Infection with Chlamydia trachomatis ( Ctr ; MOI = 1 for 30 h ; IL -

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Infection with Chlamydia trachomatis (Ctr; MOI=1 for 30 h; IL-6


Infection with Chlamydia trachomatis ( Ctr ; MOI = 1 for 30 h ; , IL - 8 ) .

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Infection with Chlamydia trachomatis (Ctr; MOI=1 for 30 h; , IL-8).


Infection with Salmonella Typhimurium ( STy ; MOI = 50 for 24 h ; IL -

Infection with Salmonella Typhimurium (STy; MOI=50 for 24 h; IL-6


Infection with Salmonella Typhimurium ( STy ; MOI = 50 for 24 h ; CXCL - 1 ) .

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Infection with Salmonella Typhimurium (STy; MOI=50 for 24 h; CXCL-1).


Infection with Toxoplasma gondii ( MOI = 5 for 16 h ; IL -

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Infection with Toxoplasma gondii (MOI=5 for 16 h; IL-6


Infection with Toxoplasma gondii ( MOI = 5 for 16 h ; , IL -

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Infection with Toxoplasma gondii (MOI=5 for 16 h; , IL-8


Infection with Toxoplasma gondii ( MOI = 5 for 16 h ; CXCL -

Infection with Toxoplasma gondii (MOI=5 for 16 h; CXCL-1


Infection with Toxoplasma gondii ( MOI = 5 for 16 h ; FGF - 2 ) .

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Infection with Toxoplasma gondii (MOI=5 for 16 h; FGF-2).


( A ) HeLa cell lines with an intact ( CTRL , carrying a non - coding gRNA ) or inactive ( Bax / Bak - double deficient ) mitochondrial apoptosis apparatus were infected with Chlamydia trachomatis expressing GFP ( MOI = 0 . 2 ) . Cells were analyzed by flow cytometry at the indicated time points . Cells were gated on the GFP - positive ( infected ) host cell population , and mean fluorescence intensity was recorded . Data are means / SEM of six experiments . ( A ) Growth of C . trachomatis was measured in HeLa cells as increase in GFP - fluorescence between 18 and 38 h . The data show means / SEM of six independent experiments ( genotype effect ( significance of the difference of GFP - expression between the cell lines overall , p = 0 . 08 ) . Red line indicates the fold change calculation of the individual different time points and cell lines ( significance tested ( one sample t - test ) for each time point for a difference between Bax / Bak vs CTRL ) .

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(A) HeLa cell lines with an intact (CTRL, carrying a non-coding gRNA) or inactive (Bax/Bak-double deficient) mitochondrial apoptosis apparatus were infected with Chlamydia trachomatis expressing GFP (MOI=0.2). Cells were analyzed by flow cytometry at the indicated time points. Cells were gated on the GFP-positive (infected) host cell population, and mean fluorescence intensity was recorded. Data are means/SEM of six experiments. (A) Growth of C. trachomatis was measured in HeLa cells as increase in GFP-fluorescence between 18 and 38 h. The data show means/SEM of six independent experiments (genotype effect (significance of the difference of GFP-expression between the cell lines overall, p = 0.08). Red line indicates the fold change calculation of the individual different time points and cell lines (significance tested (one sample t-test) for each time point for a difference between Bax/Bak vs CTRL).


AGS cell lines with an intact ( CTRL , carrying a non - coding gRNA ) or inactive ( Bax / Bak - double deficient ) mitochondrial apoptosis apparatus were infected with Chlamydia trachomatis expressing GFP ( MOI = 0 . 2 ) . Cells were analyzed by flow cytometry at the indicated time points . Cells were gated on the GFP - positive ( infected ) host cell population , and mean fluorescence intensity was recorded . Data are means / SEM of 4 - 5 experiments . ( B ) Growth of C . trachomatis was measured in AGS cells as increase in GFP - fluorescence . The data show means / SEM of 4 to 5 independent experiments ( genotype effect : p < 0 . 0001 ; time - genotype interaction : p < 0 . 0001 ) ( 12 h and 38 h , n = 4 ; 18 h , n = 5 ) .

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AGS cell lines with an intact (CTRL, carrying a non-coding gRNA) or inactive (Bax/Bak-double deficient) mitochondrial apoptosis apparatus were infected with Chlamydia trachomatis expressing GFP (MOI=0.2). Cells were analyzed by flow cytometry at the indicated time points. Cells were gated on the GFP-positive (infected) host cell population, and mean fluorescence intensity was recorded. Data are means/SEM of 4-5 experiments. (B) Growth of C. trachomatis was measured in AGS cells as increase in GFP-fluorescence. The data show means/SEM of 4 to 5 independent experiments (genotype effect: p<0.0001; time-genotype interaction: p<0.0001) (12 h and 38 h, n=4; 18 h, n=5).


C Chlamydial genomes per cell culture ( expressed as chlamydial DNA in 50 ng of total DNA , determined by quantitative PCR ) . * , p < 0 . 05 between control and mutant cells . ( n = 3 )

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C Chlamydial genomes per cell culture (expressed as chlamydial DNA in 50 ng of total DNA, determined by quantitative PCR). *, p<0.05 between control and mutant cells. (n=3)


D CTRL , Bax / Bak - deficient or Bcl - XL - over - expressing HeLa cells were infected with Salmonella Typhimurium ( MOI = 50 ) . Extracellular bacteria were killed using non cell - permeable antibiotic . At the indicated time points cells were lysed , and aliquots were plated on agar plates . Recovered colony forming units were calculated . Data are means / SEM of four independent experiments . Significant genotype effects were found between mutant and control cells ( Bax / Bak p < 0 . 0001 ; Bcl - XL p 0 . 0001 ) .

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D CTRL, Bax/Bak-deficient or Bcl-XL-over-expressing HeLa cells were infected with Salmonella Typhimurium (MOI=50). Extracellular bacteria were killed using non cell-permeable antibiotic. At the indicated time points cells were lysed, and aliquots were plated on agar plates. Recovered colony forming units were calculated. Data are means/SEM of four independent experiments. Significant genotype effects were found between mutant and control cells (Bax/Bak p<0.0001; Bcl-XL p 0.0001).


E CTRL , Bax / Bak - deficient or Apaf - 1 - deficient HeLa cells were infected with Salmonella Typhimurium ( MOI = 50 ) . CFU / well were calculated as in ( D ) . n = 3 .

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E CTRL, Bax/Bak-deficient or Apaf-1-deficient HeLa cells were infected with Salmonella Typhimurium (MOI=50). CFU/well were calculated as in (D). n=3.


A Actin expression levels shown by western blotting for strains expressing S . cerevisiae ' s actin protein from various coding sequences , with tubulin ( Tub1p ) as a loading control . B Quantification of actin expression levels , showing a decrease when more silent mutations are present . Data are presented as mean + / - SD ( n = 4 for Sc , n = 8 for ScNI ; n = 12 for Sc [ Ca ] , Sc [ Sp ] and Sc [ At ] ; 2 biological replicates with n / 2 technical replicates each ) . * P < 0 . 05 ( Brown - Forsythe and Welch ANOVA tests , with Dunnett ' s T3 multiple comparisons tests ) .

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A Actin expression levels shown by western blotting for strains expressing S. cerevisiae's actin protein from various coding sequences, with tubulin (Tub1p) as a loading control. B Quantification of actin expression levels, showing a decrease when more silent mutations are present. Data are presented as mean +/- SD (n = 4 for Sc, n = 8 for ScNI; n = 12 for Sc[Ca], Sc[Sp] and Sc[At]; 2 biological replicates with n/2 technical replicates each). *P<0.05 (Brown-Forsythe and Welch ANOVA tests, with Dunnett's T3 multiple comparisons tests).


F In vivo actin network deviation indexes , defined to evaluate the patch - cable balance compared to S . cerevisiae haploid cells ( value is 0 in S . cerevisiae ' s cells , 1 when cells contain only actin patches and - 1 when cells contain only cables ) . Data are presented as mean + / - SD ( n = 30 for all conditions ) . * * * P < 0 . 001 ( Brown - Forsythe and Welch ANOVA tests , with Dunnett ' s T3 multiple comparisons tests ) .

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F In vivo actin network deviation indexes, defined to evaluate the patch-cable balance compared to S. cerevisiae haploid cells (value is 0 in S. cerevisiae's cells, 1 when cells contain only actin patches and -1 when cells contain only cables). Data are presented as mean +/- SD (n = 30 for all conditions). ***P<0.001 (Brown-Forsythe and Welch ANOVA tests, with Dunnett's T3 multiple comparisons tests).


E Phalloidin staining of F - actin organization . Images are maximum intensity projections of 3D stacks and contrasts were adapted due to the fact that phalloidin labeling had a very different efficiency depending on the actin ortholog expressed . Micrographs of Sc and Sc _ NI cells are reproduced from Fig 2E . Scale bar : 3 µm .

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E Phalloidin staining of F-actin organization. Images are maximum intensity projections of 3D stacks and contrasts were adapted due to the fact that phalloidin labeling had a very different efficiency depending on the actin ortholog expressed. Micrographs of Sc and Sc_NI cells are reproduced from Fig 2E. Scale bar: 3 µm.


F In vivo actin network deviation indexes . Data are presented as mean + / - SD ( n = 30 for all conditions ) . * P < 0 . 05 , * * * P < 0 . 001 ( Brown - Forsythe and Welch ANOVA tests , with Dunnett ' s T3 multiple comparisons tests ) .

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F In vivo actin network deviation indexes. Data are presented as mean +/- SD (n = 30 for all conditions). *P<0.05, ***P<0.001 (Brown-Forsythe and Welch ANOVA tests, with Dunnett's T3 multiple comparisons tests).


I Effect of CK - 666 ( 75 µM ) on the organization of the actin cytoskeleton . Cells were stained with phalloidin after 30 min incubation with CK - 666 . Images are maximum intensity projections of 3D stacks . Scale bar : 3 µm J Quantification of actin patch resistance to CK - 666 treatment . Bar graphs represent the percentage of cells with a given number of visible actin patches after CK - 666 treatment . ( n = 41 for ScNI , 55 for N2 , 31 for Ca , 41 for Op , 55 for Hs ) .

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I Effect of CK-666 (75 µM) on the organization of the actin cytoskeleton. Cells were stained with phalloidin after 30 min incubation with CK-666. Images are maximum intensity projections of 3D stacks. Scale bar: 3 µm J Quantification of actin patch resistance to CK-666 treatment. Bar graphs represent the percentage of cells with a given number of visible actin patches after CK-666 treatment. (n = 41 for ScNI, 55 for N2, 31 for Ca, 41 for Op, 55 for Hs).


A ( Top ) Snapshots of actin networks assembled from three different actins sources : Act _ Sc , Act _ N2 and Act _ Ca . ( Bottom left ) Quantification of actin fluorescence on beads . Data are presented as mean + / - SD ( n = 52 for Act _ Sc branched , n = 70 for Act _ N2 branched , n = 81 for Act _ Ca branched , n = 32 for Act _ Sc linear , n = 34 for Act _ N2 linear and n = 47 for Act _ Ca linear ) . * * P < 0 . 01 , * * * P < 0 . 001 ( Kruskal - Wallis test , with multiple comparisons ) . ( Bottom right ) In vitro actin network deviation indexes .

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A (Top) Snapshots of actin networks assembled from three different actins sources: Act_Sc, Act_N2 and Act_Ca. (Bottom left) Quantification of actin fluorescence on beads. Data are presented as mean +/- SD (n = 52 for Act_Sc branched, n = 70 for Act_N2 branched, n = 81 for Act_Ca branched, n = 32 for Act_Sc linear, n = 34 for Act_N2 linear and n = 47 for Act_Ca linear). **P<0.01, ***P<0.001 (Kruskal-Wallis test, with multiple comparisons). (Bottom right) In vitro actin network deviation indexes.


B ( Left ) Snapshots of representative actin networks assembled in the presence of 600 nM Alexa - 488 - labeled ADF / cofilin ( replacement of unlabeled ADF / cofilin ) . ( Right ) Quantification of ADF / cofilin ' s fluorescence intensity relative to actin . Data are presented as mean + / - SD ( n = 34 for Act _ Sc branched , n = 28 for Act _ N2 branched , n = 43 for Act _ Ca branched , n = 24 for Act _ Sc linear , n = 15 for Act _ N2 linear and n = 27 for Act _ Ca linear ) . * * * P < 0 . 001 ( Kruskal - Wallis test , with multiple comparisons ) .

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B (Left) Snapshots of representative actin networks assembled in the presence of 600 nM Alexa-488-labeled ADF/cofilin (replacement of unlabeled ADF/cofilin). (Right) Quantification of ADF/cofilin's fluorescence intensity relative to actin. Data are presented as mean +/- SD (n = 34 for Act_Sc branched, n = 28 for Act_N2 branched, n = 43 for Act_Ca branched, n = 24 for Act_Sc linear, n = 15 for Act_N2 linear and n = 27 for Act_Ca linear). ***P<0.001 (Kruskal-Wallis test, with multiple comparisons).


C ( Left ) Snapshots of representative actin networks assembled in the presence 1 µM Alexa 488 - tropomyosin . ( Right ) Quantification of tropomyosin ' s fluorescence intensity relative to actin . Data are presented as mean + / - SD ( n = 45 for Act _ Sc branched , n = 63 for Act _ N2 branched , n = 57 for Act _ Ca branched , n = 22 for Act _ Sc linear , n = 32 for Act _ N2 linear and n = 27 for Act _ Ca linear ) . * P < 0 . 05 , * * * P < 0 . 001 ( Brown - Forsythe and Welch ANOVA tests , with Dunnett ' s T3 multiple comparisons tests ) .

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C (Left) Snapshots of representative actin networks assembled in the presence 1 µM Alexa 488-tropomyosin. (Right) Quantification of tropomyosin's fluorescence intensity relative to actin. Data are presented as mean +/- SD (n = 45 for Act_Sc branched, n = 63 for Act_N2 branched, n = 57 for Act_Ca branched, n = 22 for Act_Sc linear, n = 32 for Act_N2 linear and n = 27 for Act_Ca linear). *P<0.05, ***P<0.001 (Brown-Forsythe and Welch ANOVA tests, with Dunnett's T3 multiple comparisons tests).


C Phalloidin staining depicting F - actin organization . Images are maximum intensity projections of 3D stacks . Scale bar : 3 µm . D In vivo actin network deviation indexes . Data are presented as mean + / - SD ( n = 30 for all conditions ) . * P < 0 . 05 , * * * P < 0 . 001 ( Brown - Forsythe and Welch ANOVA tests , with Dunnett ' s T3 multiple comparisons tests ) . E Polarity Indexes . Data are presented as mean + / - SD ( n = 30 for all conditions ) . * * * P < 0 . 001 ( Kruskal - Wallis test , with multiple comparisons ) .

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C Phalloidin staining depicting F-actin organization. Images are maximum intensity projections of 3D stacks. Scale bar: 3 µm. D In vivo actin network deviation indexes. Data are presented as mean +/- SD (n = 30 for all conditions). *P<0.05, ***P<0.001 (Brown-Forsythe and Welch ANOVA tests, with Dunnett's T3 multiple comparisons tests). E Polarity Indexes. Data are presented as mean +/- SD (n = 30 for all conditions). ***P<0.001 (Kruskal-Wallis test, with multiple comparisons).


F Effect of CK - 666 ( 75 µM ) on the organization of the actin cytoskeleton . Cells were stained with phalloidin after 30 min incubation with CK - 666 . Images are maximum intensity projections of 3D stacks . Scale bar : 3 µm . G In vivo actin network deviation indexes of cells treated with DMSO or CK - 666 . Data are presented as mean + / - SD ( n = 30 for all conditions ) . * * * P < 0 . 001 ( Brown - Forsythe and Welch ANOVA tests , with Dunnett ' s T3 multiple comparisons tests ) .

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F Effect of CK-666 (75 µM) on the organization of the actin cytoskeleton. Cells were stained with phalloidin after 30 min incubation with CK-666. Images are maximum intensity projections of 3D stacks. Scale bar: 3 µm. G In vivo actin network deviation indexes of cells treated with DMSO or CK-666. Data are presented as mean +/- SD (n = 30 for all conditions). ***P<0.001 (Brown-Forsythe and Welch ANOVA tests, with Dunnett's T3 multiple comparisons tests).


( C ) The 3 . 1 Å 2Fo - Fc map ( 1 . 2 σ ) around the DNA of the final refined ATPγS - MjMR - DNA complex structure .

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(C) The 3.1 Å 2Fo-Fc map (1.2 σ) around the DNA of the final refined ATPγS-MjMR-DNA complex structure.


( D ) A ribbon representation of the 3 . 1 Å ATPγS - MjMR - DNA structure showing the Mre11 nuclease , capping , and C - terminal three helix - bundle domain ( light pink ) , Rad50 molecules ( green and light blue ) , and the DNA ( yellow and orange ) . ATPγS is shown in spheres . See Movie EV 1 for the movement of the complex .

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(D) A ribbon representation of the 3.1 Å ATPγS-MjMR-DNA structure showing the Mre11 nuclease, capping, and C-terminal three helix-bundle domain (light pink), Rad50 molecules (green and light blue), and the DNA (yellow and orange). ATPγS is shown in spheres. See Movie EV 1 for the movement of the complex.


( A ) A ribbon diagram showing the overall DNA recognition by the ATPγS - Rad50 dimer .

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(A) A ribbon diagram showing the overall DNA recognition by the ATPγS-Rad50 dimer.


( B ) Close - up view of the interaction between the internal segment ( major and minor grooves ) of the DNA strand and the edge of lobe I of Rad50A ( green ) . Loop α1 - α2 wedge faces the minor groove between the 10th and 13 ' th phosphates of the template ( orange ) and non - template ( yellow ) strands , respectively . ( C ) Close - up view of the interaction between the DNA strand and the α1 - α2 loop in an orientation different from Fig 2B .

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(B) Close-up view of the interaction between the internal segment (major and minor grooves) of the DNA strand and the edge of lobe I of Rad50A (green). Loop α1-α2 wedge faces the minor groove between the 10th and 13' th phosphates of the template (orange) and non-template (yellow) strands, respectively. (C) Close-up view of the interaction between the DNA strand and the α1-α2 loop in an orientation different from Fig 2B.


( B , C ) DNA binding analysis for the Interactions between WT or four MjMRcd mutants and a closed circular dsDNA ( φx174RFII ) in the absence ( B ) or presence ( C ) of AMP - PNP / Mg2 + . Each protein sample was incubated with φx174RFII dsDNA ( 3 . 5 nM ) for 30 min on 4 ° C . The following molar ratio of protein : DNA is used in Fig 3B - 3E ; lane 2 , 50 : 1 ; lane 3 , 100 : 1 ; lane 4 , 200 : 1 ; lane 5 , 500 : 1 ; lane 6 , 1000 : 1 ; lane 7 , 2000 : 1 . We note that some MRcd variants may form a second form of the complex with DNA .

O O O O O B-SMALL_MOLECULE O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE I-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 B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O B-SMALL_MOLECULE O

(B, C) DNA binding analysis for the Interactions between WT or four MjMRcd mutants and a closed circular dsDNA (φx174RFII) in the absence (B) or presence (C) of AMP-PNP/Mg2+. Each protein sample was incubated with φx174RFII dsDNA (3.5 nM) for 30 min on 4°C. The following molar ratio of protein: DNA is used in Fig 3B-3E; lane 2, 50:1; lane 3, 100:1; lane 4, 200:1; lane 5, 500:1; lane 6, 1000:1; lane 7, 2000:1. We note that some MRcd variants may form a second form of the complex with DNA.


( D , E ) Interactions of the WT or five full - length TmMR mutants with φx174RFII were examined in the absence ( D ) or presence of AMP - PNP / Mg2 + ( E ) . Reaction conditions are same as those in Fig 3B .

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(D, E) Interactions of the WT or five full-length TmMR mutants with φx174RFII were examined in the absence (D) or presence of AMP-PNP/Mg2+ (E). Reaction conditions are same as those in Fig 3B.


( F ) Nuclease activities of free MjMre11 ( lane 3 , 4 ) , nucleotide unbound - ( 5 , 6 ) and - bound MjMRcd complex ( 7 , 8 ) towards a hairpin DNA . Each protein sample was incubated with a hairpin DNA ( 10 nM ) in a 1 : 10 or 1 : 20 molar ratio ( protein : DNA ) for 30 min on 55 ° C . The error bars for the quantified values of uncleaved substrates on the right panel are calculated from the standard deviation from three repetitions of each experiment .

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

(F) Nuclease activities of free MjMre11 (lane 3, 4), nucleotide unbound- (5, 6) and -bound MjMRcd complex (7, 8) towards a hairpin DNA. Each protein sample was incubated with a hairpin DNA (10 nM) in a 1:10 or 1:20 molar ratio (protein: DNA) for 30 min on 55°C. The error bars for the quantified values of uncleaved substrates on the right panel are calculated from the standard deviation from three repetitions of each experiment.


( G ) Nuclease activities of free TmMre11 alone ( lane 3 , 4 ) , nucleotide unbound - ( lane 5 , 6 ) and - bound ( lane 7 , 8 ) full - length TmMR complex towards a hairpin DNA .

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(G) Nuclease activities of free TmMre11 alone (lane 3, 4), nucleotide unbound- (lane 5, 6) and -bound (lane 7, 8) full-length TmMR complex towards a hairpin DNA.


( A ) Superposition of the ATPγS - MjMR - DNA complex ( green ) and apo - MjMR ( 3AV0 , magenta ) structures . Alignment was done using their NBDs ( 1 . 3 Å rmsd in the positions of 311 Cα atoms ) . The coiled - coils of the DNA - bound Rad50 molecules are shifted ( arrow ) to more parallel orientation . The entire structures can be superimposed with a 1 . 9 Å rmsd in 692 Cα positions .

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

(A) Superposition of the ATPγS-MjMR-DNA complex (green) and apo-MjMR (3AV0, magenta) structures. Alignment was done using their NBDs (1.3 Å rmsd in the positions of 311 Cα atoms). The coiled-coils of the DNA-bound Rad50 molecules are shifted (arrow) to more parallel orientation. The entire structures can be superimposed with a 1.9 Å rmsd in 692 Cα positions.


( B , C ) Comparison of the local structures between the apo ( B ) and the DNA - bound MjMR complex ( C ) . Upon DNA binding , helix α6 translates to DNA backbone , resulting in the tighter packing of Leu155 and Leu156 against a hydrophobic pocket formed by lobe I and II . Arrows indicate the direction of the helix movement by DNA binding . See Fig EV5 for a close - up view .

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

(B, C) Comparison of the local structures between the apo (B) and the DNA-bound MjMR complex (C). Upon DNA binding, helix α6 translates to DNA backbone, resulting in the tighter packing of Leu155 and Leu156 against a hydrophobic pocket formed by lobe I and II. Arrows indicate the direction of the helix movement by DNA binding. See Fig EV5 for a close-up view.


( A ) Stereo view of the superposition of the ADP - MjRad50 ( 3AUX ) complex ( black ) onto the ATPγS - MjMR - DNA complex ( green and blue ) by aligning their lobe II domains . The α1 - α2 loop that could collide with parts of the DNA molecule is indicated . ( B ) Aligning the lobe II domains of two structures in Fig 5A shows that rotation of the lobe I opens the gate for Mre11 , which could allow the access of the unwound DNA . Color schemes are same as those in Fig 5A .

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

(A) Stereo view of the superposition of the ADP-MjRad50 (3AUX) complex (black) onto the ATPγS-MjMR-DNA complex (green and blue) by aligning their lobe II domains. The α1-α2 loop that could collide with parts of the DNA molecule is indicated. (B) Aligning the lobe II domains of two structures in Fig 5A shows that rotation of the lobe I opens the gate for Mre11, which could allow the access of the unwound DNA. Color schemes are same as those in Fig 5A.


( C ) DNA - unwinding activities of TmMR . Lane 2 , no protein ; lanes 3 and 4 , no ATP ; lanes 5 and 6 , with ATPγS ; lanes 7 and 8 , with ATP . The DNA : protein ratio was 1 : 2 for lanes 3 , 5 , and 7 , and 1 : 5 for lanes 4 , 6 , and 8 . Plotted data represent the mean of three experiments , with error bars indicating one standard deviation . A graph of the quantified data is shown in the lower panel .

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

(C) DNA-unwinding activities of TmMR. Lane 2, no protein; lanes 3 and 4, no ATP; lanes 5 and 6, with ATPγS; lanes 7 and 8, with ATP. The DNA: protein ratio was 1:2 for lanes 3, 5, and 7, and 1:5 for lanes 4, 6, and 8. Plotted data represent the mean of three experiments, with error bars indicating one standard deviation. A graph of the quantified data is shown in the lower panel.


( D ) Comparison of the ATP - dependent DNA unwinding activities of WT and mutant TmMR proteins . Lane 2 , no protein ; lane 3 and 4 , WT TmMR ; lane 5 and 6 , Rad50 ( Δ54 - 56 ) mutant ; lane 7 and 8 , R87E mutant ; lane 9 and 10 , K95E mutant ; lane 11 and 12 , R94E / K95E mutant ; lane 13 and 14 , K115E mutant . For each TmMR , reaction contains 1 : 2 or 1 : 5 ratio of DNA : protein .

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(D) Comparison of the ATP-dependent DNA unwinding activities of WT and mutant TmMR proteins. Lane 2, no protein; lane 3 and 4, WT TmMR; lane 5 and 6, Rad50 (Δ54-56) mutant; lane 7 and 8, R87E mutant; lane 9 and 10, K95E mutant; lane 11 and 12, R94E/K95E mutant; lane 13 and 14, K115E mutant. For each TmMR, reaction contains 1:2 or 1:5 ratio of DNA: protein.


( A ) Endonuclease activities of the TmMR WT and mutant proteins towards a duplex DNA in the absence ( left panel ) or presence of ATP ( right panel ) . Major products are marked with dots . The 5 ' - 32P - label is indicated by a star . See Appendix Fig S4A and S4B for the quantified values .

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

(A) Endonuclease activities of the TmMR WT and mutant proteins towards a duplex DNA in the absence (left panel) or presence of ATP (right panel). Major products are marked with dots. The 5'-32P-label is indicated by a star. See Appendix Fig S4A and S4B for the quantified values.


( B ) Quantification of a bubble DNA binding by WT or mutant TmMR in the presence or absence of AMP - PNP . See Appendix Fig S3E and S3F for the original data .

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(B) Quantification of a bubble DNA binding by WT or mutant TmMR in the presence or absence of AMP-PNP. See Appendix Fig S3E and S3F for the original data.


( C ) Endonuclease activities of the WT and mutant TmMR proteins towards a bubble DNA molecule in the absence ( left panel ) or presence of ATP ( right panel ) . See Appendix Fig S4C and S4D for the quantified values .

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

(C) Endonuclease activities of the WT and mutant TmMR proteins towards a bubble DNA molecule in the absence (left panel) or presence of ATP (right panel). See Appendix Fig S4C and S4D for the quantified values.


( D ) The effects of single or multiple point mutations in the DNA - contacting or neighboring residues of S . cerevisiae rad50 on the sensitivity to chemical drugs . Sensitivities of rad50 expressing the mutants towards indicated concentrations of CPT , PHL , and MMS were examined . ( E ) The effects of Rad50 mutation on recombination stimulated by inverted Alu repeats . See Appendix Table S1 for the quantified values .

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

(D) The effects of single or multiple point mutations in the DNA-contacting or neighboring residues of S. cerevisiae rad50 on the sensitivity to chemical drugs. Sensitivities of rad50 expressing the mutants towards indicated concentrations of CPT, PHL, and MMS were examined. (E) The effects of Rad50 mutation on recombination stimulated by inverted Alu repeats. See Appendix Table S1 for the quantified values.


( E ) The effects of Rad50 mutation on recombination stimulated by inverted Alu repeats . See Appendix Table S1 for the quantified values .

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(E) The effects of Rad50 mutation on recombination stimulated by inverted Alu repeats. See Appendix Table S1 for the quantified values.


B ) Single point activity assays of USP1WT and deletion mutants on Ub - Rho show signifi cantly increased activity in mutants where insert L1 and L3 are both deleted .

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

B) Single point activity assays of USP1WT and deletion mutants on Ub-Rho show significantly increased activity in mutants where insert L1 and L3 are both deleted.


C ) Single point activity assays of USP1WT and deletion mutants ( ± UAF1 ) on Ub - Rho show the loss in hyper - activation of USP1ΔL1L3 upon addition of UAF1 .

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

C) Single point activity assays of USP1WT and deletion mutants (± UAF1) on Ub-Rho show the loss in hyper-activation of USP1ΔL1L3 upon addition of UAF1.


D ) Michaelis - Menten analysis of USP1 deletion mutants against ubiquitin - rhodamine ( Ub - Rho ) shows that USP1ΔL1L3 has significantly higher activity compared to WT and other mutants ( n = 2 , biological replicates ) .

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

D) Michaelis-Menten analysis of USP1 deletion mutants against ubiquitin-rhodamine (Ub-Rho) shows that USP1ΔL1L3 has significantly higher activity compared to WT and other mutants (n=2, biological replicates).


E ) Michaelis - Menten analysis of USP1 deletion mutants ( + UAF1 ) against Ub - Rho shows that all deletions mutants have similar catalytic activity ( n = 2 , biological replicates ) .

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E) Michaelis-Menten analysis of USP1 deletion mutants (+UAF1) against Ub-Rho shows that all deletions mutants have similar catalytic activity (n=2, biological replicates).


F ) Comparing activity of USP1WT and USP1ΔL1L3 on a peptide substrate i . e . LRGG - AMC ( 100µM ) . USP1ΔL1L3 cleaves the peptide substrate more efficiently compared to USP1WT ( n = 2 , technical replicates ) .

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

F) Comparing activity of USP1WT and USP1ΔL1L3 on a peptide substrate i.e. LRGG-AMC (100µM). USP1ΔL1L3 cleaves the peptide substrate more efficiently compared to USP1WT (n=2, technical replicates).


G ) Comparing activity of USP1WT and USP1ΔL1L3 ( + UAF1 ) on a peptide substrate i . e . LRGG - AMC ( 100µM ) . Addition of UAF1 allows cleavage of LRGG - AMC by both USP1WT and USP1ΔL1L3 ( n = 2 , technical replicates ) .

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G) Comparing activity of USP1WT and USP1ΔL1L3 (+UAF1) on a peptide substrate i.e. LRGG-AMC (100µM). Addition of UAF1 allows cleavage of LRGG-AMC by both USP1WT and USP1ΔL1L3 (n=2, technical replicates).


A ) Quantification of gel - based activity assays where the activity of USP1WT and deletion mutants ( + UAF1 ) on PCNA - UbTAMRA is compared . The USP1ΔL1 mutant has reduced activity on PCNA - UbTAMRA compared to the other USP1 variants tested ( n = 2 , biological replicates ) .

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A) Quantification of gel-based activity assays where the activity of USP1WT and deletion mutants (+UAF1) on PCNA-UbTAMRA is compared. The USP1ΔL1 mutant has reduced activity on PCNA-UbTAMRA compared to the other USP1 variants tested (n=2, biological replicates).


C ) Quantification of gel - based activity assays where the activity of USP1 PIP mutants ( + UAF1 ) on PCNA - UbTAMRA is compared . Both the PIP mutants have reduced activity on PCNA - Ub compared to USP1WT ( n = 2 , biological replicates ) .

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

C) Quantification of gel-based activity assays where the activity of USP1 PIP mutants (+UAF1) on PCNA-UbTAMRA is compared. Both the PIP mutants have reduced activity on PCNA-Ub compared to USP1WT (n=2, biological replicates).


D ) FP based binding assays of USP1 mutants ( + UAF1 ) with PCNA - UbTAMRA show reduced binding of USP1ΔL1 and USP1PIP1 compared to USP1WT ( n = 2 , technical replicates ) .

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

D) FP based binding assays of USP1 mutants (+ UAF1) with PCNA-UbTAMRA show reduced binding of USP1ΔL1 and USP1PIP1 compared to USP1WT (n=2, technical replicates).


E ) Quantification of gel - based activity assays where the activity of USP1WT + UAF1 on PCNA - UbWT is compared with PCNA - UbPIM1 shows that PCNA - UbWT is cleaved much faster compared to PCNA - UbPIM1 ( n = 2 , technical replicates ( example shown here from 1 of 2 biological replicates ) )

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E) Quantification of gel-based activity assays where the activity of USP1WT+UAF1 on PCNA-UbWT is compared with PCNA-UbPIM1 shows that PCNA-UbWT is cleaved much faster compared to PCNA-UbPIM1 (n=2, technical replicates (example shown here from 1 of 2 biological replicates))


F ) Comparing activity of USP1WT and USP1PIP1 ( + UAF1 ) on PCNA - UbWT and PCNA - UbPIM1 in gel - based assays shows that the activity of USP1WT is severely reduced by the PIM1 mutation in PCNA , whereas USP1PIP1 activity is not further affected indicating that PIP1 and PIM mutation affect the same interaction ( n = 2 , technical replicates ( example shown here from 1 of 2 biological replicates ) ) .

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F) Comparing activity of USP1WT and USP1PIP1 (+UAF1) on PCNA-UbWT and PCNA-UbPIM1 in gel-based assays shows that the activity of USP1WT is severely reduced by the PIM1 mutation in PCNA, whereas USP1PIP1 activity is not further affected indicating that PIP1 and PIM mutation affect the same interaction (n=2, technical replicates (example shown here from 1 of 2 biological replicates)).


A ) Schematic representation of loading and purification of PCNA - Ub on nicked circular DNA . The DNA - loaded PCNA - Ub elutes in the void of the SEC column and this sample is collected and used for studying activity of USP1 on DNA - loaded PCNA - Ub .

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A) Schematic representation of loading and purification of PCNA-Ub on nicked circular DNA. The DNA-loaded PCNA-Ub elutes in the void of the SEC column and this sample is collected and used for studying activity of USP1 on DNA-loaded PCNA-Ub.


B ) Michaelis - Menten analysis of USP1 + UAF1 with and without DNA ( 65bp dsDNA ) shows that DNA binding alone has no effect on USP1 activity ( n = 2 , technical replicates ( example shown here from 1 of 2 biological replicates ) ) .

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B) Michaelis-Menten analysis of USP1+UAF1 with and without DNA (65bp dsDNA) shows that DNA binding alone has no effect on USP1 activity (n=2, technical replicates (example shown here from 1 of 2 biological replicates)).


C ) Coomassie stained gel of in vitro activity assay showing increased activity of USP1 - UAF1 on DNA - loaded PCNA - Ub compared to PCNA - Ub ( - DNA ) and PCNA - Ub ( + DNA , + RFC and not ATP ) .

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C) Coomassie stained gel of in vitro activity assay showing increased activity of USP1-UAF1 on DNA-loaded PCNA-Ub compared to PCNA-Ub (-DNA) and PCNA-Ub (+DNA, +RFC and not ATP).


D ) Quantification of gel - based activity assays showing enhanced activity of USP1 + UAF1 on DNA - loaded PCNA - Ub compared to free PCNA - Ub ( n = 3 , technical replicates ( example shown here from 1 of 2 biological replicates ) ) .

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

D) Quantification of gel-based activity assays showing enhanced activity of USP1+UAF1 on DNA-loaded PCNA-Ub compared to free PCNA-Ub (n=3, technical replicates (example shown here from 1 of 2 biological replicates)).


E ) Quantification of gel - based activity assays showing enhanced activity of USP1 on DNA - loaded PCNA - Ub compared to free PCNA - Ub ( n = 3 , technical replicates ( example shown here from 1 of 2 biological replicates ) ) .

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

E) Quantification of gel-based activity assays showing enhanced activity of USP1 on DNA-loaded PCNA-Ub compared to free PCNA-Ub (n=3, technical replicates (example shown here from 1 of 2 biological replicates)).


A ) Gel based quantification of USP1 - UAF1 activity on PCNA - Ub ( DNA loaded and free ) for three different concentration of enzyme and substrate .

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

A) Gel based quantification of USP1-UAF1 activity on PCNA-Ub (DNA loaded and free) for three different concentration of enzyme and substrate.


B ) FP based activity assays on free PCNA - Ub ( three concentrations of USP1 and PCNA - Ub ) and activity of USP1 on increasing concentrations of Ub - Rho .

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

B) FP based activity assays on free PCNA-Ub (three concentrations of USP1 and PCNA-Ub) and activity of USP1 on increasing concentrations of Ub-Rho.


A ) Comparison of USP1WT and deletion mutants ( + UAF1 ) for activity on DNA - loaded PCNA - Ub ( solid lines ) and free PCNA - Ub ( dashed lines ) shows no increase in USP1ΔL1 activity on the loaded substrate . Left panel : Quantification of gel - based activity assays showing percentage of cleaved PCNA - Ub at the mentioned time points ( n = 2 , biological replicates ) , Right panel : Quantification of the activation fold observed in USP1WT and mutants on DNA - loaded PCNA - Ub versus free PCNA - Ub ( n = 2 , biological replicates ) .

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

A) Comparison of USP1WT and deletion mutants (+UAF1) for activity on DNA-loaded PCNA-Ub (solid lines) and free PCNA-Ub (dashed lines) shows no increase in USP1ΔL1 activity on the loaded substrate. Left panel: Quantification of gel-based activity assays showing percentage of cleaved PCNA-Ub at the mentioned time points (n=2, biological replicates), Right panel: Quantification of the activation fold observed in USP1WT and mutants on DNA-loaded PCNA-Ub versus free PCNA-Ub (n=2, biological replicates).


B ) Multiple sequence alignment of USP1 insert L1 which highlights the DNA binding region and the conservation of the positively charged residues across species . Three groups of positively charged residues ( * * * ) are mutated separately and tested for binding to double stranded DNA ( 65bp ) using SPR which shows the importance of this region for DNA binding of USP1 .

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

B) Multiple sequence alignment of USP1 insert L1 which highlights the DNA binding region and the conservation of the positively charged residues across species. Three groups of positively charged residues (***) are mutated separately and tested for binding to double stranded DNA (65bp) using SPR which shows the importance of this region for DNA binding of USP1.


C ) Comparison of USP1DM1 , USP1PIP1 and USP1PIP1 + DM1 ( + UAF1 ) activity on DNA - loaded PCNA - Ub ( solid lines ) and free PCNA - Ub ( dashed lines ) shows reduced increase in activity of these mutants compared to USP1WT . Left panel : Quantification of gel based activity assays showing percentage of cleaved PCNA - Ub at the mentioned time points ( n = 2 , technical replicates ) , Right panel : Quantification of the activation fold observed in USP1WT , USP1PIP1 , USP1DM1 and USP1PIP1 + DM1 on DNA - loaded PCNA - Ub versus free PCNA - Ub ( n = 2 , technical replicates ) .

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

C) Comparison of USP1DM1, USP1PIP1 and USP1PIP1+DM1 (+UAF1) activity on DNA-loaded PCNA-Ub (solid lines) and free PCNA-Ub (dashed lines) shows reduced increase in activity of these mutants compared to USP1WT. Left panel: Quantification of gel based activity assays showing percentage of cleaved PCNA-Ub at the mentioned time points (n=2, technical replicates), Right panel: Quantification of the activation fold observed in USP1WT, USP1PIP1, USP1DM1 and USP1PIP1+DM1 on DNA-loaded PCNA-Ub versus free PCNA-Ub (n=2, technical replicates).


D ) EMSA based DNA binding experiment shows that USP1PIP1 and USP1WT has similar DNA binding capability while USP1DM1 has lost its ability to bind DNA .

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

D) EMSA based DNA binding experiment shows that USP1PIP1 and USP1WT has similar DNA binding capability while USP1DM1 has lost its ability to bind DNA.


A . BMDMM were treated as indicated with either 60ng / mL ultra - pure ( UP ) LPS , 50ng / mL human derived recombinant GM - CSF , 40µM WEHD - FMK for 16 hours or 5µM nigericin for the last hour . Supernatants were then analysed for IL - 1β secretion by ELISA . Data information : n ≥ 3 biological replicates ( every dot represents one biological replicate ) . For all panels , error bars are SEM . Significance was calculated using one way ANOVA with multiple comparisons or two - way ANOVA to compare across genotypes . p values are shown .

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

A. BMDMM were treated as indicated with either 60ng/mL ultra-pure (UP) LPS, 50ng/mL human derived recombinant GM-CSF, 40µM WEHD-FMK for 16 hours or 5µM nigericin for the last hour. Supernatants were then analysed for IL-1β secretion by ELISA. Data information: n≥ 3 biological replicates (every dot represents one biological replicate). For all panels, error bars are SEM. Significance was calculated using one way ANOVA with multiple comparisons or two-way ANOVA to compare across genotypes. p values are shown.


B . HoxB8 macrophages were treated with the indicated dilutions of GM - CSF using supernatants from GM - CSF expressing cells . Shown are ELISA analysis of IL - 1β secretion in supernatants and western blots indicating IL - 1β expression in lysates . . Data information : n ≥ 3 biological replicates ( every dot represents one biological replicate ) . For all panels , error bars are SEM . Significance was calculated using one way ANOVA with multiple comparisons or two - way ANOVA to compare across genotypes . p values are shown .

O O B-GENEPROD B-CELL_TYPE O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O B-EXP_ASSAY O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. HoxB8 macrophages were treated with the indicated dilutions of GM-CSF using supernatants from GM-CSF expressing cells. Shown are ELISA analysis of IL-1β secretion in supernatants and western blots indicating IL-1β expression in lysates.. Data information: n≥ 3 biological replicates (every dot represents one biological replicate). For all panels, error bars are SEM. Significance was calculated using one way ANOVA with multiple comparisons or two-way ANOVA to compare across genotypes. p values are shown.


C . HoxB8 macrophages of the indicated genotypes were treated as indicated with 60ng / mL UP - LPS , 1 % GM - CSF containing supernatant ( approximately 2ng / mL GM - CSF ) , 40µM WEHD . FMK , 0 , 5µM CRT - 0066101 for 16 hours , 5µM nigericin was added for the last hour . Supernatants were analysed by ELISA for IL - 1β . Inlay panels show western blots for levels of IL - 1β expression in the indicated knockouts . Data information : n ≥ 3 biological replicates ( every dot represents one biological replicate ) . For all panels , error bars are SEM . Significance was calculated using one way ANOVA with multiple comparisons or two - way ANOVA to compare across genotypes . p values are shown .

O O B-GENEPROD B-CELL_TYPE O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE O O O O O O O O O O O B-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-EXP_ASSAY I-EXP_ASSAY O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. HoxB8 macrophages of the indicated genotypes were treated as indicated with 60ng/mL UP-LPS, 1% GM-CSF containing supernatant (approximately 2ng/mL GM-CSF), 40µM WEHD.FMK, 0,5µM CRT-0066101 for 16 hours, 5µM nigericin was added for the last hour. Supernatants were analysed by ELISA for IL-1β. Inlay panels show western blots for levels of IL-1β expression in the indicated knockouts. Data information: n≥ 3 biological replicates (every dot represents one biological replicate). For all panels, error bars are SEM. Significance was calculated using one way ANOVA with multiple comparisons or two-way ANOVA to compare across genotypes. p values are shown.


D . HoxB8 cells were treated with 60ng / mL of UP - LPS , 1 % GM - CSF supernatant , 50mM KCl , and 5µM nigericin as indicated for 16 hours before supernatants were analysed for IL - 1β by ELISA . Data information : n ≥ 3 biological replicates ( every dot represents one biological replicate ) . For all panels , error bars are SEM . Significance was calculated using one way ANOVA with multiple comparisons or two - way ANOVA to compare across genotypes . p values are shown .

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

D. HoxB8 cells were treated with 60ng/mL of UP-LPS, 1% GM-CSF supernatant, 50mM KCl, and 5µM nigericin as indicated for 16 hours before supernatants were analysed for IL-1β by ELISA. Data information: n≥ 3 biological replicates (every dot represents one biological replicate). For all panels, error bars are SEM. Significance was calculated using one way ANOVA with multiple comparisons or two-way ANOVA to compare across genotypes. p values are shown.


A . BMDMM ( left panel ) and HoxB8 macrophages ( right panel ) were treated as indicated with either LPS or LPS + GM - CSF . Cells were harvested and proteins extracted and analysed by western blot for levels of NRF2 . Shown is the densitometric quantification of Nrf2 protein level from three independent experiments . Data information : n ≥ 3 biological replicates ( every dot represents one biological replicate ) . For all panels , error bars are SEM . Significance was calculated using one way ANOVA with multiple comparisons , or two - way ANOVA to compare across genotypes . For the western blot quantification , significance was calculated using unpaired t - test . p values are shown .

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

A. BMDMM (left panel) and HoxB8 macrophages (right panel) were treated as indicated with either LPS or LPS+ GM-CSF. Cells were harvested and proteins extracted and analysed by western blot for levels of NRF2. Shown is the densitometric quantification of Nrf2 protein level from three independent experiments. Data information: n≥ 3 biological replicates (every dot represents one biological replicate). For all panels, error bars are SEM. Significance was calculated using one way ANOVA with multiple comparisons, or two-way ANOVA to compare across genotypes. For the western blot quantification, significance was calculated using unpaired t-test. p values are shown.


B . HoxB8 macrophages were treated as in A . and levels of IL - 1β in the supernatant were analysed by ELISA Data information : n ≥ 3 biological replicates ( every dot represents one biological replicate ) . For all panels , error bars are SEM . Significance was calculated using one way ANOVA with multiple comparisons , or two - way ANOVA to compare across genotypes . For the western blot quantification , significance was calculated using unpaired t - test . p values are shown .

O O B-GENEPROD B-CELL_TYPE O O O O O O O O O B-GENEPROD I-GENEPROD I-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 O O O O O O O O O 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. HoxB8 macrophages were treated as in A. and levels of IL-1β in the supernatant were analysed by ELISA Data information: n≥ 3 biological replicates (every dot represents one biological replicate). For all panels, error bars are SEM. Significance was calculated using one way ANOVA with multiple comparisons, or two-way ANOVA to compare across genotypes. For the western blot quantification, significance was calculated using unpaired t-test. p values are shown.


C . HoxB8 macrophages were treated for 12 hours with LPS or LPS + GM - CSF and RNA extracted and analysed by qPCR for the indicated targets . Data information : n ≥ 3 biological replicates ( every dot represents one biological replicate ) . For all panels , error bars are SEM . Significance was calculated using one way ANOVA with multiple comparisons , or two - way ANOVA to compare across genotypes . For the western blot quantification , significance was calculated using unpaired t - test . p values are shown .

O O B-GENEPROD B-CELL_TYPE O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O B-GENEPROD I-GENEPROD I-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 O O O O O O O O O O O O O O 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. HoxB8 macrophages were treated for 12 hours with LPS or LPS+GM-CSF and RNA extracted and analysed by qPCR for the indicated targets. Data information: n≥ 3 biological replicates (every dot represents one biological replicate). For all panels, error bars are SEM. Significance was calculated using one way ANOVA with multiple comparisons, or two-way ANOVA to compare across genotypes. For the western blot quantification, significance was calculated using unpaired t-test. p values are shown.


D . Wild type and Tnf - / - HoxB8 cells were treated as indicated . Cells were harvested and proteins extracted and analysed by western blot for levels of NRF2 and HMOX1 . For wild type cells , MitoTEMPO ( 50µM ) was also added as indicated . Shown are the densitometric quantifications of NRF2 and HMOX1 protein levels of three independent experiments ( right panel ) . Data information : n ≥ 3 biological replicates ( every dot represents one biological replicate ) . For all panels , error bars are SEM . Significance was calculated using one way ANOVA with multiple comparisons , or two - way ANOVA to compare across genotypes . For the western blot quantification , significance was calculated using unpaired t - test . p values are shown .

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-EXP_ASSAY I-EXP_ASSAY O O O B-GENEPROD O B-GENEPROD O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O B-EXP_ASSAY O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Wild type and Tnf-/- HoxB8 cells were treated as indicated. Cells were harvested and proteins extracted and analysed by western blot for levels of NRF2 and HMOX1. For wild type cells, MitoTEMPO (50µM) was also added as indicated. Shown are the densitometric quantifications of NRF2 and HMOX1 protein levels of three independent experiments (right panel). Data information: n≥ 3 biological replicates (every dot represents one biological replicate). For all panels, error bars are SEM. Significance was calculated using one way ANOVA with multiple comparisons, or two-way ANOVA to compare across genotypes. For the western blot quantification, significance was calculated using unpaired t-test. p values are shown.


E . HoxB8 cells of the indicated genotypes were treated with LPS for 8 hours followed by staining with mitoSOX red . The fluorescent signal was analyzed using flow cytometry . Shown are average geometric mean fluorescence intensity of mitoSOX red . Data information : n ≥ 3 biological replicates ( every dot represents one biological replicate ) . For all panels , error bars are SEM . Significance was calculated using one way ANOVA with multiple comparisons , or two - way ANOVA to compare across genotypes . For the western blot quantification , significance was calculated using unpaired t - test . p values are shown .

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

E. HoxB8 cells of the indicated genotypes were treated with LPS for 8 hours followed by staining with mitoSOX red. The fluorescent signal was analyzed using flow cytometry. Shown are average geometric mean fluorescence intensity of mitoSOX red. Data information: n≥ 3 biological replicates (every dot represents one biological replicate). For all panels, error bars are SEM. Significance was calculated using one way ANOVA with multiple comparisons, or two-way ANOVA to compare across genotypes. For the western blot quantification, significance was calculated using unpaired t-test. p values are shown.


F . Tnf - / - HoxB8 macrophages were treated with LPS with or without 50ng / ml recombinant TNF for 16 hours . Cells were harvested and proteins extracted and analysed by western blot for levels of NRF2 and HMOX1 ( upper panel ) . Shown are the densitometric quantifications of indicated protein levels of three independent experiments ( lower panel ) . Data information : n ≥ 3 biological replicates ( every dot represents one biological replicate ) . For all panels , error bars are SEM . Significance was calculated using one way ANOVA with multiple comparisons , or two - way ANOVA to compare across genotypes . For the western blot quantification , significance was calculated using unpaired t - test . p values are shown .

O O B-GENEPROD O O O B-GENEPROD B-CELL_TYPE O O O B-SMALL_MOLECULE 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 I-EXP_ASSAY O O O B-GENEPROD O B-GENEPROD O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Tnf-/- HoxB8 macrophages were treated with LPS with or without 50ng/ml recombinant TNF for 16 hours. Cells were harvested and proteins extracted and analysed by western blot for levels of NRF2 and HMOX1 (upper panel). Shown are the densitometric quantifications of indicated protein levels of three independent experiments (lower panel). Data information: n≥ 3 biological replicates (every dot represents one biological replicate). For all panels, error bars are SEM. Significance was calculated using one way ANOVA with multiple comparisons, or two-way ANOVA to compare across genotypes. For the western blot quantification, significance was calculated using unpaired t-test. p values are shown.


A . HoxB8 macrophages were seeded in an 8 - well Ibidi chamber and treated as indicated for 12 hours . Cells were fixed and stained for ASC ( Red ) and with DAPI ( blue ) . For nigericin treatments nigericin was added for only 30 minutes . Example cells containing ASC Specks are shown with white arrows . Scale bars represent 50µM . Data information : Microscopy pictures are representative images from 3 biological replicates .

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

A. HoxB8 macrophages were seeded in an 8-well Ibidi chamber and treated as indicated for 12 hours. Cells were fixed and stained for ASC (Red) and with DAPI (blue). For nigericin treatments nigericin was added for only 30 minutes. Example cells containing ASC Specks are shown with white arrows. Scale bars represent 50µM. Data information: Microscopy pictures are representative images from 3 biological replicates.


B . HoxB8 cells were treated as indicated with LPS or LPS + GM - CSF . The cross linker DSS was added to the cells as described in methods . Samples were analysed by western blot for ASC oligomerization . Black vertical lines indicate that non - relevant lanes were removed during figure preparation but samples were run on the same gel . Data information : westerns are representative images from 3 biological replicates .

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

B. HoxB8 cells were treated as indicated with LPS or LPS+GM-CSF. The cross linker DSS was added to the cells as described in methods. Samples were analysed by western blot for ASC oligomerization. Black vertical lines indicate that non-relevant lanes were removed during figure preparation but samples were run on the same gel. Data information: westerns are representative images from 3 biological replicates.


C . Wild type and caspase - 1 - / - HoxB8 macrophages were treated as indicated for 16 hours . Cell lysates were made and analysed by western blot for levels of caspase - 1 and gasdermin D cleavage . Data information : westerns are representative images from 3 biological replicates .

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

C. Wild type and caspase-1-/- HoxB8 macrophages were treated as indicated for 16 hours. Cell lysates were made and analysed by western blot for levels of caspase-1 and gasdermin D cleavage. Data information: westerns are representative images from 3 biological replicates.


D . HoxB8 macrophages were treated as indicated with LPS or LPS + GM - CSF for 12 hours . Cells were lysed and NLRP3 was immune precipitated using a magnetic bead coupled antibody . As a negative control , beads without NLRP3 antibody were used . Beads were boiled and analysed for recruitment of NLRP3 , and caspase - 1 . * indicates light chain from the antibody . Molecular weight markers shown in middle of NLRP3 blot were embedded in image using Chemostar imaging software ( INTAS ) at time of image acquisition . Data information : westerns are representative images from 3 biological replicates .

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

D. HoxB8 macrophages were treated as indicated with LPS or LPS+GM-CSF for 12 hours. Cells were lysed and NLRP3 was immune precipitated using a magnetic bead coupled antibody. As a negative control, beads without NLRP3 antibody were used. Beads were boiled and analysed for recruitment of NLRP3, and caspase-1. * indicates light chain from the antibody. Molecular weight markers shown in middle of NLRP3 blot were embedded in image using Chemostar imaging software (INTAS) at time of image acquisition. Data information: westerns are representative images from 3 biological replicates.


E . HoxB8 macrophages were incubated with combinations of LPS , GM - CSF , nigericin + / - Z - VAD . FMK as indicated . Proteins were extracted and analysed by western blot for caspase - 1 processing . Data information : westerns are representative images from 3 biological replicates .

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

E. HoxB8 macrophages were incubated with combinations of LPS, GM-CSF, nigericin +/- Z-VAD.FMK as indicated. Proteins were extracted and analysed by western blot for caspase-1 processing. Data information: westerns are representative images from 3 biological replicates.


F . HoxB8 macrophages were seeded in a 96 well microplate and co - treated with either 10µM or 50µM z - VAD with LPS + GM - CSF for 16 hours . Then 100nM of Sytox Green was added to the cells . The number of sytox green + cells per image was quantified . Data information : Significance was calculated using one way ANOVA with multiple comparisons . Every dot represents one biological replicate . P values are shown .

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

F. HoxB8 macrophages were seeded in a 96 well microplate and co-treated with either 10µM or 50µM z-VAD with LPS+GM-CSF for 16 hours. Then 100nM of Sytox Green was added to the cells. The number of sytox green+ cells per image was quantified. Data information: Significance was calculated using one way ANOVA with multiple comparisons. Every dot represents one biological replicate. P values are shown.


A . HoxB8 macrophages were treated for 16 hours with LPS + / - GM - CSF as indicated . Proteins were extracted and analysed for levels of IL - 1β by western blot .

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

A. HoxB8 macrophages were treated for 16 hours with LPS +/- GM-CSF as indicated. Proteins were extracted and analysed for levels of IL-1β by western blot.


B . HoxB8 macrophages were treated with LPS or LPS + GM - CSF as indicated for 16 hours . Cells were lysed in buffer containing FLAG tagged K63 TUBE and K63 ubiquitylated proteins were precipitated using anti FLAG pull down . Pulled down proteins were analysed for IL - 1β and ubiquitin by western blot .

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

B. HoxB8 macrophages were treated with LPS or LPS+GM-CSF as indicated for 16 hours. Cells were lysed in buffer containing FLAG tagged K63 TUBE and K63 ubiquitylated proteins were precipitated using anti FLAG pull down. Pulled down proteins were analysed for IL-1β and ubiquitin by western blot.


C . IL - 1β - / - hoxB8 macrophages were infected with lentivirus expressing IL - 1β - FLAG - 6xHis or the indicated mutants from the IL - 1β promoter . Cells were treated as indicated with LPS or LPS + GM - CSF or LPS + Nigericin and levels of secreted IL - 1β were analysed by ELISA ( top panel ) . GM - CSF treated cells were also lysed in urea buffer and nickel NTA affinity pull down was performed . Nickel NTA pull down was also performed on supernatants ( bottom panel ) . Pulled down proteins form the His tag pull down were analysed for the presence of IL - 1β by western blot . Molecular weight markers shown in middle of blot were embedded in image using Chemostar imaging software ( INTAS ) at time of image acquisition . Data information : n ≥ 3 biological replicates ( every dot represents one biological replicate ) . For all panels , error bars are SEM . Significance was calculated using one way ANOVA with multiple comparisons , or two - way ANOVA to compare across genotypes . p values are shown .

O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-GENEPROD B-CELL_TYPE O O O B-ORGANISM O B-GENEPROD I-GENEPROD I-GENEPROD 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 B-SMALL_MOLECULE O B-SMALL_MOLECULE O B-GENEPROD I-GENEPROD I-GENEPROD O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-EXP_ASSAY O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O B-SMALL_MOLECULE O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O B-GENEPROD O B-EXP_ASSAY I-EXP_ASSAY O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-EXP_ASSAY I-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 O O O O O O O O O O O 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. IL-1β-/- hoxB8 macrophages were infected with lentivirus expressing IL-1β-FLAG-6xHis or the indicated mutants from the IL-1β promoter. Cells were treated as indicated with LPS or LPS+GM-CSF or LPS+Nigericin and levels of secreted IL-1β were analysed by ELISA (top panel). GM-CSF treated cells were also lysed in urea buffer and nickel NTA affinity pull down was performed. Nickel NTA pull down was also performed on supernatants (bottom panel). Pulled down proteins form the His tag pull down were analysed for the presence of IL-1β by western blot. Molecular weight markers shown in middle of blot were embedded in image using Chemostar imaging software (INTAS) at time of image acquisition. Data information: n≥ 3 biological replicates (every dot represents one biological replicate). For all panels, error bars are SEM. Significance was calculated using one way ANOVA with multiple comparisons, or two-way ANOVA to compare across genotypes. p values are shown.


D . HoxB8 macrophages were treated as in A with addition of 50µM mitoTEMPO or 10mM NAC as indicated . Proteins were extracted and analysed for IL - 1β by western blot .

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

D. HoxB8 macrophages were treated as in A with addition of 50µM mitoTEMPO or 10mM NAC as indicated. Proteins were extracted and analysed for IL-1β by western blot.


E . Wild type and TNF - / - HoxB8 macrophages were treated as indicated with LPS or LPS + GM - CSF . Proteins were extracted and analysed for IL - 1β by western blot . Black vertical lines indicate that non - relevant lanes were removed during figure preparation but samples were run on the same gel .

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

E. Wild type and TNF-/- HoxB8 macrophages were treated as indicated with LPS or LPS+GM-CSF. Proteins were extracted and analysed for IL-1β by western blot. Black vertical lines indicate that non-relevant lanes were removed during figure preparation but samples were run on the same gel.


F . HoxB8 macrophages were treated as in A but with the addition of 25µM Punicalagin throughout the 16 hour time course . Proteins were extracted and analysed by western blot ( left panel ) . Supernatants were also analysed by ELISA for secreted IL - 1β ( right panel ) . Data information : n ≥ 3 biological replicates ( every dot represents one biological replicate ) . For all panels , error bars are SEM . Significance was calculated using one way ANOVA with multiple comparisons , or two - way ANOVA to compare across genotypes . p values are shown .

O O B-GENEPROD B-CELL_TYPE O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O B-EXP_ASSAY O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. HoxB8 macrophages were treated as in A but with the addition of 25µM Punicalagin throughout the 16 hour time course. Proteins were extracted and analysed by western blot (left panel). Supernatants were also analysed by ELISA for secreted IL-1β (right panel). Data information: n≥ 3 biological replicates (every dot represents one biological replicate). For all panels, error bars are SEM. Significance was calculated using one way ANOVA with multiple comparisons, or two-way ANOVA to compare across genotypes. p values are shown.


A . Wild type and GsdmD - / - HoxB8 macrophages were treated as indicated for 16 hours . Supernatants were analyzed for IL - 1β secretion by ELISA . Data information : n ≥ 3 biological replicates ( every dot represents one biological replicate ) . For all panels , error bars are SEM . Significance was calculated using one way ANOVA with multiple comparisons , or two - way ANOVA to compare across genotypes . p values are shown .

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

A. Wild type and GsdmD-/- HoxB8 macrophages were treated as indicated for 16 hours. Supernatants were analyzed for IL-1β secretion by ELISA. Data information: n≥ 3 biological replicates (every dot represents one biological replicate). For all panels, error bars are SEM. Significance was calculated using one way ANOVA with multiple comparisons, or two-way ANOVA to compare across genotypes. p values are shown.


B . HoxB8 macrophages were seeded in an 8 - well Ibidi chamber and treated for 14 hours with LPS and GM - CSF . Cells were fixed and stained for GasderminD ( green ) , IL - 1β ( red ) and DAPI ( blue ) . Images were taken at the Zeiss LSM880 Confocal microscope Data information : shown . Microscopy pictures are representative images from 3 biological replicates . Scale bars in microscopy images represent 10µM .

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

B. HoxB8 macrophages were seeded in an 8-well Ibidi chamber and treated for 14 hours with LPS and GM-CSF. Cells were fixed and stained for GasderminD (green), IL-1β (red) and DAPI (blue). Images were taken at the Zeiss LSM880 Confocal microscope Data information: shown. Microscopy pictures are representative images from 3 biological replicates. Scale bars in microscopy images represent 10µM.


C . HoxB8 macrophages were co - stimulated with three different PEG 3mM for 16 hours as indicated . Supernatants were analyzed for IL - 1β secretion by ELISA . Data information : n ≥ 3 biological replicates ( every dot represents one biological replicate ) . For all panels , error bars are SEM . Significance was calculated using one way ANOVA with multiple comparisons , or two - way ANOVA to compare across genotypes . p values are shown .

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

C. HoxB8 macrophages were co-stimulated with three different PEG 3mM for 16 hours as indicated. Supernatants were analyzed for IL-1β secretion by ELISA. Data information: n≥ 3 biological replicates (every dot represents one biological replicate). For all panels, error bars are SEM. Significance was calculated using one way ANOVA with multiple comparisons, or two-way ANOVA to compare across genotypes. p values are shown.


D . HoxB8 macrophages were seeded in 96 well microplate and treated as indicated . Before treatment , sytox green was added and cells were imaged in an incucyte incubator microscope every hour for 16 hours . Shown is the sytox green signal normalized to the total cell area . Data information : n ≥ 3 biological replicates ( every dot represents one biological replicate ) . For all panels , error bars are SEM . Significance was calculated using one way ANOVA with multiple comparisons , or two - way ANOVA to compare across genotypes . p values are shown .

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

D. HoxB8 macrophages were seeded in 96 well microplate and treated as indicated. Before treatment, sytox green was added and cells were imaged in an incucyte incubator microscope every hour for 16 hours. Shown is the sytox green signal normalized to the total cell area. Data information: n≥ 3 biological replicates (every dot represents one biological replicate). For all panels, error bars are SEM. Significance was calculated using one way ANOVA with multiple comparisons, or two-way ANOVA to compare across genotypes. p values are shown.


E . Wild type and GsdmD - / - HoxB8 macrophages were treated as indicated for 16 hours . Proteins were extracted and analysed for IL - 1β ubiquitination by western blot . Data information : westerns are representative images from 3 biological replicates .

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

E. Wild type and GsdmD-/- HoxB8 macrophages were treated as indicated for 16 hours. Proteins were extracted and analysed for IL-1β ubiquitination by western blot. Data information: westerns are representative images from 3 biological replicates.


( a ) Representative confocal micrographs of mitochondrial morphology in MEFs of the indicated genotype 24 h after transfection with mtYFP . Where indicated , cells were starved for 2 . 5 h . Scale bar , 20 μm . ( b ) Morphometric analysis of mitochondrial shape . Experiments were carried out as in a . Data represent mean ± s . e . m . of three independent experiments ( n = 100 cells per condition in each experiment ) .

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

(a) Representative confocal micrographs of mitochondrial morphology in MEFs of the indicated genotype 24 h after transfection with mtYFP. Where indicated, cells were starved for 2.5 h. Scale bar, 20 μm. (b) Morphometric analysis of mitochondrial shape. Experiments were carried out as in a. Data represent mean ± s.e.m. of three independent experiments (n=100 cells per condition in each experiment).


( c ) Top left , 48 h after transfection with the indicated siRNA , MEFs were lysed and 25 μg samples of proteins were separated by DS - polyacrylamide gel electrophoresis ( PAGE ) and immunoblotted with the indicated antibodies . Top right and bottom , representative images show mitochondrial morphology of MEFs transfected with the indicated siRNA and after 24 h with mtYFP . After a further 24 h , confocal micrographs were acquired . Scale bar , 20 μm . Uncropped images of blots are shown in Supplementary Fig . S8 . ( d ) Morphometric analysis of mitochondrial shape . Experiments were carried out as in c . Data represent mean ± s . e . m . of five independent experiments ( n = 100 cells per condition in each experiment ) .

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

(c) Top left, 48 h after transfection with the indicated siRNA, MEFs were lysed and 25 μg samples of proteins were separated by DS-polyacrylamide gel electrophoresis (PAGE) and immunoblotted with the indicated antibodies. Top right and bottom, representative images show mitochondrial morphology of MEFs transfected with the indicated siRNA and after 24 h with mtYFP. After a further 24 h, confocal micrographs were acquired. Scale bar, 20 μm. Uncropped images of blots are shown in Supplementary Fig. S8. (d) Morphometric analysis of mitochondrial shape. Experiments were carried out as in c. Data represent mean ± s.e.m. of five independent experiments (n=100 cells per condition in each experiment).


( e ) Representative images of mitochondrial fusion over time ( indicated in min ) . MEFs were co - transfected with mtPAGFP and mtRFP and after 24 h , mtPAGFP was photoactivated in a region of interest and cells were imaged by real - time confocal microscopy . Where indicated , MEFs were starved for 2 . 5 h . Scale bar , 20 μm . See also Supplementary Movies S1 and S2 . ( f , g ) Quantification of mitochondrial fusion in MEFs of the indicated genotype . Experiments were carried out as in e . Data represent mean ± s . e . m . of four independent experiments .

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

(e) Representative images of mitochondrial fusion over time (indicated in min). MEFs were co-transfected with mtPAGFP and mtRFP and after 24 h, mtPAGFP was photoactivated in a region of interest and cells were imaged by real-time confocal microscopy. Where indicated, MEFs were starved for 2.5 h. Scale bar, 20 μm. See also Supplementary Movies S1 and S2. (f,g) Quantification of mitochondrial fusion in MEFs of the indicated genotype. Experiments were carried out as in e. Data represent mean ± s.e.m. of four independent experiments.


( h ) Representative electron micrographs of muscle ( longitudinal sections ; four top micrographs ) and liver ( four bottom micrographs ) from CD1 mice . Where indicated , mice were fasted for 12 h . The outlined regions of micrographs from muscle samples are magnified × 7 in the images below . Scale bars , 2 μm .

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

(h) Representative electron micrographs of muscle (longitudinal sections; four top micrographs) and liver (four bottom micrographs) from CD1 mice. Where indicated, mice were fasted for 12 h. The outlined regions of micrographs from muscle samples are magnified ×7 in the images below. Scale bars, 2 μm.


( a - d ) Levels of mitochondria - shaping proteins during starvation . Protein samples ( 20 μg ) from MEFs of the indicated genotype were separated by SDS - PAGE and immunoblotted with the indicated antibodies . Cells were starved for the indicated times .

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

(a-d) Levels of mitochondria-shaping proteins during starvation. Protein samples (20 μg) from MEFs of the indicated genotype were separated by SDS-PAGE and immunoblotted with the indicated antibodies. Cells were starved for the indicated times.


( e ) Association of DRP1 with mitochondria during starvation . Mitochondria were isolated from MEFs starved for the indicated times and 25 μg samples of proteins were separated by SDS - PAGE and immunoblotted with the indicated antibodies . ( f ) Levels of Ser 637 phosphorylation of DRP1 during starvation . Equal amounts of cell lysates from wild - type MEFs starved for the indicated times were immunoprecipitated with the indicated antibody and the immunoprecipitated proteins were separated by SDS - PAGE and immunoblotted with the indicated antibodies . ( g ) Quantitative analysis of Ser 637 phosphorylation of DRP1 during starvation . Experiments were carried out as in f . Data are normalized to total levels of DRP1 and represent the mean ± s . e . m . of three independent experiments .

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

(e) Association of DRP1 with mitochondria during starvation. Mitochondria were isolated from MEFs starved for the indicated times and 25 μg samples of proteins were separated by SDS-PAGE and immunoblotted with the indicated antibodies. (f) Levels of Ser 637 phosphorylation of DRP1 during starvation. Equal amounts of cell lysates from wild-type MEFs starved for the indicated times were immunoprecipitated with the indicated antibody and the immunoprecipitated proteins were separated by SDS-PAGE and immunoblotted with the indicated antibodies. (g) Quantitative analysis of Ser 637 phosphorylation of DRP1 during starvation. Experiments were carried out as in f. Data are normalized to total levels of DRP1 and represent the mean ± s.e.m. of three independent experiments.


( h ) MEFs were treated for the indicated times with 100 nM rapamycin or with 25 μM forskolin ( FRSK ) for 0 . 5 h , lysed and equal amounts ( 50 μg ) of proteins were separated by SDS - PAGE and immunoblotted using the indicated antibodies .

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

(h) MEFs were treated for the indicated times with 100 nM rapamycin or with 25 μM forskolin (FRSK) for 0.5 h, lysed and equal amounts (50 μg) of proteins were separated by SDS-PAGE and immunoblotted using the indicated antibodies.


( i ) HeLa cells were transfected for 2 days with the indicated siRNA or treated with 25 μM forskolin for 0 . 5 h and lysed . Equal amounts ( 50 μg ) of proteins were separated by SDS - PAGE and immunoblotted using the indicated antibodies . Uncropped images of all blots in this figure are shown in Supplementary Fig . S8 .

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

(i) HeLa cells were transfected for 2 days with the indicated siRNA or treated with 25 μM forskolin for 0.5 h and lysed. Equal amounts (50 μg) of proteins were separated by SDS-PAGE and immunoblotted using the indicated antibodies. Uncropped images of all blots in this figure are shown in Supplementary Fig. S8.


( a ) Pseudocolour - coded images of EPACI - camps FRET from real - time imaging of wild - type MEFs transfected with EPACI - camps . Where indicated , cells were perfused with the starvation solution for the indicated times ( in min ) or with 25 μM forskolin ( FRSK ) . Scale bar , 20 μm . Colour scale indicates F1 / F0 ratio . See also Supplementary Movie S3 . ( b ) Quantitative analysis of CFP / YFP FRET ratio . Experiments were carried out as in a . Where indicated , cells were perfused with the starvation solution or with 25 μM forskolin . Data represent mean ± s . e . m . of 13 independent experiments .

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

(a) Pseudocolour-coded images of EPACI-camps FRET from real-time imaging of wild-type MEFs transfected with EPACI-camps. Where indicated, cells were perfused with the starvation solution for the indicated times (in min) or with 25 μM forskolin (FRSK). Scale bar, 20 μm. Colour scale indicates F1/F0 ratio. See also Supplementary Movie S3. (b) Quantitative analysis of CFP/YFP FRET ratio. Experiments were carried out as in a. Where indicated, cells were perfused with the starvation solution or with 25 μM forskolin. Data represent mean ± s.e.m. of 13 independent experiments.


( c ) Lysate samples ( 50 μg ) from MEFs of the indicated genotypes were analysed by SDS - PAGE and immunoblotting using the indicated antibodies . Where indicated , MEFs were starved , or treated with 25 μM forskolin . Where indicated , 20 μM H89 was added during starvation . Uncropped images of all blots in this figure are shown in Supplementary Fig . S8 .

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

(c) Lysate samples (50 μg) from MEFs of the indicated genotypes were analysed by SDS-PAGE and immunoblotting using the indicated antibodies. Where indicated, MEFs were starved, or treated with 25 μM forskolin. Where indicated, 20 μM H89 was added during starvation. Uncropped images of all blots in this figure are shown in Supplementary Fig. S8.