or six (Mta3) replicate pulldowns.


ChIP - seq peaks identified for Mta1 and Mta2 ( left ) , Mta1 and Mta3 ( middle ) or Mta2 and Mta3 ( right ) are plotted by enrichment for each protein . Peaks called for both proteins are indicated in the overlap .

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ChIP-seq peaks identified for Mta1 and Mta2 (left), Mta1 and Mta3 (middle) or Mta2 and Mta3 (right) are plotted by enrichment for each protein. Peaks called for both proteins are indicated in the overlap.


ChIP - seq enrichment for indicated proteins or histone modifications is plotted across different subsets of Mta - bound sites . Mta1 / 2 / 3 refers to peaks identified in all three ChIP - seq datasets , whereas " Mta1 Only " , " Mta2 Only " or " Mta3 Only " refer to peaks only called for that protein .

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ChIP-seq enrichment for indicated proteins or histone modifications is plotted across different subsets of Mta-bound sites. Mta1/2/3 refers to peaks identified in all three ChIP-seq datasets, whereas "Mta1 Only", "Mta2 Only" or "Mta3 Only" refer to peaks only called for that protein.


Average enrichment of density plots are plotted for different subsets of Mta ChIP - seq peaks .

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Average enrichment of density plots are plotted for different subsets of Mta ChIP-seq peaks.


Western blots of wild type ( Control ) , double or triple - null ES cell nuclear extract was probed with antibodies indicated at right . LaminB1 acts as a loading control . Approximate sizes are indicated in kDa .

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Western blots of wild type (Control), double or triple-null ES cell nuclear extract was probed with antibodies indicated at right. LaminB1 acts as a loading control. Approximate sizes are indicated in kDa.


Phase - contrast images of wild type or Mta123 ∆ ES cells in 2i + LIF conditions . Scale bars represent

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Phase-contrast images of wild type or Mta123∆ ES cells in 2i+LIF conditions. Scale bars represent 100µ


Western blots of anti - Chd4 immunoprecipitation of nuclear extract from indicated cell lines ( top ) probed with antibodies indicated at right . For the Gatad2b and Mbd3 blots , arrowheads indicate target proteins , while an asterisk marks a non - specific IgG bands . 10 % of the input was loaded into " Input " lanes . Approximate sizes are indicated in kDa .

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Western blots of anti-Chd4 immunoprecipitation of nuclear extract from indicated cell lines (top) probed with antibodies indicated at right. For the Gatad2b and Mbd3 blots, arrowheads indicate target proteins, while an asterisk marks a non-specific IgG bands. 10% of the input was loaded into "Input" lanes. Approximate sizes are indicated in kDa.


Western blot of a time course of Mta3 deletion in Mta1 ∆ Mta2 ∆ Mta3Flox / Flox : Cre - ER ( Mta12 ∆ 3F / F ) or Control ( Mta12 ∆ 3F / F without Cre - ER ) ES cells probed for indicated NuRD component proteins or RNA Polymerase II as a loading control ( αRNAPII ) . The time course is indicated at the top as Days + tamoxifen .

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Western blot of a time course of Mta3 deletion in Mta1∆Mta2∆Mta3Flox/Flox: Cre-ER (Mta12∆3F/F) or Control (Mta12∆3F/F without Cre-ER) ES cells probed for indicated NuRD component proteins or RNA Polymerase II as a loading control (αRNAPII). The time course is indicated at the top as Days + tamoxifen.


Western blot showing rescue of Mta123 ∆ ES cells by ectopic expression of Mta1 , Mta2 or Mta3 from a transgene ( TG ) . Total RNA Polymerase II acts as a loading control ( αRNAPII ) . For all western blots molecular weight is indicated at left in kDa .

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Western blot showing rescue of Mta123∆ ES cells by ectopic expression of Mta1, Mta2 or Mta3 from a transgene (TG). Total RNA Polymerase II acts as a loading control (αRNAPII). For all western blots molecular weight is indicated at left in kDa.


Comparison of gene expression in Mta123 ∆ ES cells and wild type ( WT ) ES cells . Each circle represents a gene : red indicates spike - in controls , blue indicates genes that are not differentially expressed to a significant degree , and green indicates differentially expressed genes ( 2404 increased , 1293 decreased ) defined with an adjusted p - value < 0 . 05 and a log2 fold - change > 1 . N = 3 for each genotype .

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

Comparison of gene expression in Mta123∆ ES cells and wild type (WT) ES cells. Each circle represents a gene: red indicates spike-in controls, blue indicates genes that are not differentially expressed to a significant degree, and green indicates differentially expressed genes (2404 increased, 1293 decreased) defined with an adjusted p-value < 0.05 and a log2 fold-change > 1. N=3 for each genotype.


Fold change in gene expression is plotted for different subsets of genes . All genes are plotted in black , while subsets of genes located nearest to ChIP - seq peaks for the indicated proteins + Chd4 and which show significant changes in expression compared to wild type cells are plotted in dashed coloured lines as indicated . The number of genes in each of the Mta categories are : all genes ( 32271 ) , all differentially expressed ( n = 3701 ) , Mta1 + 2 + 3 ( n = 1738 ) , Mta1 + 2 ( n = 1460 ) , Mta1 ( n = 1020 ) and Mta2 ( n = 924 ) .

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

Fold change in gene expression is plotted for different subsets of genes. All genes are plotted in black, while subsets of genes located nearest to ChIP-seq peaks for the indicated proteins +Chd4 and which show significant changes in expression compared to wild type cells are plotted in dashed coloured lines as indicated. The number of genes in each of the Mta categories are: all genes (32271), all differentially expressed (n=3701), Mta1+2+3 (n=1738), Mta1+2 (n=1460), Mta1 (n=1020) and Mta2 (n=924).


Principal component analysis of RNAseq data from ES cell lines of indicated genotypes in either self - renewing conditions ( 2i ) or after 48 hours in the absence of two inhibitors and LIF ( Diff48 ) . Each point represents a biological replicate .

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Principal component analysis of RNAseq data from ES cell lines of indicated genotypes in either self-renewing conditions (2i) or after 48 hours in the absence of two inhibitors and LIF (Diff48). Each point represents a biological replicate.


Genes associated with indicated GO terms plotted by fold change in expression in Mta123 ∆ ES cells ( x - axis ) or Mbd3 ∆ ES cells ( y - axis ) . Genes are coloured if they are differentially expressed ( log2 fold - change > 1 and adjusted p - value < 0 . 05 ) in either comparison as indicated . The dotted lines show the fold - change cut - off of 2 . GO - terms were identified using David v . 6 . 8 using a Benjamini score with a cutoff of 0 . 05 .

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

Genes associated with indicated GO terms plotted by fold change in expression in Mta123∆ ES cells (x-axis) or Mbd3∆ ES cells (y-axis). Genes are coloured if they are differentially expressed (log2 fold-change > 1 and adjusted p-value < 0.05) in either comparison as indicated. The dotted lines show the fold-change cut-off of 2. GO-terms were identified using David v.6.8 using a Benjamini score with a cutoff of 0.05.


Genes associated with core NuRD peaks , with an MTA - only peak , with a Chd4 peak and an MTA - peak but not an Mbd3 peak ( Chd4 + Mta - Mbd3 genes ) , or with an MTA - peak but not a Chd4 peak ( MTA - only genes ) were divided into quartiles based upon expression levels in wild type cells . Change in gene expression is plotted on the x - axis and calculated significance of the expression change on the y - axis . Those genes significantly misexpressed ( | log2FC | > 1 ; p ≤ 0 . 05 ) are indicated in red , genes showing no significant change are plotted in black . The percentage of genes within each quartile which is significantly misexpressed is indicated at the top of each plot .

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

Genes associated with core NuRD peaks, with an MTA-only peak, with a Chd4 peak and an MTA-peak but not an Mbd3 peak (Chd4+Mta-Mbd3 genes), or with an MTA-peak but not a Chd4 peak (MTA-only genes) were divided into quartiles based upon expression levels in wild type cells. Change in gene expression is plotted on the x-axis and calculated significance of the expression change on the y-axis. Those genes significantly misexpressed (|log2FC|>1; p≤0.05) are indicated in red, genes showing no significant change are plotted in black. The percentage of genes within each quartile which is significantly misexpressed is indicated at the top of each plot.


Phase contrast pictures of ES cells of indicated genotypes after 5 days in neuroectoderm differentiation conditions ( N2B27 ) . White arrowheads in the Mbd3 ∆ panel indicate pockets of ES - like cells . Scale bars represent 100 µm .

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Phase contrast pictures of ES cells of indicated genotypes after 5 days in neuroectoderm differentiation conditions (N2B27). White arrowheads in the Mbd3∆ panel indicate pockets of ES-like cells. Scale bars represent 100 µm.


Expression of indicated genes in the neuroectoderm differentiation time course was measured by RT - qPCR in Control ( green ) Mta123 ∆ ( magenta ) or Mbd3 ∆ ( blue ) ES cells . N ≥ 3 biological replicates , error bars indicate SEM .

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Expression of indicated genes in the neuroectoderm differentiation time course was measured by RT-qPCR in Control (green) Mta123∆ (magenta) or Mbd3∆ (blue) ES cells. N ≥ 3 biological replicates, error bars indicate SEM.


Phase contrast images of embryoid body outgrowths of indicated genotypes after 12 days in differentiation conditions ( N2B27 + 10 % serum ) . Scale bars represent 100µm .

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Phase contrast images of embryoid body outgrowths of indicated genotypes after 12 days in differentiation conditions (N2B27 + 10% serum). Scale bars represent 100µm.


Expression of indicated genes in embryoid body cultures over time was measured by RT -

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Expression of indicated genes in embryoid body cultures over time was measured by RT-qPCR


Comparison of expression data for wild type , Mbd3 ∆ or Mta123 ∆ ES cells in self - renewing ( 2i ) conditions or after 48 hours in differentiation conditions ( 48h N2B27 ) with mouse embryonic single cell RNA - seq data Larger shading encloses biological replicates , smaller circles represent individual cells .

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Comparison of expression data for wild type, Mbd3∆ or Mta123∆ ES cells in self-renewing (2i) conditions or after 48 hours in differentiation conditions (48h N2B27) with mouse embryonic single cell RNA-seq data Larger shading encloses biological replicates, smaller circles represent individual cells.


Composite images of representative chimaeric embryos made with control ( Mta1Flox / ∆ Mta2 + / + Mta3Flox / Flox ) or Mta123 ∆ ES cells . ES - derived cells express the Kusabira Orange fluorescent marker . Sox2 indicates epiblast cells and Cdx2 is expressed in trophectoderm cells . Arrows indicate examples of K - Orange expressing cells in the mutant embryos . Scale bars = 20µm .

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Composite images of representative chimaeric embryos made with control (Mta1Flox/∆Mta2+/+Mta3Flox/Flox) or Mta123∆ ES cells. ES-derived cells express the Kusabira Orange fluorescent marker. Sox2 indicates epiblast cells and Cdx2 is expressed in trophectoderm cells. Arrows indicate examples of K-Orange expressing cells in the mutant embryos. Scale bars = 20µm.


( Top ) Number of K - Orange expressing cells observed in chimaeric embryos obtained using control ES cells , Mta123 ∆ ES cells , or Mta123 ∆ ES cells in which Mta2 was reintroduced on a constitutively expressed transgene ( Mta123 ∆ + Mta2TG ) . P - values calculated using a two - tailed t - test . ( Middle ) Mean number of K - Orange cells per embryo separated by Sox2 expression . P - values calculated using a two - tailed t - test . ( Bottom ) Number of K - Orange and Caspase - 3 positive cells per embryo . P - values calculated using a one - tailed t - test : * P < 0 . 05 , * * * * P < 0 . 0001 , " ns " = not significant .

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(Top) Number of K-Orange expressing cells observed in chimaeric embryos obtained using control ES cells, Mta123∆ ES cells, or Mta123∆ ES cells in which Mta2 was reintroduced on a constitutively expressed transgene (Mta123∆+Mta2TG). P-values calculated using a two-tailed t-test. (Middle) Mean number of K-Orange cells per embryo separated by Sox2 expression. P-values calculated using a two-tailed t-test. (Bottom) Number of K-Orange and Caspase-3 positive cells per embryo. P-values calculated using a one-tailed t-test: *P < 0.05, ****P < 0.0001, "ns" = not significant.


RNAPII - RBP - RNA complex was immunoprecipitated by anti - RNAPII antibody or control antibody ( cont ) , and subjected to immunoblotting . The input lanes contained 0 . 4 % of lysates used in the immunoprecipitation experiments .

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RNAPII-RBP-RNA complex was immunoprecipitated by anti-RNAPII antibody or control antibody (cont), and subjected to immunoblotting. The input lanes contained 0.4% of lysates used in the immunoprecipitation experiments.


Generation of tRIP - seq libraries of RNAPII - RBP - RNA complexes . After UV crosslinking , protein - RNA complexes were immunoprecipitated with anti - RNAPII ( 1st IP ) . RNA of the isolated RNAPII - RBP - RNA complexes was partially digested with RNase on beads , and RBP - RNA complexes dissociated from the bead were further immunoprecipitated with anti - FUS or anti - U1C antibody bound to the other beads ( 2nd IP ) . The immunoprecipitated FUS - RNA or U1C - RNA complex was treated with TEX , and then subjected to tRIP - seq library generation . The bottom panels show PCR - amplification of tRIP - seq libraries generated from RNAPII - FUS - RNA complex ( left panel ) and RNAPII - U1C - RNA complex ( right panel ) .

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

Generation of tRIP-seq libraries of RNAPII-RBP-RNA complexes. After UV crosslinking, protein-RNA complexes were immunoprecipitated with anti-RNAPII (1st IP). RNA of the isolated RNAPII-RBP-RNA complexes was partially digested with RNase on beads, and RBP-RNA complexes dissociated from the bead were further immunoprecipitated with anti-FUS or anti-U1C antibody bound to the other beads (2nd IP). The immunoprecipitated FUS-RNA or U1C-RNA complex was treated with TEX, and then subjected to tRIP-seq library generation. The bottom panels show PCR-amplification of tRIP-seq libraries generated from RNAPII-FUS-RNA complex (left panel) and RNAPII-U1C-RNA complex (right panel).


PolyA - seq was performed using N2A cells to examine the effect of downregulation of FUS and U1 snRNP on polyA site usage . Cells were treated with siRNA against Fus ( siFus ) and / or U1 antisense morpholino ( U1AS ) . Control cells ( Cont ) were treated with both control siRNA and control antisense morpholino . A . Correlation of changes in polyA site usage between U1 - inhibition and Fus - silencing . Fold change in the RPM of a polyA site between siFus and Cont ( siFus / Cont ) was plotted against that between U1AS and Cont ( U1AS / Cont ) . Pearson ' s correlation coefficient ( r ) and the slope of the regression line are shown . B . Correlation of changes in polyA site usage between U1 - inhibition alone and the combination of Fus - silencing and U1 - inhibition . Fold change in the RPM of a polyA site between siFus + U1AS and Cont ( siFus + U1AS / Cont ) was plotted against that between U1AS and Cont ( U1AS / Cont ) . Pearson ' s correlation coefficient ( r ) and the slope of the regression line are shown . A slope of 1 . 0 indicates that Fus - silencing has no additive effect on U1 - inhibition .

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

PolyA-seq was performed using N2A cells to examine the effect of downregulation of FUS and U1 snRNP on polyA site usage. Cells were treated with siRNA against Fus (siFus) and/or U1 antisense morpholino (U1AS). Control cells (Cont) were treated with both control siRNA and control antisense morpholino. A. Correlation of changes in polyA site usage between U1-inhibition and Fus-silencing. Fold change in the RPM of a polyA site between siFus and Cont (siFus/Cont) was plotted against that between U1AS and Cont (U1AS/Cont). Pearson's correlation coefficient (r) and the slope of the regression line are shown. B. Correlation of changes in polyA site usage between U1-inhibition alone and the combination of Fus-silencing and U1-inhibition. Fold change in the RPM of a polyA site between siFus+U1AS and Cont (siFus+U1AS/Cont) was plotted against that between U1AS and Cont (U1AS/Cont). Pearson's correlation coefficient (r) and the slope of the regression line are shown. A slope of 1.0 indicates that Fus-silencing has no additive effect on U1-inhibition.


Localization of FUS in R495X _ N2A cells ( R495X ) , and wild - type N2A cells ( WT ) . Cells were fractionated to separate nucleus and cytoplasm . Equal proportion of cell extracts from nuclear and cytoplasmic fractions were immunoblotted with indicated antibodies .

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Localization of FUS in R495X_N2A cells (R495X), and wild-type N2A cells (WT). Cells were fractionated to separate nucleus and cytoplasm. Equal proportion of cell extracts from nuclear and cytoplasmic fractions were immunoblotted with indicated antibodies.


Interactions between FUS and U1 snRNP in the R495X _ N2A cells ( R495X ) and wild - type N2A cells ( WT ) . Total cell lysates were immunoprecipitated with anti - U1 - 70K antibody or a control antibody ( cont ) , and immunoblotted with indicated antibodies .

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Interactions between FUS and U1 snRNP in the R495X_N2A cells (R495X) and wild-type N2A cells (WT). Total cell lysates were immunoprecipitated with anti-U1-70K antibody or a control antibody (cont), and immunoblotted with indicated antibodies.


Effects of the R495X mutation on four categories of polyA sites . Four categories of polyA sites are comprised of ( 1 ) FUS - repressed ( rep ) / U1 - repressed sites , ( 2 ) FUS - activated ( act ) / U1 - repressed sites , ( 3 ) FUS - repressed / U1 - activated sites , and ( 4 ) FUS - activated / U1 - activated sites , according to Fig 4A . Fold change of each polyA site usage by the R495X mutation ( R495X / WT ) is shown in violin plots . Bars indicate the median and 95 % confidential intervals for each category . * * * p < 0 . 001 , * * * * p < 0 . 0001 , based on the Steel - Dwass test for comparing all 6 pairs of categories .

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

Effects of the R495X mutation on four categories of polyA sites. Four categories of polyA sites are comprised of (1) FUS-repressed (rep) /U1-repressed sites, (2) FUS-activated (act) /U1-repressed sites, (3) FUS-repressed/U1-activated sites, and (4) FUS-activated/U1-activated sites, according to Fig 4A. Fold change of each polyA site usage by the R495X mutation (R495X/WT) is shown in violin plots. Bars indicate the median and 95% confidential intervals for each category. *** p < 0.001, **** p < 0.0001, based on the Steel-Dwass test for comparing all 6 pairs of categories.


A . qPCR analysis of Ahr expression in B cell subsets purified from C57Bl / 6 mice . Ahr expression was normalized to Hprt1 . n = 3 - 5 independent experiments ; mean ± SD .

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A. qPCR analysis of Ahr expression in B cell subsets purified from C57Bl/6 mice. Ahr expression was normalized to Hprt1. n = 3-5 independent experiments; mean ± SD.


B . qPCR analysis of Ahr expression in splenic CD19 + cells isolated from C57Bl / 6 mice and cultured for 4h as indicated . Ahr expression was normalized to Hprt1 . Ahr expression among groups was normalized to Medium . n = 4 independent experiments ; mean ± SEM ; one - way ANOVA , Dunnett ' s multiple comparison test .

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B. qPCR analysis of Ahr expression in splenic CD19+ cells isolated from C57Bl/6 mice and cultured for 4h as indicated. Ahr expression was normalized to Hprt1. Ahr expression among groups was normalized to Medium. n = 4 independent experiments; mean ± SEM; one-way ANOVA, Dunnett's multiple comparison test.


C . qPCR analysis of Ahr expression in splenic CD19 + cells isolated from C57Bl / 6 mice and cultured for 4h with 20 ng / ml IL - 4 and / or 10 μg / ml α - IgM . Ahr expression was normalized to Hprt1 . Ahr expression among groups was normalized to Medium . n = 3 independent experiments ; mean ± SEM ; one - way ANOVA , Tukey ' s multiple comparison test .

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

C. qPCR analysis of Ahr expression in splenic CD19+ cells isolated from C57Bl/6 mice and cultured for 4h with 20 ng/ml IL-4 and/or 10 μg/ml α-IgM. Ahr expression was normalized to Hprt1. Ahr expression among groups was normalized to Medium. n = 3 independent experiments; mean ± SEM; one-way ANOVA, Tukey's multiple comparison test.


D . Western blot of protein extracts from splenic CD19 + cells isolated from C57Bl / 6 mice and cultured for 4h with 20 ng / ml IL - 4 and / or 10 μg / ml α - IgM . Values above the blots indicate AhR protein quantification obtained by densitometry , normalized to β - actin and compared to Medium sample . Sample A and B indicate 2 independent replicates .

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

D. Western blot of protein extracts from splenic CD19+ cells isolated from C57Bl/6 mice and cultured for 4h with 20 ng/ml IL-4 and/or 10 μg/ml α-IgM. Values above the blots indicate AhR protein quantification obtained by densitometry, normalized to β-actin and compared to Medium sample. Sample A and B indicate 2 independent replicates.


E . Quantification by densitometric analysis of the results shown in C . n = 2 independent experiments ; mean ± range .

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

E. Quantification by densitometric analysis of the results shown in C. n = 2 independent experiments; mean ± range.


F . qPCR analysis of Ahr expression in purified splenic B cell subsets isolated from C57Bl / 6 mice and cultured as indicated for 4h . Ahr expression was normalized to Hprt1 . n = 2 independent experiments ; mean ± range .

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

F. qPCR analysis of Ahr expression in purified splenic B cell subsets isolated from C57Bl/6 mice and cultured as indicated for 4h. Ahr expression was normalized to Hprt1. n = 2 independent experiments; mean ± range.


G . qPCR analysis of Ahr expression in splenic CD19 + cells isolated from C57Bl / 6 mice and cultured for the indicated time points with 20 ng / ml IL - 4 and / or 10 μg / ml α - IgM . Ahr expression was normalized to Hprt1 . Ahr expression among groups was normalized to Medium . n = 5 independent experiments ; mean ± SEM ; one - way ANOVA , Dunnett ' s multiple comparison test .

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

G. qPCR analysis of Ahr expression in splenic CD19+ cells isolated from C57Bl/6 mice and cultured for the indicated time points with 20 ng/ml IL-4 and/or 10 μg/ml α-IgM. Ahr expression was normalized to Hprt1. Ahr expression among groups was normalized to Medium. n = 5 independent experiments; mean ± SEM; one-way ANOVA, Dunnett's multiple comparison test.


A . Western blot of nuclear and cytoplasmic protein extracts from splenic CD19 + cells isolated from C57Bl / 6 mice and cultured with anti - IgM ( α - IgM ) for 4h , followed by the indicated treatment for 2h . Values above the blots indicate AhR protein quantification obtained by densitometry , normalized to Sam68 or Tubulin and compared to the DMSO - treated sample . Representative data of n = 3 independent experiments .

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

A. Western blot of nuclear and cytoplasmic protein extracts from splenic CD19+ cells isolated from C57Bl/6 mice and cultured with anti-IgM (α-IgM) for 4h, followed by the indicated treatment for 2h. Values above the blots indicate AhR protein quantification obtained by densitometry, normalized to Sam68 or Tubulin and compared to the DMSO-treated sample. Representative data of n = 3 independent experiments.


B . qPCR analysis of Cyp1a1 expression in splenic CD19 + cells isolated from C57Bl / 6 mice and cultured for 24h as indicated . Cyp1a1 expression was normalized to Hprt1 . Cyp1a1 expression among groups was normalized to Medium DMSO . n = 2 independent experiments ; mean ± range .

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

B. qPCR analysis of Cyp1a1 expression in splenic CD19+ cells isolated from C57Bl/6 mice and cultured for 24h as indicated. Cyp1a1 expression was normalized to Hprt1. Cyp1a1 expression among groups was normalized to Medium DMSO. n = 2 independent experiments; mean ± range.


C . Flow cytometry analysis of Cyp1a1 expression , reported by eYFP , in splenic CD19 + cells isolated from Cyp1a1Cre R26R eYFP mice and cultured for 72h as indicated . Representative data of n = 3 independent experiments .

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

C. Flow cytometry analysis of Cyp1a1 expression, reported by eYFP, in splenic CD19+ cells isolated from Cyp1a1Cre R26R eYFP mice and cultured for 72h as indicated. Representative data of n = 3 independent experiments.


D . qPCR analysis of Cyp1a1 expression in purified splenic B cell subsets isolated from C57Bl / 6 mice and cultured as indicated for 24h . Cyp1a1 expression was normalized to Hprt1 . n = 2 independent experiments ; mean ± range .

D. qPCR analysis of Cyp1a1 expression in purified splenic B cell subsets isolated from C57Bl/6 mice and cultured as indicated for 24h. Cyp1a1 expression was normalized to Hprt1. n = 2 independent experiments; mean ± range.


B . Representative flow cytometry analysis of Cyp1a1 expression , reported by eYFP , in CD45 . 1 + cells harvested from mice challenged as in A . Gating is indicated above the plots . Representative data of n = 3 independent experiments . GC cells : CD95 + GL - 7 + ; non - GC cells : CD95 - GL - 7 - . C , D . Summary of percentage of GC ( C ) and non - GC eYFP + ( D ) cells from flow cytometry analysis . n = 3 independent experiments ; lines indicate mean ; one - way ANOVA , Tukey ' s multiple comparison test .

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

B. Representative flow cytometry analysis of Cyp1a1 expression, reported by eYFP, in CD45.1+ cells harvested from mice challenged as in A. Gating is indicated above the plots. Representative data of n = 3 independent experiments. GC cells: CD95+ GL-7+; non-GC cells: CD95- GL-7-.C, D. Summary of percentage of GC (C) and non-GC eYFP+ (D) cells from flow cytometry analysis. n = 3 independent experiments; lines indicate mean; one-way ANOVA, Tukey's multiple comparison test.


A . Flow cytometry analysis of CTV dilution in splenic CD19 + cells sorted from non - immune Ahrfl / + mb1Cre + ( Ahr + / - B cells , solid grey ) and Ahrfl / - mb1Cre + ( Ahr - / - B cells , black ) mice stimulated for 72h as indicated . Treatment of Ahr + / - CD19 + cells with CH223191 is indicated in red . Representative data of n = 4 - 5 independent experiments . B - D . Flow cytometry analysis of expansion index ( B ) , % of divided cells ( C ) and replication index ( D ) of splenic CD19 + cells sorted from non - immune Ahrfl / + mb1Cre + ( black ) and Ahrfl / - mb1Cre + ( white ) mice stimulated for 72h as indicated . Treatment of Ahr + / - CD19 + cells with CH223191 is indicated in red . n = 4 - 5 independent experiments ; mean ± SEM ; 2 - way ANOVA , Sidak ' s multiple comparison test .

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

A. Flow cytometry analysis of CTV dilution in splenic CD19+ cells sorted from non-immune Ahrfl/+mb1Cre+ (Ahr+/- B cells, solid grey) and Ahrfl/-mb1Cre+ (Ahr-/- B cells, black) mice stimulated for 72h as indicated. Treatment of Ahr+/- CD19+ cells with CH223191 is indicated in red. Representative data of n = 4-5 independent experiments.B-D. Flow cytometry analysis of expansion index (B), % of divided cells (C) and replication index (D) of splenic CD19+ cells sorted from non-immune Ahrfl/+mb1Cre+ (black) and Ahrfl/-mb1Cre+ (white) mice stimulated for 72h as indicated. Treatment of Ahr+/- CD19+ cells with CH223191 is indicated in red. n = 4-5 independent experiments; mean ± SEM; 2-way ANOVA, Sidak's multiple comparison test.


E . Flow cytometry analysis of cell cycle distribution of splenic CD19 + cells purified from non - immune Ahrfl / + mb1Cre + ( black ) and Ahrfl / - mb1Cre + ( white ) mice stimulated for 48h with 5 µg / ml α - IgM . Data representative of n = 2 independent experiments ; lines indicate mean ; 2 - way ANOVA , Sidak ' s multiple comparison test .

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

E. Flow cytometry analysis of cell cycle distribution of splenic CD19+ cells purified from non-immune Ahrfl/+mb1Cre+ (black) and Ahrfl/-mb1Cre+ (white) mice stimulated for 48h with 5 µg/ml α-IgM. Data representative of n = 2 independent experiments; lines indicate mean; 2-way ANOVA, Sidak's multiple comparison test.


A , B . Flow cytometry analysis of distribution of CD45 . 1 + and CD45 . 2 + cells in indicated cell subsets purified from bone marrow ( A ) and lymph node ( B ) of sub lethally - irradiated Rag1 - / - mice 8 weeks after reconstitution with equal numbers of bone marrow cells from Ahr + / + ( CD45 . 1 + ) and Ahr - / - ( CD45 . 2 + ) mice . Dashed lines indicate 50 % level . Representative data of n = 2 independent experiments ; two - tailed paired t test .

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

A, B. Flow cytometry analysis of distribution of CD45.1+ and CD45.2+ cells in indicated cell subsets purified from bone marrow (A) and lymph node (B) of sub lethally-irradiated Rag1-/- mice 8 weeks after reconstitution with equal numbers of bone marrow cells from Ahr+/+ (CD45.1+) and Ahr-/- (CD45.2+) mice. Dashed lines indicate 50% level. Representative data of n = 2 independent experiments; two-tailed paired t test.


D . Flow cytometry analysis of distribution of CD45 . 1 + CD45 . 2 + and CD45 . 2 + cells harvested from host mice challenged as indicated above the dot plots . Representative data of n = 3 independent experiments .

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

D. Flow cytometry analysis of distribution of CD45.1+CD45.2+ and CD45.2+ cells harvested from host mice challenged as indicated above the dot plots. Representative data of n = 3 independent experiments.


E . Flow cytometry analysis of distribution of Ahr + / + CD45 . 1 + CD45 . 2 + ( black ) and Ahr - / - CD45 . 2 + ( white ) cells harvested from host mice . Indicated distribution is quantified relative to transferred cells . X symbols indicate SRBC - mock - treated control . Dashed line indicates 50 % threshold . Representative data of n = 3 independent experiments ; two - tailed paired t test .

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

E. Flow cytometry analysis of distribution of Ahr+/+ CD45.1+CD45.2+ (black) and Ahr-/- CD45.2+ (white) cells harvested from host mice. Indicated distribution is quantified relative to transferred cells. X symbols indicate SRBC-mock-treated control. Dashed line indicates 50% threshold. Representative data of n = 3 independent experiments; two-tailed paired t test.


F , G . Flow cytometry analysis of distribution ( F ) and cell numbers ( G ) of IgG1 + and IgG1 - HEL3x - binding Ahr + / + CD45 . 1 + CD45 . 2 + ( black ) and Ahr - / - CD45 . 2 + ( white ) cells harvested from host mice . Representative data of n = 2 independent experiments ; two - tailed paired t test .

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

F, G. Flow cytometry analysis of distribution (F) and cell numbers (G) of IgG1+ and IgG1- HEL3x-binding Ahr+/+ CD45.1+CD45.2+ (black) and Ahr-/- CD45.2+ (white) cells harvested from host mice. Representative data of n = 2 independent experiments; two-tailed paired t test.


A , B . Tables showing top 20 down - regulated ( A ) genes in Ahr - / - B cells as compared to Ahr + / + cells , after analysis by RNA sequencing . B cells were activated with 10 µg / ml α - IgM + 20 ng / ml IL - 4 for 8h ; 250 nM FICZ was added to the culture during the last 4h . The average read counts are directly proportional to the extent of expression of a given gene in Ahr + / + or Ahr - / - cells . Data from n = 3 mice per group . Average read counts for the housekeeping gene Hprt1 in both Ahr + / + and Ahr - / - B cells are indicated below the table in figure 6A .

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

A, B. Tables showing top 20 down-regulated (A) genes in Ahr-/- B cells as compared to Ahr+/+ cells, after analysis by RNA sequencing. B cells were activated with 10 µg/ml α-IgM + 20 ng/ml IL-4 for 8h; 250 nM FICZ was added to the culture during the last 4h. The average read counts are directly proportional to the extent of expression of a given gene in Ahr+/+ or Ahr-/- cells. Data from n = 3 mice per group. Average read counts for the housekeeping gene Hprt1 in both Ahr+/+ and Ahr-/- B cells are indicated below the table in figure 6A.


A , B . Tables showing top up - regulated ( B ) genes in Ahr - / - B cells as compared to Ahr + / + cells , after analysis by RNA sequencing . B cells were activated with 10 µg / ml α - IgM + 20 ng / ml IL - 4 for 8h ; 250 nM FICZ was added to the culture during the last 4h . The average read counts are directly proportional to the extent of expression of a given gene in Ahr + / + or Ahr - / - cells . Data from n = 3 mice per group . Average read counts for the housekeeping gene Hprt1 in both Ahr + / + and Ahr - / - B cells are indicated below the table in figure 6A .

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

A, B. Tables showing top up-regulated (B) genes in Ahr-/- B cells as compared to Ahr+/+ cells, after analysis by RNA sequencing. B cells were activated with 10 µg/ml α-IgM + 20 ng/ml IL-4 for 8h; 250 nM FICZ was added to the culture during the last 4h. The average read counts are directly proportional to the extent of expression of a given gene in Ahr+/+ or Ahr-/- cells. Data from n = 3 mice per group. Average read counts for the housekeeping gene Hprt1 in both Ahr+/+ and Ahr-/- B cells are indicated below the table in figure 6A.


C . qPCR analysis of Ccno expression in splenic CD19 + cells isolated from non - immune Ahrfl / + mb1Cre + ( Ahr + / - B cells , black ) and Ahrfl / - mb1Cre + ( Ahr - / - B cells , white ) mice , stimulated with 10 µg / ml anti - IgM for the indicated time points . Ccno expression was normalized to Hprt1 . n = 3 independent experiments ; mean ± SEM ; two - way ANOVA , Sidak ' s multiple comparison test .

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

C. qPCR analysis of Ccno expression in splenic CD19+ cells isolated from non-immune Ahrfl/+mb1Cre+ (Ahr+/- B cells, black) and Ahrfl/-mb1Cre+ (Ahr-/- B cells, white) mice, stimulated with 10 µg/ml anti-IgM for the indicated time points. Ccno expression was normalized to Hprt1. n = 3 independent experiments; mean ± SEM; two-way ANOVA, Sidak's multiple comparison test.


D . qPCR analysis of Ccno expression in splenic CD19 + cells isolated from C57Bl / 6 mice and cultured for 8h with 20 ng / ml IL - 4 and / or 10 μg / ml α - IgM . Ccno expression was normalized to Hprt1 . n = 3 independent experiments ; mean ± SEM ; one - way ANOVA , Tukey ' s multiple comparison test .

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

D. qPCR analysis of Ccno expression in splenic CD19+ cells isolated from C57Bl/6 mice and cultured for 8h with 20 ng/ml IL-4 and/or 10 μg/ml α-IgM. Ccno expression was normalized to Hprt1. n = 3 independent experiments; mean ± SEM; one-way ANOVA, Tukey's multiple comparison test.


E . qPCR analysis of Ccno expression in splenic CD19 + cells isolated from C57Bl / 6 mice and cultured for the indicated time points with 20 ng / ml IL - 4 and / or 10 μg / ml α - IgM in presence or absence of 250 nM FICZ or 1 μM 3 - MC . Ccno expression was normalized to Hprt1 . n = 3 independent experiments ; mean ± SEM ; one - way ANOVA , Tukey ' s multiple comparison test .

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

E. qPCR analysis of Ccno expression in splenic CD19+ cells isolated from C57Bl/6 mice and cultured for the indicated time points with 20 ng/ml IL-4 and/or 10 μg/ml α-IgM in presence or absence of 250 nM FICZ or 1 μM 3-MC. Ccno expression was normalized to Hprt1. n = 3 independent experiments; mean ± SEM; one-way ANOVA, Tukey's multiple comparison test.


F . Chromatin Immunoprecipitation ( ChIP ) analysis of AhR interaction with the Ccno promoter and an irrelevant region ( - 3 . 3 Kb from Ccno transcription starting site ) in Ahr + / + ( black ) and Ahr - / - ( white ) B cells 5h after activation with 10 µg / ml α - IgM + 20 ng / ml IL - 4 . 250 nM FICZ was added in the last hour of culture . Representative data of n = 2 independent experiments ; mean ± SEM ; two - way ANOVA , Sidak ' s multiple comparison test .

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

F. Chromatin Immunoprecipitation (ChIP) analysis of AhR interaction with the Ccno promoter and an irrelevant region (-3.3 Kb from Ccno transcription starting site) in Ahr+/+ (black) and Ahr-/- (white) B cells 5h after activation with 10 µg/ml α-IgM + 20 ng/ml IL-4. 250 nM FICZ was added in the last hour of culture. Representative data of n = 2 independent experiments; mean ± SEM; two-way ANOVA, Sidak's multiple comparison test.


A Tissue distribution of RIPK2 determined by anti - FLAG immunoprecipitation . Organ homogenates from WT ( wild - type ) and KI ( homozygous FLAG - RIPK2 ) mice were subjected to anti - FLAG immunoprecipitation and immunoblotting .

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

A Tissue distribution of RIPK2 determined by anti-FLAG immunoprecipitation. Organ homogenates from WT (wild-type) and KI (homozygous FLAG-RIPK2) mice were subjected to anti-FLAG immunoprecipitation and immunoblotting.


B Inflammatory signaling in wild - type ( WT / WT ) , RIPK2 CRISPR KO ( KO / KO ) and FLAG - RIPK2 heterozygous ( WT / KI ) and homozygous ( KI / KI ) BMDMs . BMDMs were primed with IFNy , stimulated with MDP for indicated times and analysed by immunoblotting .

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

B Inflammatory signaling in wild-type (WT/WT), RIPK2 CRISPR KO (KO/KO) and FLAG-RIPK2 heterozygous (WT/KI) and homozygous (KI/KI) BMDMs. BMDMs were primed with IFNy, stimulated with MDP for indicated times and analysed by immunoblotting.


C Cytokine production of RIPK2 CRISPR KO ( KO ) , wild - type ( WT ) and FLAG - RIPK2 heterozygous ( WT / KI ) and homozygous ( KI / KI ) BMDMs in response to MDP . BMDMs were left untreated or treated with IFNy or IFNy and MDP over - night and cytokines were measured by ELISA . N = 5 - 8 mice . Shown is average ± SEM . ns = P > 0 . 05 ; * * = P ≤ 0 . 01 ; * * * * = P ≤ 0 . 0001 ; two - way ANOVA .

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

C Cytokine production of RIPK2 CRISPR KO (KO), wild-type (WT) and FLAG-RIPK2 heterozygous (WT/KI) and homozygous (KI/KI) BMDMs in response to MDP. BMDMs were left untreated or treated with IFNy or IFNy and MDP over-night and cytokines were measured by ELISA. N = 5-8 mice. Shown is average ± SEM. ns = P > 0.05; ** = P ≤ 0.01; **** = P ≤ 0.0001; two-way ANOVA.


D Serum cytokines in RIPK2 CRISPR KO ( KO ) , wild - type ( WT ) and FLAG - RIPK2 heterozygous ( WT / KI ) or homozygous ( KI / KI ) mice after i . p . MDP administration . Mice were injected i . p . with PBS or MDP , sacrificed after 4h and serum cytokines were measured by ELISA . N = 3 - 6 mice . Shown is average ± SEM . ns = P > 0 . 05 ; * * = P ≤ 0 . 01 ; two - way ANOVA .

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

D Serum cytokines in RIPK2 CRISPR KO (KO), wild-type (WT) and FLAG-RIPK2 heterozygous (WT/KI) or homozygous (KI/KI) mice after i.p. MDP administration. Mice were injected i.p. with PBS or MDP, sacrificed after 4h and serum cytokines were measured by ELISA. N = 3-6 mice. Shown is average ± SEM. ns = P > 0.05; ** = P ≤ 0.01; two-way ANOVA.


A Two step enrichment to isolate ubiquitinated RIPK2 from BMDMs . Protein lysates from FLAG - RIPK2 BMDMs ( A ) were sequentially subjected to ubiquitin enrichment ( UBA , B ) and FLAG pulldown ( C ) prior to protein elution and subsequent mass spectrometry analysis .

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

A Two step enrichment to isolate ubiquitinated RIPK2 from BMDMs. Protein lysates from FLAG-RIPK2 BMDMs (A) were sequentially subjected to ubiquitin enrichment (UBA, B) and FLAG pulldown (C) prior to protein elution and subsequent mass spectrometry analysis.