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

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

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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 indicated concentrations for 300 s , followed by dipping in buffer for 600 s . response curves ( black ) were fitted using either a 1 : 1 ( for VHH - 72 ) or 2 : 1 ( for Re6B06 Re9F06 ) binding models to obtain KDs . Fitted curves are shown in red . For Re6B06 Re9F06 , some secondary binding with weaker affinity at high VHH concentrations was observed ( denoted as KD2 ) .

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

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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 were infected as in A . Viral in culture supernatants was quantified by reverse transcription ( RT ) - qPCR . signals are normalized to respective minus - VHH controls ( 100 % ) . Note log10 scale of y - axis . " Inoculum " ( red dotted ) marks level detected at 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 indicated VHH antibodies at nanomolar ( A B ) or picomolar ( C D ) concentrations . neutralization experiment was performed ; n " indicates 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 were transiently transfected to express SARS - CoV - 2 Spike . Following fixation , were stained for 1 hour with fluorophore - labeled Re10B10 ( 5 nM , green ) Re7E02 ( 15 nM , red ) in presence of indicated unlabeled VHH competitors . Competitor ( 150 nM ) was added 20 min prior to labeled nanobodies . weakly binding competitors Re5A08 , Re9F06 , Re6B06 , , Re6D06 were added as trimers ( see below ) . were imaged by CLSM . For each competitor , class binding site on 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 thermostable Re5D06 variants R15 R28 at indicated concentrations . 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 trimer variants ( 20 nM ) were analyzed by BLI for RBD - binding , with 900 s association 1500 s dissociation steps . Note 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 indicated VHH antibodies . neutralization experiment was performed Note gain in neutralization efficacy when a VHH is trimerized . Re6B06 , for example , is 30 000 - fold more potent when symmetry - matched with 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 quadruple RBD mutant was monitored by BLI . Immobilized RBD was incubated with 100 nM of nanobodies for 450 s , followed by 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 B Neutralization of SARS - CoV - 2 B . 1 . 351 / Beta by indicated VHH antibody constructs . neutralization experiment was performed as described in Fig 3A , with difference being that a mutant - optimized anti - RBD / S1 VHH cocktail was used for immunofluorescence Two titrations for Re9F06 trimer are shown , corresponding to standard ( upper row ) or a five - fold higher 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 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 - monoubiquitin resembles SARS PLpro activity ( Békés et al , 2015 ; 2016 ) . See Source Data for uncropped gels , Appendix Fig S1B - D for gel - cleavage .

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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 ) polarisation assays against ubiquitin - TAMRA ISG15 - TAMRA using indicated SARS2 PLpro variants performed in technical triplicate n = 2 for each mutant , 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 of PLpro variant activity against Lys48 - triubiquitin proISG15 . Experiments were performed in duplicate , see Fig EV3 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 polarisation with PLpro wild - type ( reproduced from Appendix Fig S1A ) , PLpro F69S . ( Left ) Using same range of SARS2 PLpro , F69S mutant activity could not be fitted ; ( middle ) a higher 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 S2 site contributes difference in binding for N - terminal Ubl - fold . Experiments for F69S were performed in technical triplicate 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 showing hydrolysis time course of triubiquitin ( D ) , proISG15CTD ( E ) 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 ) reduces proISG15 cleavage to same 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 compound rac5c ( see Fig 5 ) , were further assessed in 10 - point IC50 titrations using Ub - Rhodamine , using a starting of 100 µM serially diluted in 1 : 3 steps . Degree of inhibition is shown as a titration heat - map from ( full inhibition ) to blue ( low / no inhibition ) . catalytic domain of human USP21 ( Ye et al , 2011 ) was used as a counterscreen . Each PLpro hit compound showed either no activity in titration , or an identical inhibition profile against PLpro USP21 , suggesting interference . Rac5c was specific for SARS2 PLpro did not inhibit USP21 even at highest 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 HTS ( 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 - nsp3 was expressed from a C - terminally GFP - tagged vector in HEK293T treated with increasing concentrations of rac5c for 24 h . GFP is released from 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 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 ) hydroxychloroquine ( HCQ ) treatment . DMSO 0 . 3 % ( v / v ) was required to keep 33 µM rac5c in solution ( see Fig EV6A , B ) . Mean ( black ) is provided for 18 samples in each group , representing 3 independent experiments with 6 biological replicates per experiment across different concentrations of rac5c . HCQ data is pooled from 2 independent experiments RDV from 1 experiment using 6 biological repeats . P values were calculated using a one - way ANOVA , with regular Dunnet ' s post - hoc for multiple comparisons between treatment arms 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 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 in heterozygously targeted lines were immunoprecipitated subsequent western blots probed with antibodies indicated at right . Solid black triangles indicate locations of untagged open triangles show position of epitope - tagged in anti - Mta1 , Mta2 or Mta3 panels ; a grey triangle shows location of IgG bands in anti - Mbd3 panel . 10 % of 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.


co - purifying with Mta1 , Mta2 or Mta3 in IP - mass spectrometry experiments in wild type ( top ) or Mbd3 - null ( bottom ) . showing significant enrichment with bait are located outside dotted lines . For panels being immunoprecipitated is indicated in red , other MTA in purple , other NuRD components in blue . Each IP / Mass spec experiment set comprised three independent immunoprecipitations from one 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 in MTA pulldowns from wild type ( WT ) or Mbd3 - null ( Mbd3KO ) ES , normalised to 2x MTA . NuRD components comprising remodelling subunit are labelled in blue , those comprising deacetylase subunit in red . As MTA are bait in these experiments , they may be isolated more efficiently than their interaction partners . Error bars represent standard deviation from three ( Mta1 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 Mta2 ( left ) , Mta1 Mta3 ( middle ) or Mta2 Mta3 ( right ) are plotted by enrichment for each . Peaks called for both are indicated in 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 or histone modifications is plotted across different subsets of Mta - bound sites . Mta1 / 2 / 3 refers to peaks identified in three ChIP - seq datasets , whereas " Mta1 Only " , " Mta2 Only " or " Mta3 Only " refer to peaks only called for that .

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

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 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 of wild type or Mta123 ∆ ES 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 extract from indicated lines ( top ) probed with antibodies indicated at right . For Gatad2b Mbd3 blots , arrowheads indicate target , while an asterisk marks a non - specific IgG bands . 10 % of 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 probed for indicated NuRD component or Polymerase II as a loading control ( αRNAPII ) . time course is indicated at 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 by ectopic expression of Mta1 , Mta2 or Mta3 from a transgene ( TG ) . Total Polymerase II acts as a loading control ( αRNAPII ) . For western blots molecular 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 wild type ( WT ) ES . Each circle represents a gene : red indicates spike - in controls , blue indicates genes that are not differentially expressed to a significant degree , green indicates differentially expressed genes ( 2404 increased , 1293 decreased ) defined with an adjusted p - value < 0 . 05 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 B-CELL_TYPE 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 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

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 . genes are plotted in black , while subsets of genes located nearest to ChIP - seq peaks for indicated + Chd4 which show significant changes in expression compared to wild type are plotted in dashed coloured lines as indicated . number of genes in each of Mta categories are : genes ( 32271 ) , differentially expressed ( n = 3701 ) , Mta1 + 2 + 3 ( n = 1738 ) , Mta1 + 2 ( n = 1460 ) , Mta1 ( n = 1020 ) 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 B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O B-GENEPROD 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 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 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 of RNAseq data from ES lines of indicated genotypes in either self - renewing conditions ( 2i ) or after 48 hours in absence of two inhibitors 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 ( x - axis ) or Mbd3 ∆ ES ( y - axis ) . Genes are coloured if they are differentially expressed ( log2 fold - change > 1 adjusted p - value < 0 . 05 ) in either comparison as indicated . dotted lines show 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 .

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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 NuRD peaks , with an MTA - only peak , with a Chd4 peak 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 upon expression in wild type . Change in gene expression is plotted on x - axis calculated significance of expression change on y - axis . Those genes significantly misexpressed ( | log2FC | > 1 ; p ≤ 0 . 05 ) are indicated in red , genes showing no significant change are plotted in black . of genes within each quartile which is significantly misexpressed is indicated at top of each plot .

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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 of indicated genotypes after 5 days in neuroectoderm differentiation conditions ( N2B27 ) . arrowheads in Mbd3 ∆ panel indicate pockets of ES - like . 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 neuroectoderm differentiation time course was measured by RT - qPCR in Control ( green ) Mta123 ∆ ( magenta ) or Mbd3 ∆ ( blue ) ES . N ≥ 3 biological replicates , error bars indicate .

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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 of embryoid 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 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 in self - renewing ( 2i ) conditions or after 48 hours in differentiation conditions ( 48h N2B27 ) with mouse embryonic single - seq data Larger shading encloses biological replicates , smaller circles represent .

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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 of representative chimaeric made with control ( Mta1Flox / ∆ Mta2 + / + Mta3Flox / Flox ) or Mta123 ∆ ES . ES - derived express Kusabira Orange marker . Sox2 indicates epiblast Cdx2 is expressed in trophectoderm . Arrows indicate examples of K - Orange expressing in mutant . 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 observed in chimaeric obtained using control ES , Mta123 ∆ ES , or Mta123 ∆ ES in which Mta2 was reintroduced on a constitutively expressed transgene ( Mta123 ∆ + Mta2TG ) . P - values calculated using a two - t - . ( Middle ) Mean number of K - Orange per separated by Sox2 expression . P - values calculated using a two - t - . ( Bottom ) Number of K - Orange Caspase - 3 positive per . P - values calculated using a one - t - : * P < 0 . 05 , * * * * P < 0 . 0001 , " ns " = not significant .

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

(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 - was immunoprecipitated by anti - RNAPII antibody or control antibody ( cont ) , subjected to immunoblotting . input lanes contained 0 . 4 % of lysates used in 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 - complexes . After UV crosslinking , - complexes were immunoprecipitated with anti - RNAPII ( 1st IP ) . of isolated RNAPII - RBP - complexes was partially digested with RNase on beads , RBP - complexes dissociated from bead were further immunoprecipitated with anti - FUS or anti - U1C antibody bound to other beads ( 2nd IP ) . immunoprecipitated FUS - or U1C - was treated with TEX , then subjected to tRIP - seq library generation . bottom panels show PCR - amplification of tRIP - seq libraries generated from RNAPII - FUS - ( left panel ) RNAPII - U1C - ( right panel ) .

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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 to examine effect of downregulation of FUS U1 snRNP on polyA site usage . were treated with siRNA against Fus ( siFus ) / or U1 antisense morpholino ( U1AS ) . Control ( Cont ) were treated with both control siRNA control antisense morpholino . A . Correlation of changes in polyA site usage between U1 - inhibition Fus - silencing . Fold change in RPM of a polyA site between siFus Cont ( siFus / Cont ) was plotted against that between U1AS Cont ( U1AS / Cont ) . Pearson ' s correlation coefficient ( r ) slope of regression are shown . B . Correlation of changes in polyA site usage between U1 - inhibition alone combination of Fus - silencing U1 - inhibition . Fold change in RPM of a polyA site between siFus + U1AS Cont ( siFus + U1AS / Cont ) was plotted against that between U1AS Cont ( U1AS / Cont ) . Pearson ' s correlation coefficient ( r ) slope of regression 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 B-GENEPROD I-GENEPROD 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 O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O B-GENEPROD O O O O 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 B-GENEPROD O O B-GENEPROD 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 B-GENEPROD O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O 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 ( R495X ) , wild - type N2A ( WT ) . were fractionated to separate nucleus cytoplasm . Equal proportion of extracts from 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 U1 snRNP in R495X _ N2A ( R495X ) wild - type N2A ( WT ) . Total lysates were immunoprecipitated with anti - U1 - 70K antibody or a control antibody ( cont ) , 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 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 , ( 4 ) FUS - activated / U1 - activated sites , according to Fig 4A . Fold change of each polyA site usage by R495X mutation ( R495X / WT ) is shown in violin plots . Bars indicate median 95 % confidential intervals for each category . * * * p < 0 . 001 , * * * * p < 0 . 0001 , on Steel - Dwass for comparing 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 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 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

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

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

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

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

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 of Ahr expression in splenic CD19 + isolated from C57Bl / 6 mice cultured for 4h with 20 ng / ml IL - 4 / 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 ± ; one - way ANOVA , Tukey ' s multiple comparison .

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

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

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

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

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


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

O O B-EXP_ASSAY O B-GENEPROD O O O B-TISSUE B-CELL_TYPE O O O B-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM 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 of Ahr expression in splenic CD19 + isolated from C57Bl / 6 mice cultured for indicated time points with 20 ng / ml IL - 4 / 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 ± ; one - way ANOVA , Dunnett ' s multiple comparison .

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

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

O O B-EXP_ASSAY I-EXP_ASSAY O O O B-TISSUE B-GENEPROD O O O B-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM 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 B-GENEPROD O O O O O O B-GENEPROD O B-GENEPROD 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 of Cyp1a1 expression in splenic CD19 + isolated from C57Bl / 6 mice 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 B-GENEPROD O O B-TISSUE B-GENEPROD O O O B-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM 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 of Cyp1a1 expression , reported by eYFP , in splenic CD19 + isolated from Cyp1a1Cre R26R eYFP mice cultured for 72h as indicated . Representative data of n = 3 independent experiments .

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

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 of Cyp1a1 expression in purified splenic B subsets isolated from C57Bl / 6 mice 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 of Cyp1a1 expression , reported by eYFP , in CD45 . 1 + harvested from mice challenged as in A . Gating is indicated above plots . Representative data of n = 3 independent experiments . GC : CD95 + GL - 7 + ; non - GC : CD95 - GL - 7 - . C , D . Summary of of GC ( C ) non - GC eYFP + ( D ) from flow cytometry . n = 3 independent experiments ; lines indicate mean ; one - way ANOVA , Tukey ' s multiple comparison .

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

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

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 of cycle distribution of splenic CD19 + purified from non - immune Ahrfl / + mb1Cre + ( black ) Ahrfl / - mb1Cre + ( ) 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 .

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

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

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

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 of distribution of CD45 . 1 + CD45 . 2 + CD45 . 2 + harvested from host mice challenged as indicated above dot plots . Representative data of n = 3 independent experiments .

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

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

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 of distribution ( F ) numbers ( G ) of IgG1 + IgG1 - HEL3x - binding Ahr + / + CD45 . 1 + CD45 . 2 + ( black ) Ahr - / - CD45 . 2 + ( ) harvested from host mice . Representative data of n = 2 independent experiments ; two - paired t .

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

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 as compared to Ahr + / + , after by sequencing . B were activated with 10 µg / ml α - IgM + 20 ng / ml IL - 4 for 8h ; 250 nM FICZ was added to culture during last 4h . average read are directly proportional to extent of expression of a given gene in Ahr + / + or Ahr - / - . Data from n = 3 mice per group . Average read for housekeeping gene Hprt1 in both Ahr + / + Ahr - / - B are indicated below 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 O O O B-GENEPROD O O O O O O B-EXP_ASSAY O B-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 B-GENEPROD O O O O B-GENEPROD O O O O O O O O O B-ORGANISM O O O O O O O O B-GENEPROD O O B-GENEPROD O O O B-GENEPROD O O O B-CELL_TYPE 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 as compared to Ahr + / + , after by sequencing . B were activated with 10 µg / ml α - IgM + 20 ng / ml IL - 4 for 8h ; 250 nM FICZ was added to culture during last 4h . average read are directly proportional to extent of expression of a given gene in Ahr + / + or Ahr - / - . Data from n = 3 mice per group . Average read for housekeeping gene Hprt1 in both Ahr + / + Ahr - / - B are indicated below 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 O O O B-GENEPROD O O O O O O B-EXP_ASSAY O B-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 B-GENEPROD O O O O B-GENEPROD O O O O O O O O O B-ORGANISM O O O O O O O O B-GENEPROD O O B-GENEPROD O O O B-GENEPROD O O O B-CELL_TYPE 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 of Ccno expression in splenic CD19 + isolated from non - immune Ahrfl / + mb1Cre + ( Ahr + / - B , black ) Ahrfl / - mb1Cre + ( Ahr - / - B , ) mice , stimulated with 10 µg / ml anti - IgM for indicated time points . Ccno expression was normalized to Hprt1 . n = 3 independent experiments ; mean ± ; two - way ANOVA , Sidak ' s multiple comparison .

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

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

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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 of Ccno expression in splenic CD19 + isolated from C57Bl / 6 mice cultured for indicated time points with 20 ng / ml IL - 4 / 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 ± ; one - way ANOVA , Tukey ' s multiple comparison .

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

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

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

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 ) KI ( homozygous FLAG - RIPK2 ) mice were subjected to anti - FLAG immunoprecipitation immunoblotting .

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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 ) FLAG - RIPK2 heterozygous ( WT / KI ) homozygous ( KI / KI ) BMDMs . BMDMs were primed with IFNy , stimulated with MDP for indicated times analysed by immunoblotting .

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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 ) FLAG - RIPK2 heterozygous ( WT / KI ) homozygous ( KI / KI ) BMDMs in response to MDP . BMDMs were left untreated or treated with IFNy or IFNy MDP over - night cytokines were measured by ELISA . N = 5 - 8 mice . Shown is average ± . 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 B-GENEPROD O B-GENEPROD 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 B-SMALL_MOLECULE 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

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 ) 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 serum cytokines were measured by ELISA . N = 3 - 6 mice . Shown is average ± . ns = P > 0 . 05 ; * * = P ≤ 0 . 01 ; two - way ANOVA .

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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 . lysates from FLAG - RIPK2 BMDMs ( A ) were sequentially subjected to ubiquitin enrichment ( UBA , B ) FLAG pulldown ( C ) prior to elution subsequent mass spectrometry .

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


A Activation of NF - κB pathway by RIPK2 Lysine - phosphosite mutants . RIPK2 - deficient THP - 1 reconstituted with wild - type RIPK2 or RIPK2 mutants were stimulated with L18 - MDP , harvested at indicated time points activation of NF - κB pathway was analyzed by immunoblotting .

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A Activation of the NF-κB pathway by RIPK2 Lysine- and phosphosite mutants. RIPK2-deficient THP-1 cells reconstituted with wild-type RIPK2 or RIPK2 mutants were stimulated with L18-MDP, harvested at indicated time points and activation of the NF-κB pathway was analyzed by immunoblotting.


B IL - 8 production of wild - type THP - 1 RIPK2 - deficient THP - 1 reconstituted with wild - type RIPK2 or RIPK2 mutants stimulated with L18 - MDP was assessed by ELISA . N = 4 - 8 experiments . Shown is average ± . * = P ≤ 0 . 05 ; two - way ANOVA .

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B IL-8 production of wild-type THP-1 and RIPK2-deficient THP-1 cells reconstituted with wild-type RIPK2 or RIPK2 mutants and stimulated with L18-MDP was assessed by ELISA. N = 4-8 experiments. Shown is average ± SEM. * = P ≤ 0.05; two-way ANOVA.


C ) RIPK2 ubiquitination determined by UBA pulldown in RIPK2 - deficient reconstituted with wild - type or mutant RIPK2 after stimulation with L18 - MDP .

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C) RIPK2 ubiquitination determined by UBA pulldown in RIPK2-deficient cells reconstituted with wild-type or mutant RIPK2 after stimulation with L18-MDP.


D ) Detection of K63 - M1 - linked ubiquitin chains by UBA pulldown or pulldown with ubiquitin chain type - specific antibodies in RIPK2 - deficient reconstituted with RIPK2 K209R after stimulation with L18 - MDP .

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

D) Detection of K63- and M1-linked ubiquitin chains by UBA pulldown or pulldown with ubiquitin chain type-specific antibodies in RIPK2-deficient cells reconstituted with RIPK2 K209R after stimulation with L18-MDP.


C Activation of NF - κB pathway by RIPK2 pocket mutants . RIPK2 - deficient THP - 1 were reconstituted with wild - type RIPK2 or RIPK2 mutants , stimulated with L18 - MDP , harvested at indicated time points analyzed by immunoblotting .

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

C Activation of the NF-κB pathway by RIPK2 pocket mutants. RIPK2-deficient THP-1 cells were reconstituted with wild-type RIPK2 or RIPK2 mutants, stimulated with L18-MDP, harvested at indicated time points and analyzed by immunoblotting.


D IL - 8 production of RIPK2 - deficient THP - 1cells reconstituted with wild - type RIPK2 or RIPK2 mutants stimulated with L18 - MDP was assessed by ELISA . N = 3 - 8 experiments . Shown is average ± . ns = P > 0 . 05 ; * = P ≤ 0 . 05 ; * * * = P ≤ 0 . 001 ; * * * * = P ≤ 0 . 0001 ; two - way ANOVA .

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

D IL-8 production of RIPK2-deficient THP-1cells reconstituted with wild-type RIPK2 or RIPK2 mutants and stimulated with L18-MDP was assessed by ELISA. N = 3-8 experiments. Shown is average ± SEM. ns = P > 0.05; * = P ≤ 0.05; *** = P ≤ 0.001; **** = P ≤ 0.0001; two-way ANOVA.


E Ubiquitination of RIPK2 pocket mutants . RIPK2 - deficient THP - 1 were reconstituted with wild - type or mutant RIPK2 , left unstimulated or stimulated with L18 - MDP subjected to UBA pulldown immunoblotting .

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

E Ubiquitination of RIPK2 pocket mutants. RIPK2-deficient THP-1 cells were reconstituted with wild-type or mutant RIPK2, left unstimulated or stimulated with L18-MDP and subjected to UBA pulldown and immunoblotting.


A : Thermal stability using selected RIPK2 mutants . THP - 1 expressing RIPK2 were subjected to heat treatment non - denatured fraction was analysed by immunoblotting .

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

A: Thermal stability assay using selected RIPK2 mutants. THP-1 cells expressing RIPK2 were subjected to heat treatment and the non-denatured fraction was analysed by immunoblotting.


B : Binding of RIPK2 to XIAP - BIR2 . Lysates from RIPK2 - deficient THP - 1 reconstituted with WT or mutant RIPK2 were subjected to pulldown experiments with recombinantly expressed XIAP - BIR2 analysed by immunoblotting .

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

B: Binding of RIPK2 to XIAP-BIR2. Lysates from RIPK2-deficient THP-1 cells reconstituted with WT or mutant RIPK2 were subjected to pulldown experiments with recombinantly expressed XIAP-BIR2 and analysed by immunoblotting.


( A ) Illustration of crossing ageing scheme used to obtain total extracts from fly heads for transcriptional profiling successive qPCR assays . Expression of different Atrophin forms with GMR driver was induced , owing to a ‐ sensitive mutant Gal80 repressor . F1 flies were allowed to develop at 18 ° C ; at this Gal80 repressor keeps transgenes silent . Newly eclosed flies ( 0 - 48 h ) were collected killed immediately ( 0d ) or aged for 2 or 14 days at 29 ° C . This inactivates Gal80 transgenes are switched on by GMR ‐ Gal4 . This protocol allows comparing siblings that differ exclusively in their transgenes expression . Control flies crossed to GMR ‐ Gal4 ; UbiGal80ts in experiments are from w1118 stock in which UAS transgenes have been generated .

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

(A) Illustration of the crossing and ageing scheme used to obtain total RNA extracts from fly heads for the transcriptional profiling and all successive qPCR assays. Expression of different Atrophin forms with the GMR driver was induced, owing to a temperature‐sensitive mutant Gal80 repressor. F1 flies were allowed to develop at 18°C; at this temperature the Gal80 repressor keeps transgenes silent. Newly eclosed flies (0-48 h) were collected and killed immediately (0d) or aged for 2 or 14 days at 29°C. This inactivates Gal80 and transgenes are switched on by GMR‐Gal4. This protocol allows comparing siblings that differ exclusively in their age and transgenes expression. Control flies crossed to GMR‐Gal4; UbiGal80ts in all experiments are from the w1118 stock in which all UAS transgenes have been generated.


( B ) Tangential eye sections of flies representative of different populations used in microarray at different time points . Weak degeneration is only visible after 14 days with polyQ Atro ; in particular with Atro75QN there is an initial loss of photoreceptors ( PR , arrow ) , 30 . 7 % of ommatidia has lost at least 1 PR , that is only 5 . 1 % of neuronal PR have been lost at this stage ( N = 333 ) .

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

(B) Tangential eye sections of flies representative of all the different populations used in the microarray analysis at all different time points. Weak degeneration is only visible after 14 days with polyQ Atro; in particular with Atro75QN there is an initial loss of photoreceptors (PR, arrow), 30.7% of the ommatidia has lost at least 1 PR, that is only 5.1% of all neuronal PR have been lost at this stage (N=333).


( A ) Heat maps for three subsets of pattern clustering generated using Euclidean metric . Upregulated genes are in red downregulated genes are in green . downregulating transcriptional activity increases with time . main branch in clustering of different Atro mutants is along time points . See also Supplementary Figure 4 .

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(A) Heat maps for three subsets of the pattern clustering analysis generated using the Euclidean Distance metric. Upregulated genes are in red and downregulated genes are in green. The downregulating transcriptional activity increases with time. The main branch in clustering of the different Atro mutants is along time points. See also Supplementary Figure 4.


( A ) qPCR of fold changes ± s . d . of ft transcription . Downregulation starts at 2 days progresses at 14 days .

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

(A) qPCR analysis of the fold changes±s.d. of ft transcription. Downregulation starts at 2 days and progresses at 14 days.


( B ) qPCR of enrichment ± s . d . of different regions of ft regulatory elements in ChIP for Atro from BG3neuronal extracts . Enrichment is calculated as of DNAimmunoprecipitated from extracts of in which Atro expression has been induced , with respect to amount immunoprecipitated from uninduced extracts according to ( Atro - No Ab ) induced / ( Atro − No Ab ) uninduced formula . region from IV chromosome has been previously shown not to be immunoprecipitated in ChIP for Atro ( Haecker et al , 2007 ) has been used as a negative control .

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

(B) qPCR analysis of the enrichment±s.d. of different regions of the ft regulatory elements in ChIP for Atro from BG3neuronal cell extracts. Enrichment is calculated as the percentage of DNAimmunoprecipitated from extracts of cells in which Atro expression has been induced, with respect to the amount immunoprecipitated from uninduced cell extracts according to the (Atro-No Ab)induced/(Atro−No Ab)uninduced formula. The DNA region from IV chromosome has been previously shown not to be immunoprecipitated in ChIP for Atro ( Haecker et al, 2007) and has been used as a negative control.


( C ) Histograms showing number of ommatidia with a full complement ( 7 ) of PR in flies expressing Atro75QN with Rhodopsin1 driver in either a control ( w1118 ) or different mutant backgrounds aged at 29 ° C for 28 days . Heterozygosis for two independent ft alleles a sav allele significantly enhances loss of PR . Mild overexpression of Wts via a GMR - wts transgene , which does not display any strong phenotype per se , significantly suppresses loss of PR . No interaction was detected in this with wtsx1 ykiB5 alleles in heterozygosity . N = 430 - 963 from at least four eyes .

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

(C) Histograms showing the number of ommatidia with a full complement (7) of PR in flies expressing Atro75QN with the Rhodopsin1 driver in either a control (w1118) or different mutant backgrounds and aged at 29°C for 28 days. Heterozygosis for two independent ft alleles and a sav allele significantly enhances the loss of PR. Mild overexpression of Wts via a GMR-wts transgene, which does not display any strong phenotype per se, significantly suppresses the loss of PR. No interaction was detected in this assay with wtsx1 and ykiB5 alleles in heterozygosity. N=430-963 from at least four eyes.


( D ) Tangential eye sections histograms showing degeneration of GMR > Atro75QN , Ubi - Gal80ts flies in combination with different UAS transgenes aged 14 days as in Figure 1A . UAS - EGFP is used as a negative control . Either Ft or Wts overexpression strongly suppresses loss of PR caused by Atro75QN . Lower panels are provided as examples of . A more modest but still significant rescue is observed by overexpressing a Yki RNAi contruct or Hpo ; however , in this case it is to be considered that overexpression of Hpoper se brings about loss of at least one PR in ∼ 5 % of ommatidia ( data not shown ) . No effect is detected with overexpression of Sav . Two ‐ t ‐ : * P0 . 05 ; * * P0 . 01 . wtsEPG4808 is a previously uncharacterised EP element insertion at 5 ′ of wts gene . Rare homozygous escapers display larger eyes , whereas in combination with GMR - GMR this gives rise to smaller rough eyes , , finally , GMR of GMR > wtsEPG4808 indicates a 10 ‐ fold increase in head content of wts mRNA ( data not shown ) .

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

(D) Tangential eye sections and histograms showing the degeneration of GMR>Atro75QN, Ubi-Gal80ts flies in combination with different UAS transgenes and aged 14 days as in Figure 1A. UAS-EGFP is used as a negative control. Either Ft or Wts overexpression strongly suppresses loss of PR caused by Atro75QN. Lower panels are provided as examples of quantification. A more modest but still significant rescue is observed by overexpressing a Yki RNAi contruct or Hpo; however, in this case it is to be considered that the overexpression of Hpoper se brings about the loss of at least one PR in ∼5% of ommatidia (data not shown). No effect is detected with overexpression of Sav. Two‐tailed t‐test: *P0.05; **P0.01. The wtsEPG4808 is a previously uncharacterised EP element insertion at the 5′ of the wts gene. Rare homozygous escapers display larger eyes, whereas in combination with GMR-GMR this line gives rise to smaller rough eyes, and, finally, GMR analysis of GMR>wtsEPG4808 indicates a 10‐fold increase in the head content of wts mRNA (data not shown).


( A ) Tangential eye sections through ftfd clones aged 1 , 7 14 days , of ftfd , dDGC13 double mutant clones of ftfd clones in a ykiB5 heterozygous background aged 14 days . Clones are marked by absence of yellow pigment . Arrows point at degenerating photoreceptors , arrowheads point at intact wt photoreceptors in mosaic ommatidia . circled ommatidium is genetically wt but non ‐ autonomously flipped in its polarity has not degenerated . Lower panels are masks that show clonal borders of PR number per ommatidia . After 14 days , almost ftfd have degenerated , whereas almost ftfd , dDGC13 some ftfd , ykiB5 / + neuronalphotoreceptors have not .

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

(A) Tangential eye sections through ftfd clones aged 1, 7 and 14 days, of ftfd,dDGC13 double mutant clones and of ftfd clones in a ykiB5 heterozygous background aged 14 days. Clones are marked by the absence of yellow pigment. Arrows point at degenerating photoreceptors, arrowheads point at intact wt photoreceptors in mosaic ommatidia. The circled ommatidium is genetically wt but non‐autonomously flipped in its polarity and has not degenerated. Lower panels are masks that show clonal borders and quantification of PR number per ommatidia. After 14 days, almost all ftfd cells have degenerated, whereas almost all ftfd,dDGC13 and some ftfd,ykiB5/+ neuronalphotoreceptors have not.


( B ) Histograms showing of degeneration in ftfd , ftfddDGC13 ftfdykiB5 / + clones . Loss of ft leads to statistically significant loss of neurons this is suppressed at 14 days by ykiB5and much more dramatically by dDGC13 . * * * P0 . 001 , * * P0 . 01 ; * P0 . 05 in two ‐ t ‐ .

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(B) Histograms showing the quantification of degeneration in ftfd, ftfddDGC13 and ftfdykiB5/+ clones. Loss of ft leads to statistically significant loss of neurons and this is suppressed at 14 days by ykiB5and much more dramatically by dDGC13. ***P0.001, **P0.01; *P0.05 in two‐tailed t‐test.


( C ) Degeneration in mosaic ommatidia in ftfd clones . At this stage of degeneration approximately half of non ‐ pigmented ( genotypically mutant ) PR display a degenerative phenotype , whereas virtually pigmented ( genotypically wt ) PR are normal . χ2 ‐ : 82 . 38 ; P0 . 001 . Total PR N = 310 ( pigmented ) 327 ( not pigmented ) .

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

(C) Degeneration in mosaic ommatidia in ftfd clones. At this stage of degeneration approximately half of the non‐pigmented (genotypically mutant) PR cells display a degenerative phenotype, whereas virtually all pigmented (genotypically wt) PR cells are normal. χ2‐test: 82.38; P0.001. Total PR N=310 (pigmented) and 327 (not pigmented).


( D ) Tangential eye sections through MARCM clone mutants for ftfd that express either no transgene or an RNAi construct against yki ( UAS - ykiIR ) with Tub - Gal4 aged 14 days at 29 ° C . Because transgene UAS - ykiIR is on chromosomal arm 2 L , same as ft , clones generated with this system carry two copies of UAS - ykiIR therefore downregulate yki very effectively . No other outside clones expresses construct is therefore wt for yki . Mask panel is presented on right of each section . A of photoreceptor numbers ( far right ) displays a very significant increase in number of wt ommatidia ( N = 210 219 from four eyes ) . * * * P0 . 001 in two ‐ t ‐ . If classes of ommatidia are considered , χ2 ‐ = 232 . 30 ; P0 . 001 for 3 degrees of freedom .

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

(D) Tangential eye sections through MARCM clone mutants for ftfd that express either no transgene or an RNAi construct against yki (UAS-ykiIR) with Tub-Gal4 and aged 14 days at 29°C. Because the transgene UAS-ykiIR is on chromosomal arm 2 L, the same as ft, clones generated with this system carry two copies of UAS-ykiIR and therefore downregulate yki very effectively. No other cell outside the clones expresses the construct and is therefore wt for yki. Mask panel is presented on the right of each section. A quantification of photoreceptor numbers (far right) displays a very significant increase in the number of wt ommatidia (N=210 and 219 from four eyes). ***P0.001 in two‐tailed t‐test. If all classes of ommatidia are considered, χ2‐test=232.30; P0.001 for 3 degrees of freedom.


( A ) Tangential eye sections through wtsX1 mutant fly eyes . In clones for null wtsX1 allele , degeneration inside clones is at severe stages at 14 days .

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

(A) Tangential eye sections through wtsX1 mutant fly eyes. In clones for the null wtsX1 allele, degeneration inside the clones is at severe stages at 14 days.


( B ) Tangential eye sections through sav3 clones . In sav3 after enclosure many photoreceptors are intact but after 14 days at 29 ° C most sav mutant photoreceptors have degenerated .

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(B) Tangential eye sections through sav3 clones. In sav3 after enclosure many photoreceptors are intact but after 14 days at 29°C most sav mutant photoreceptors have degenerated.