Figure 7 ( A , B ) Expression of quantitative RT - PCR + ( A ) using quantitative RT - PCR of RNA extracted from cells grown either in rich medium ( dark gray ) or starved for 2 hours ( light grey ) . Cells of the following genotypes were analyzed : wild - type isogenic controls ( WT ) and taf12 - 5A mutants . act1 + served as a control for normalization across samples . Values from a WT strain grown in rich medium were set at 1 to allow comparisons across culture conditions and mutant strains . Each column represents the mean value of 6 independent experiments , overlaid with individual data points and SE . Statistical significance was determined by 2 - way ANOVA followed by Bonferroni ' s multiple comparison tests ( n = 6 ) . mei2 + ( B ) using quantitative RT - PCR of RNA extracted from cells grown either in rich medium ( dark gray ) or starved for 2 hours ( light grey ) . Cells of the following genotypes were analyzed : wild - type isogenic controls ( WT ) and taf12 - 5A mutants . act1 + served as a control for normalization across samples . Values from a WT strain grown in rich medium were set at 1 to allow comparisons across culture conditions and mutant strains . Each column represents the mean value of 6 independent experiments , overlaid with individual data points and SE . Statistical significance was determined by 2 - way ANOVA followed by Bonferroni ' s multiple comparison tests ( n = 6 ) . ( C , E ) Zygotes and tetrads , which correspond to differentiated cells , were counted from cultures of homothallic cells grown to mid - log phase in rich medium ( t0 ) and shifted to starvation medium for up to 24 hours ( C ) Cells of the following genotypes were analyzed : wild - type isogenic controls ( WT ) , taf12 - 5A , taf12 - 5DE , tor1 - 1972A , and taf12 - 5A tor1 - 1972A . Each value represents the mean percentage and SE of differentiating cells to the total number of cells , averaged from 6 independent experiments . At least 200 cells from the indicated genotypes were counted in each experiment . Statistical significance was determined by 2 - way ANOVA followed by Tukey ' s multiple comparison tests ( n = 6 ) . ( D ) P - Taf12 was followed by anti - FLAG IB of protein extracts from WT and tor1 - 1972A strains , grown in rich medium and shifted to starved conditions for 60 minutes . An anti - Tubulin IB is shown as a control for loading . Shown is an IB which is representative of 2 independent experiments . ( C , E ) taf12 and tetrads , which correspond to differentiated cells , were counted from heterothallic cells mixed and incubated for 2 days on mating medium ( E ) . Cells of the following genotypes were analyzed : wild - type isogenic controls ( WT ) , taf12 - 5A , taf12 - 5DE , tor1 - 1972A , and taf12 - 5A tor1 - 1972A . Each value represents the mean percentage and SE of differentiating cells to the total number of cells , averaged from 6 independent experiments . At least 200 cells from the indicated genotypes were counted in each experiment . Statistical significance was determined by 2 - way ANOVA followed by Tukey ' s multiple comparison tests ( n = 6 ) .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 7(A,B) Expression of quantitative RT-PCR+ (A) using quantitative RT-PCR of RNA extracted from cells grown either in rich medium (dark gray) or starved for 2 hours (light grey). Cells of the following genotypes were analyzed: wild-type isogenic controls (WT) and taf12-5A mutants. act1+ served as a control for normalization across samples. Values from a WT strain grown in rich medium were set at 1 to allow comparisons across culture conditions and mutant strains. Each column represents the mean value of 6 independent experiments, overlaid with individual data points and SE. Statistical significance was determined by 2-way ANOVA followed by Bonferroni's multiple comparison tests (n = 6).mei2+ (B) using quantitative RT-PCR of RNA extracted from cells grown either in rich medium (dark gray) or starved for 2 hours (light grey). Cells of the following genotypes were analyzed: wild-type isogenic controls (WT) and taf12-5A mutants. act1+ served as a control for normalization across samples. Values from a WT strain grown in rich medium were set at 1 to allow comparisons across culture conditions and mutant strains. Each column represents the mean value of 6 independent experiments, overlaid with individual data points and SE. Statistical significance was determined by 2-way ANOVA followed by Bonferroni's multiple comparison tests (n = 6).(C,E) Zygotes and tetrads, which correspond to differentiated cells, were counted from cultures of homothallic cells grown to mid-log phase in rich medium (t0) and shifted to starvation medium for up to 24 hours (C) Cells of the following genotypes were analyzed: wild-type isogenic controls (WT), taf12-5A, taf12-5DE, tor1-1972A, and taf12-5A tor1-1972A. Each value represents the mean percentage and SE of differentiating cells to the total number of cells, averaged from 6 independent experiments. At least 200 cells from the indicated genotypes were counted in each experiment. Statistical significance was determined by 2-way ANOVA followed by Tukey's multiple comparison tests (n = 6).(D) P-Taf12 was followed by anti-FLAG IB of protein extracts from WT and tor1-1972A strains, grown in rich medium and shifted to starved conditions for 60 minutes. An anti-Tubulin IB is shown as a control for loading. Shown is an IB which is representative of 2 independent experiments.(C,E) taf12 and tetrads, which correspond to differentiated cells, were counted from heterothallic cells mixed and incubated for 2 days on mating medium (E). Cells of the following genotypes were analyzed: wild-type isogenic controls (WT), taf12-5A, taf12-5DE, tor1-1972A, and taf12-5A tor1-1972A. Each value represents the mean percentage and SE of differentiating cells to the total number of cells, averaged from 6 independent experiments. At least 200 cells from the indicated genotypes were counted in each experiment. Statistical significance was determined by 2-way ANOVA followed by Tukey's multiple comparison tests (n = 6).


Figure 2A ) IL - 8 production after 6 h of treatment with digitonin and βHBP at indicated concentrations or digitonin and wt S . flexneri lysate in HeLa cells ( Hoechst in blue , IL - 8 in red ) after 6 h of treatment with digitonin and βHBP at indicated concentrations or digitonin and wt S . flexneri lysate in HeLa cell B ) Quantification of IL - 8 awith digitonin and βHBP at indicated concentrations or digitonin and wt S . flexneri lysat C ) TIFA oligomerization at 6 h in TIFA - GFP - expressing HeLa cells treatewith digitonin and βHBP at indicated concentrations or digitonin and wt S . flexneri lysat at 6 h in TIFA - GFP - expressing HeLa cells treate D ) Quantification of TIFA oligomerizatiowith digitonin and βHBP at indicated concentrations or digitonin and wt S . flexneri lysat E ) TIFA oligomerization at 30 min in cells treateF ) Kinetics of TIFA - GFP oligomerization upon treatment with digitonin alone or in combination with βHBP or S . flexneri

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Figure 2A) IL-8 production after 6 h of treatment with digitonin and βHBP at indicated concentrations or digitonin and wt S. flexneri lysate in HeLa cells (Hoechst in blue, IL-8 in red)after 6 h of treatment with digitonin and βHBP at indicated concentrations or digitonin and wt S. flexneri lysate in HeLa cell B) Quantification of IL-8 awith digitonin and βHBP at indicated concentrations or digitonin and wt S. flexneri lysat C) TIFA oligomerization at 6 h in TIFA-GFP-expressing HeLa cells treatewith digitonin and βHBP at indicated concentrations or digitonin and wt S. flexneri lysat at 6 h in TIFA-GFP-expressing HeLa cells treate D) Quantification of TIFA oligomerizatiowith digitonin and βHBP at indicated concentrations or digitonin and wt S. flexneri lysat E) TIFA oligomerization at 30 min in cells treateF) Kinetics of TIFA-GFP oligomerization upon treatment with digitonin alone or in combination with βHBP or S. flexneri lysate


Figure 3B ) TIFA - GFP oligomerization at 30 min in HeLa cells treated with digitonin ( mock ) or digitonin and a lysate from indicated S . flexneri strains ( pHldA / C means plasmid - encoded HldA and HldC ) with digitonin ( mock ) or digitonin and a lysate from indicated S . flexneri strain ( pHldA / C means plasmid - encoded HldA and HldC ) C ) TIFA - GFP oligomerization at 6 h in cells treatewith digitonin ( mock ) or digitonin and a lysate from indicated S . flexneri strains ( pHldA / C means plasmid - encoded HldA and HldC ) D ) IL - 8 production at 6 h in cells were treateE ) TIFA - GFP oligomerization in cells treated for 30 min with digitonin alone , digitonin and wild - type S . flexneri lysate or digitonin and ADP - L - β - d - heptose at indicated concentrationswith digitonin alone , digitonin and wild - type S . flexneri lysate or digitonin and ADP - L - β - d - heptose at indicated concentrations . F ) IL - 8 production after 6 h in cells

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Figure 3B) TIFA-GFP oligomerization at 30 min in HeLa cells treated with digitonin (mock) or digitonin and a lysate from indicated S. flexneri strains (pHldA/C means plasmid-encoded HldA and HldC)with digitonin (mock) or digitonin and a lysate from indicated S. flexneri strain (pHldA/C means plasmid-encoded HldA and HldC) C) TIFA-GFP oligomerization at 6 h in cells treatewith digitonin (mock) or digitonin and a lysate from indicated S. flexneri strains (pHldA/C means plasmid-encoded HldA and HldC) D) IL-8 production at 6 h in cells were treateE) TIFA-GFP oligomerization in cells treated for 30 min with digitonin alone, digitonin and wild-type S. flexneri lysate or digitonin and ADP-L-β-d-heptose at indicated concentrationswith digitonin alone, digitonin and wild-type S. flexneri lysate or digitonin and ADP-L-β-d-heptose at indicated concentrations. F) IL-8 production after 6 h in cells treate


Figure 4A ) Representative images of the formation of TIFA oligomers in HeLa cells infected for 30 min with dsRed - expressing wt or indicated mutants of S . flexneri ( MOI 50 ) . pHldA / C means plasmid - encoded HldA and HldC . Fluorescence intensity was adjusted between strains to optimize visualization of bacteria . Scale bar , 20 μmB ) Infectivity of indicated S . flexneri strainsC ) TIFA - GFP oligomerization after 30 min of infection with selected mutants of the LPS biosynthesis pathway ( MOI 10 ) of the LPS biosynthesis pathway ( MOI 10 ) . D ) TIFA - GFP oligomerization after 4 h of infection with the S . flexneri mutantE ) IL - 8 production 4 h post infection ( MOI 5 )

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Figure 4A) Representative images of the formation of TIFA oligomers in HeLa cells infected for 30 min with dsRed-expressing wt or indicated mutants of S. flexneri (MOI 50). pHldA/C means plasmid-encoded HldA and HldC. Fluorescence intensity was adjusted between strains to optimize visualization of bacteria. Scale bar, 20 μmB) Infectivity of indicated S. flexneri strainsC) TIFA-GFP oligomerization after 30 min of infection with selected mutants of the LPS biosynthesis pathway (MOI 10)of the LPS biosynthesis pathway (MOI 10). D) TIFA-GFP oligomerization after 4 h of infection with the S. flexneri mutantE) IL-8 production 4 h post infection (MOI 5)


Figure 5A ) ADP - heptose - induced TIFA - GFP oligomerization depends on ALPK1 . After siRNA transfection , HeLa cells were transfected with empty pCMV or pCMV - ALPK1 as indicated . They were then infected with wt S . flexneri ( MOI 10 ) or treated with digitonin and ADP - heptose ( 10 - 5 M ) for 30 min . As control , cells were left untreated ( mock ) or treated with digitoninADP - heptose - induced cytokine secretion is ALPK1 - dependent . HeLa cells were transfected with control or ALPK1 - targeting siRNAs and infected with wt S . flexneri ( MOI 2 ) or treated with ADP - heptose ( 10 - 5 M ) . As control , cells were left untreated ( mock ) or treated with digitonin . Cytokines were measured in cell culture supernatants after 8

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Figure 5A) ADP-heptose-induced TIFA-GFP oligomerization depends on ALPK1. After siRNA transfection, HeLa cells were transfected with empty pCMV or pCMV-ALPK1 as indicated. They were then infected with wt S. flexneri (MOI 10) or treated with digitonin and ADP-heptose (10-5 M) for 30 min. As control, cells were left untreated (mock) or treated with digitoninADP-heptose-induced cytokine secretion is ALPK1-dependent. HeLa cells were transfected with control or ALPK1-targeting siRNAs and infected with wt S. flexneri (MOI 2) or treated with ADP-heptose (10-5 M). As control, cells were left untreated (mock) or treated with digitonin. Cytokines were measured in cell culture supernatants after 8 h


Figure 1Representative flow cytometry data showing surface phenotypes of DCs sorted from spleens of wild type ( WT ) or Nlrc3 - / - mice and treated with LPS ( 100 ng / ml ) for 48 hours . ELISA of cytokines in culture supernatants of DCs treatedThe intracellular production of IFN - γ and IL - 17 by CD4 + T cells among naive 2D2 CD4 + T cells stimulated with MOG ( 35 - 55 ) plus DCs treatedcytokines in culture supernatants among naive 2D2 CD4 + T cells stimulated with MOG ( 35 - 55 ) plus DCs treatedthymidine incorporation proliferation assay among naive 2D2 CD4 + T cells stimulated with MOG ( 35 - 55 ) plus DCs treatedCFSE proliferation assay among naive 2D2 CD4 + T cells stimulated with MOG ( 35 - 55 ) plus DCs

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Figure 1Representative flow cytometry data showing surface phenotypes of DCs sorted from spleens of wild type (WT) or Nlrc3-/- mice and treated with LPS (100 ng/ml) for 48 hours.ELISA of cytokines in culture supernatants of DCs treatedThe intracellular production of IFN-γ and IL-17 by CD4+ T cells among naive 2D2 CD4+ T cells stimulated with MOG(35-55) plus DCs treatedcytokines in culture supernatants among naive 2D2 CD4+ T cells stimulated with MOG(35-55) plus DCs treatedthymidine incorporation proliferation assay among naive 2D2 CD4+ T cells stimulated with MOG(35-55) plus DCs treatedCFSE proliferation assay among naive 2D2 CD4+ T cells stimulated with MOG(35-55) plus DCs treated


Figure 2WT and Nlrc3 - / - mice were immunized with MOG ( 35 - 55 ) peptide in CFA adjuvant and pertussis toxin to induce EAE . Mean clinical scores of EAE in immunized WT ( n = 10 ) and Nlrc3 - / - mice ( n = 10 ) . Representative flow cytometry data showing intracellular production of IFN - γ and IL - 17A by CD4 + T cells from the spinal cord and brain of WT or Nlrc3 - / - mice 26 d after EAE induction after restimulation with MOG ( 35 - 55 ) peptide . Pooled data are presented in the right panel . Representative flow cytometry data showing surface phenotypes of DCs from spleens of WT or Nlrc3 - / - mice 26 d after EAE induction . Expression of Nlrc3 , Il12 , Il6 , Il23 and Il27 mRNA in DCs sorted from WT and Nlrc3 - / - mice 26 d after EAE induction , presented relative to that of Gapdh .

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Figure 2WT and Nlrc3-/- mice were immunized with MOG(35-55) peptide in CFA adjuvant and pertussis toxin to induce EAE. Mean clinical scores of EAE in immunized WT (n= 10) and Nlrc3-/- mice (n= 10).Representative flow cytometry data showing intracellular production of IFN-γ and IL-17A by CD4+ T cells from the spinal cord and brain of WT or Nlrc3-/- mice 26 d after EAE induction after restimulation with MOG(35-55) peptide. Pooled data are presented in the right panel.Representative flow cytometry data showing surface phenotypes of DCs from spleens of WT or Nlrc3-/- mice 26 d after EAE induction.Expression of Nlrc3, Il12, Il6, Il23 and Il27 mRNA in DCs sorted from WT and Nlrc3-/- mice 26 d after EAE induction, presented relative to that of Gapdh.


Figure 3DC ( WT ) and DC ( NLRC3 - KO ) mice were immunized with MOG ( 35 - 55 ) peptide in CFA adjuvant and pertussis toxin to induce EAE . Mean clinical scores of EAE in immunized DC ( WT ) ( n = 5 ) and DC ( NLRC3 - KO ) mice ( n = 5 ) . DC ( WT ) and DC ( NLRC3 - KO ) mice were immunized with MOG ( 35 - 55 ) peptide in CFA adjuvant and pertussis toxin to induce EAE . Representative flow cytometry data showing intracellular production of IFN - γ and IL - 17A by CD4 + T cells in the spinal cord and brain from DC ( WT ) and DC ( NLRC3 - KO ) mice 26 d after EAE induction after restimulation with MOG ( 35 - 55 ) peptide . DC ( WT ) and DC ( NLRC3 - KO ) mice were immunized with MOG ( 35 - 55 ) peptide in CFA adjuvant and pertussis toxin to induce EAE . Recall response to MOG ( 35 - 55 ) by splenocytes isolated from DC ( WT ) and DC ( NLRC3 - KO ) mice 26 d after EAE induction . DC ( WT ) and DC ( NLRC3 - KO ) mice were immunized with MOG ( 35 - 55 ) peptide in CFA adjuvant and pertussis toxin to induce EAE . Expression of Il12 , Il6 , Il23 and Il27 mRNA in DCs sorted from DC ( WT ) and DC ( NLRC3 - KO ) mice 26 d after EAE induction , presented relative to that of Gapdh . DC ( WT ) and DC ( NLRC3 - KO ) mice were immunized with MOG ( 35 - 55 ) peptide in CFA adjuvant and pertussis toxin to induce EAE . CFSE proliferation assay of naive 2D2 CD4 + T cells stimulated with MOG ( 35 - 55 ) plus DCs sorted from DC ( WT ) and DC ( NLRC3 - KO ) mice 26 d after EAE induction . DC ( WT ) and DC ( NLRC3 - KO ) mice were immunized with MOG ( 35 - 55 ) peptide in CFA adjuvant and pertussis toxin to induce EAE . cytokine secretion of naive 2D2 CD4 + T cells stimulated with MOG ( 35 - 55 ) plus DCs sorted from DC ( WT ) and DC ( NLRC3 - KO ) mice 26 d after EAE induction .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3DC(WT) and DC(NLRC3-KO) mice were immunized with MOG(35-55) peptide in CFA adjuvant and pertussis toxin to induce EAE. Mean clinical scores of EAE in immunized DC(WT) (n= 5) and DC(NLRC3-KO) mice (n= 5).DC(WT) and DC(NLRC3-KO) mice were immunized with MOG(35-55) peptide in CFA adjuvant and pertussis toxin to induce EAE. Representative flow cytometry data showing intracellular production of IFN-γ and IL-17A by CD4+ T cells in the spinal cord and brain from DC(WT) and DC(NLRC3-KO) mice 26 d after EAE induction after restimulation with MOG(35-55) peptide.DC(WT) and DC(NLRC3-KO) mice were immunized with MOG(35-55) peptide in CFA adjuvant and pertussis toxin to induce EAE. Recall response to MOG(35-55) by splenocytes isolated from DC(WT) and DC(NLRC3-KO) mice 26 d after EAE induction.DC(WT) and DC(NLRC3-KO) mice were immunized with MOG(35-55) peptide in CFA adjuvant and pertussis toxin to induce EAE. Expression of Il12, Il6, Il23 and Il27 mRNA in DCs sorted from DC(WT) and DC(NLRC3-KO) mice 26 d after EAE induction, presented relative to that of Gapdh.DC(WT) and DC(NLRC3-KO) mice were immunized with MOG(35-55) peptide in CFA adjuvant and pertussis toxin to induce EAE. CFSE proliferation assay of naive 2D2 CD4+ T cells stimulated with MOG(35-55) plus DCs sorted from DC(WT) and DC(NLRC3-KO) mice 26 d after EAE induction.DC(WT) and DC(NLRC3-KO) mice were immunized with MOG(35-55) peptide in CFA adjuvant and pertussis toxin to induce EAE. cytokine secretion of naive 2D2 CD4+ T cells stimulated with MOG(35-55) plus DCs sorted from DC(WT) and DC(NLRC3-KO) mice 26 d after EAE induction.


Figure 4DCs were sorted from spleens of WT or Nlrc3 - / - mice . Purified DCs were treated with LPS ( 100 ng / ml ) for specified time . DC lysates were probed for phosphorylated p65 ( p - p65 ) , total p65 , p - AKT , AKT , p - p38 , p38 , p - ERK , ERK , p - JNK , JNK and GAPDH . DCs were sorted from spleens of WT or Nlrc3 - / - mice . Purified DCs were treated for 48 hours with LPS ( 100 ng / ml ) in the presence or absence of the p38 inhibitor SB203580 ( 10 μM or indicated concentrations ) . Concentrations of IL - 12 , IL - 6 , IL - 23 and IL - 27 in supernatants were detected by ELISA . DCs were sorted from spleens of WT or Nlrc3 - / - mice . Purified DCs were treated for 48 hours with LPS ( 100 ng / ml ) in the presence or absence of the p38 inhibitor SB203580 ( 10 μM or indicated concentrations ) . Cytokines in culture supernatants among naive 2D2 CD4 + T cells stimulated with MOG ( 35 - 55 ) plus DCs . NC : negative control . DCs were sorted from spleens of WT or Nlrc3 - / - mice . Purified DCs were treated for 48 hours with LPS ( 100 ng / ml ) in the presence or absence of the p38 inhibitor SB203580 ( 10 μM or indicated concentrations ) . CFSE proliferation assay ( D ) among naive 2D2 CD4 + T cells stimulated with MOG ( 35 - 55 ) plus DCs . NC : negative control .

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Figure 4DCs were sorted from spleens of WT or Nlrc3-/- mice. Purified DCs were treated with LPS (100 ng/ml) for specified time. DC lysates were probed for phosphorylated p65 (p-p65), total p65, p-AKT, AKT, p-p38, p38, p-ERK, ERK, p-JNK, JNK and GAPDH.DCs were sorted from spleens of WT or Nlrc3-/- mice. Purified DCs were treated for 48 hours with LPS (100 ng/ml) in the presence or absence of the p38 inhibitor SB203580 (10 μM or indicated concentrations). Concentrations of IL-12, IL-6, IL-23 and IL-27 in supernatants were detected by ELISA.DCs were sorted from spleens of WT or Nlrc3-/- mice. Purified DCs were treated for 48 hours with LPS (100 ng/ml) in the presence or absence of the p38 inhibitor SB203580 (10 μM or indicated concentrations). Cytokines in culture supernatants among naive 2D2 CD4+ T cells stimulated with MOG(35-55) plus DCs. NC: negative control.DCs were sorted from spleens of WT or Nlrc3-/- mice. Purified DCs were treated for 48 hours with LPS (100 ng/ml) in the presence or absence of the p38 inhibitor SB203580 (10 μM or indicated concentrations). CFSE proliferation assay (D) among naive 2D2 CD4+ T cells stimulated with MOG(35-55) plus DCs. NC: negative control.


Figure 5Activity phosphorylation of p38 were detected in DCs in the spleens from DC ( WT ) and DC ( NLRC3 - KO ) mice 26 d after EAE induction . Pooled data are presented in the right panel . DC ( p38 - KO ) and DC ( p38 + NLRC3 - KO ) mice were immunized with MOG ( 35 - 55 ) peptide in CFA adjuvant and pertussis toxin to induce EAE . Mean clinical scores of EAE in immunized DC ( WT ) ( n = 5 ) and DC ( NLRC3 - KO ) mice ( n = 5 ) . DC ( p38 - KO ) and DC ( p38 + NLRC3 - KO ) mice were immunized with MOG ( 35 - 55 ) peptide in CFA adjuvant and pertussis toxin to induce EAE . Frequencies of CD4 + T cells that express IFN - γ and IL - 17A in the spinal cord and brain from DC ( WT ) and DC ( NLRC3 - KO ) mice 26 d after EAE induction after restimulation with MOG ( 35 - 55 ) peptide . Pooled data are presented in the right panel . DC ( p38 - KO ) and DC ( p38 + NLRC3 - KO ) mice were immunized with MOG ( 35 - 55 ) peptide in CFA adjuvant and pertussis toxin to induce EAE . Expression of Il12 , Il6 and Il23 mRNA in DCs sorted from spleens of DC ( WT ) , DC ( NLRC3 - KO ) , DC ( p38 - KO ) and DC ( p38 + NLRC3 - KO ) mice 26 d after EAE induction , presented relative to that of Gapdh .

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Figure 5Activity phosphorylation of p38 were detected in DCs in the spleens from DC(WT) and DC(NLRC3-KO) mice 26 d after EAE induction. Pooled data are presented in the right panel.DC(p38-KO) and DC(p38+NLRC3-KO) mice were immunized with MOG(35-55) peptide in CFA adjuvant and pertussis toxin to induce EAE. Mean clinical scores of EAE in immunized DC(WT) (n= 5) and DC(NLRC3-KO) mice (n= 5).DC(p38-KO) and DC(p38+NLRC3-KO) mice were immunized with MOG(35-55) peptide in CFA adjuvant and pertussis toxin to induce EAE. Frequencies of CD4+ T cells that express IFN-γ and IL-17A in the spinal cord and brain from DC(WT) and DC(NLRC3-KO) mice 26 d after EAE induction after restimulation with MOG(35-55) peptide. Pooled data are presented in the right panel.DC(p38-KO) and DC(p38+NLRC3-KO) mice were immunized with MOG(35-55) peptide in CFA adjuvant and pertussis toxin to induce EAE. Expression of Il12, Il6 and Il23 mRNA in DCs sorted from spleens of DC(WT), DC(NLRC3-KO), DC(p38-KO) and DC(p38+NLRC3-KO) mice 26 d after EAE induction, presented relative to that of Gapdh.


Figure 6DC ( Ctrl ) and DC ( NLRC3 - OE ) were stimulated with LPS ( 100 ng / ml ) for specified time . DC lysates were probed for p - p38 , p38 , NLRC3 and GAPDH . Densitometry quantification of band intensity were presented in the right panel . DC ( Ctrl ) and DC ( NLRC3 - OE ) were stimulated with LPS ( 100 ng / ml ) for specified time . Enzyme - linked immunosorbent assay of cytokines in culture supernatants of DCs treated with LPS for 48 hours . DC ( Ctrl ) and DC ( NLRC3 - OE ) were stimulated with LPS ( 100 ng / ml ) for specified time . Cytokines in culture supernatants among naive 2D2 CD4 + T cells stimulated with MOG ( 35 - 55 ) plus DCs treated with LPS for 48 hours . DC ( Ctrl ) and DC ( NLRC3 - OE ) were stimulated with LPS ( 100 ng / ml ) for specified time . CFSE proliferation assay ( D ) among naive 2D2 CD4 + T cells stimulated with MOG ( 35 - 55 ) plus DCs treated with LPS for 48 hours .

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Figure 6DC(Ctrl) and DC(NLRC3-OE) were stimulated with LPS (100 ng/ml) for specified time. DC lysates were probed for p-p38, p38, NLRC3 and GAPDH. Densitometry quantification of band intensity were presented in the right panel.DC(Ctrl) and DC(NLRC3-OE) were stimulated with LPS (100 ng/ml) for specified time. Enzyme-linked immunosorbent assay of cytokines in culture supernatants of DCs treated with LPS for 48 hours.DC(Ctrl) and DC(NLRC3-OE) were stimulated with LPS (100 ng/ml) for specified time. Cytokines in culture supernatants among naive 2D2 CD4+ T cells stimulated with MOG(35-55) plus DCs treated with LPS for 48 hours.DC(Ctrl) and DC(NLRC3-OE) were stimulated with LPS (100 ng/ml) for specified time. CFSE proliferation assay (D) among naive 2D2 CD4+ T cells stimulated with MOG(35-55) plus DCs treated with LPS for 48 hours.


Figure 7EAE was induced by immunization of naive B6 mice with MOG ( 35 - 55 ) , and the mice were randomly divided into five groups . BMDCs transduced with either lentiviral vector encoding GFP and NLRC3 ( LV - NLRC3 ) or only GFP ( LV - Ctrl ) were administered i . v . 4 times , once every 4 days , starting at day 10 after EAE induction . Mean clinical scores of EAE ( n = 5 mice per group ) . Arrows indicate DC vaccine administration . EAE was induced by immunization of naive B6 mice with MOG ( 35 - 55 ) , and the mice were randomly divided into five groups . BMDCs transduced with either lentiviral vector encoding GFP and NLRC3 ( LV - NLRC3 ) or only GFP ( LV - Ctrl ) were administered i . v . 4 times , once every 4 days , starting at day 10 after EAE induction . Recall proliferative to MOG ( 35 - 55 ) in splenocytes taken from DCs - treated mice 26 days after EAE induction . EAE was induced by immunization of naive B6 mice with MOG ( 35 - 55 ) , and the mice were randomly divided into five groups . BMDCs transduced with either lentiviral vector encoding GFP and NLRC3 ( LV - NLRC3 ) or only GFP ( LV - Ctrl ) were administered i . v . 4 times , once every 4 days , starting at day 10 after EAE induction . cytokine response ( D ) to MOG ( 35 - 55 ) in splenocytes taken from DCs - treated mice 26 days after EAE induction .

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Figure 7EAE was induced by immunization of naive B6 mice with MOG (35-55), and the mice were randomly divided into five groups. BMDCs transduced with either lentiviral vector encoding GFP and NLRC3 (LV-NLRC3) or only GFP (LV-Ctrl) were administered i.v. 4 times, once every 4 days, starting at day 10 after EAE induction. Mean clinical scores of EAE (n= 5 mice per group ). Arrows indicate DC vaccine administration.EAE was induced by immunization of naive B6 mice with MOG (35-55), and the mice were randomly divided into five groups. BMDCs transduced with either lentiviral vector encoding GFP and NLRC3 (LV-NLRC3) or only GFP (LV-Ctrl) were administered i.v. 4 times, once every 4 days, starting at day 10 after EAE induction. Recall proliferative to MOG (35-55) in splenocytes taken from DCs-treated mice 26 days after EAE induction.EAE was induced by immunization of naive B6 mice with MOG (35-55), and the mice were randomly divided into five groups. BMDCs transduced with either lentiviral vector encoding GFP and NLRC3 (LV-NLRC3) or only GFP (LV-Ctrl) were administered i.v. 4 times, once every 4 days, starting at day 10 after EAE induction. cytokine response (D) to MOG (35-55) in splenocytes taken from DCs-treated mice 26 days after EAE induction.


Figure 2Starvation triggers accumulation of nucleosides , bases and S7P . Wild ‐ type S . cerevisiae cells growing in minimal media were switched to minimal media containing no carbon , no nitrogen or no phosphate . After the indicated duration of starvation , the metabolome was quantified by LC ‐ MS . For media composition , see Supplementary Table S2 . For the list of absolute concentration of metabolties , see Supplementary Table S3 . Data are shown in heat map format , with each line reflecting the dynamics of a particular compound in a particular culture condition . Metabolite levels of biological duplicates were averaged , normalized to cells growing steadily in glucose ( time zero ) , and the resulting fold changes log2 transformed .

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Figure 2Starvation triggers accumulation of nucleosides, bases and S7P. Wild‐type S. cerevisiae cells growing in minimal media were switched to minimal media containing no carbon, no nitrogen or no phosphate. After the indicated duration of starvation, the metabolome was quantified by LC‐MS. For media composition, see Supplementary Table S2. For the list of absolute concentration of metabolties, see Supplementary Table S3. Data are shown in heat map format, with each line reflecting the dynamics of a particular compound in a particular culture condition. Metabolite levels of biological duplicates were averaged, normalized to cells growing steadily in glucose (time zero), and the resulting fold changes log2 transformed.


Figure 3 ( A ) Experimental design for demonstrating metabolite production via macromolecule degradation . Yeast cells were grown on 2 % unlabeled glucose , and then switched to U ‐ 13C ‐ glucose for 70 min , which completely labels free metabolite but only partially labels macromolecules . Thereafter , glucose was removed and the metabolome analyzed by LC ‐ MS . ( B ) Fraction of unlabeled nucleosides , nucleic bases and PPP intermediates as a function of starvation time , in wild ‐ type and autophagy deficient ( atg7 deletion ) yeast . The x axis represents minutes after carbon starvation , and the y axis represents fraction of unlabeled metabolites ( mean ± range of N = 2 biological replicates ) . ( C ) Ratio of metabolite levels in atg7 strain versus wild ‐ type strain in carbon starvation . ( D ) Ratio of metabolite levels in bcy1 strain and snf1 strain versus wild ‐ type strain in carbon starvation . ( E ) Ratio of metabolite levels in rapamycin treatment versus nitrogen starvation for wild ‐ type yeast . In ( C ) to ( E ) , all reported values are log2 transformed ratios ; data are mean of duplicate samples at each time point .

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Figure 3(A) Experimental design for demonstrating metabolite production via macromolecule degradation. Yeast cells were grown on 2% unlabeled glucose, and then switched to U‐13C‐glucose for 70 min, which completely labels free metabolite but only partially labels macromolecules. Thereafter, glucose was removed and the metabolome analyzed by LC‐MS.(B) Fraction of unlabeled nucleosides, nucleic bases and PPP intermediates as a function of starvation time, in wild‐type and autophagy deficient (atg7 deletion) yeast. The x axis represents minutes after carbon starvation, and the y axis represents fraction of unlabeled metabolites (mean±range of N=2 biological replicates).(C) Ratio of metabolite levels in atg7 strain versus wild‐type strain in carbon starvation.(D) Ratio of metabolite levels in bcy1 strain and snf1 strain versus wild‐type strain in carbon starvation.(E) Ratio of metabolite levels in rapamycin treatment versus nitrogen starvation for wild‐type yeast. In (C) to (E), all reported values are log2 transformed ratios; data are mean of duplicate samples at each time point.


Figure 4 ( A ) Ratio of metabolite levels in sdt1 and phm8 strains versus wild ‐ type strain in carbon starvation . All reported values are log2 transformed ratios ; data are mean of duplicate samples at each time point . ( B ) Summary of transcripts data of PHM8 , SDT1 and other enzymes in the pathway ( Gasch et al , 2000 ; Bradley et al , 2009 ; Klosinska et al , 2011 ) . All values are log2 transformed fold changes . ( C ) Screening of Phm8 ' s phosphatase activity against 90 phosphorylated compounds . Phosphatase activity was measured in the presence of 0 . 5 mM substrate and 5 mM Mg2 + ( pH = 7 . 0 , 30 ° C ) . Compounds with specific activity higher than 0 . 1 μmol / mg / min are shown . The x axis represents specific phosphatase activity ( μmol of phosphate produced per minute per mg of enzyme , mean ± range of N = 2 replicates ) . ( D ) Top table : absolute intracellular concentration of nucleotide monophosphates in carbon starvation . Bottom plots : Phosphatase activity of Phm8 and Sdt1 as a function of CMP concentration . The x axis represents CMP concentration and the y axis represents specific phosphatase activity ( μmol of phosphate produced per minute per mg of enzyme , mean ± range of N = 2 replicates ) .

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Figure 4(A) Ratio of metabolite levels in sdt1 and phm8 strains versus wild‐type strain in carbon starvation. All reported values are log2 transformed ratios; data are mean of duplicate samples at each time point.(B) Summary of transcripts data of PHM8, SDT1 and other enzymes in the pathway (Gasch et al, 2000; Bradley et al, 2009; Klosinska et al, 2011). All values are log2 transformed fold changes.(C) Screening of Phm8's phosphatase activity against 90 phosphorylated compounds. Phosphatase activity was measured in the presence of 0.5 mM substrate and 5 mM Mg2+ (pH=7.0, 30°C). Compounds with specific activity higher than 0.1 μmol/mg/min are shown. The x axis represents specific phosphatase activity (μmol of phosphate produced per minute per mg of enzyme, mean±range of N=2 replicates).(D) Top table: absolute intracellular concentration of nucleotide monophosphates in carbon starvation. Bottom plots: Phosphatase activity of Phm8 and Sdt1 as a function of CMP concentration. The x axis represents CMP concentration and the y axis represents specific phosphatase activity (μmol of phosphate produced per minute per mg of enzyme, mean±range of N=2 replicates).


Figure 5Confirmation that Pnp1 is the physiological purine nucleoside phosphorylase , Urh1 is the pyrimidine hydrolase , and Prm15 ( Pgm3 ) is the phosphoribomutase . Ratio of metabolite levels in pnp1 / urh1 , pnp1 , urh1 and prm15 ( pgm3 ) strains versus wild ‐ type strain during carbon starvation . Data are shown in heat map format , with each line reflecting the dynamics of the ratio of the metabolite levels in a particular strain versus wild ‐ type strain . All reported values are log2 transformed ratios ; data are mean of duplicate samples at each time point .

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Figure 5Confirmation that Pnp1 is the physiological purine nucleoside phosphorylase, Urh1 is the pyrimidine hydrolase, and Prm15 (Pgm3) is the phosphoribomutase. Ratio of metabolite levels in pnp1/urh1, pnp1, urh1 and prm15 (pgm3) strains versus wild‐type strain during carbon starvation. Data are shown in heat map format, with each line reflecting the dynamics of the ratio of the metabolite levels in a particular strain versus wild‐type strain. All reported values are log2 transformed ratios; data are mean of duplicate samples at each time point.


Figure 6 ( B ‐ E ) DHAP , SBP and S7P levels in wild type , tkl1 / tkl2 ( B ) , tal1 / nqm1 ( C ) and pfk1 ( E ) strains upon carbon starvation , and wild ‐ type strain upon abruptly switching from glucose to no carbon ( WT ) versus to dihydroxyacetone ( WT + DHA ) as the sole carbon source ( D ) . The x axis represents hours after carbon starvation , and the logarithmic y axis represents absolute intracellular concentration ( mean ± range of N = 2 biological replicates ) .

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Figure 6(B‐E) DHAP, SBP and S7P levels in wild type, tkl1/tkl2 (B), tal1/nqm1 (C) and pfk1 (E) strains upon carbon starvation, and wild‐type strain upon abruptly switching from glucose to no carbon (WT) versus to dihydroxyacetone (WT+DHA) as the sole carbon source (D). The x axis represents hours after carbon starvation, and the logarithmic y axis represents absolute intracellular concentration (mean±range of N=2 biological replicates).


Figure 7 ( A ) Level of hallmark metabolites and energy charge in wild type , phm8 and pnp1 / urh1 strains in starvation . The x axis represents hours after starvation , and the logarithmic y axis represents energy charge ( [ ATP ] + 0 . 5 [ ADP ] / ( [ ATP ] + [ ADP ] + [ AMP ] ) or absolute intracellular concentration ( mean ± range of N = 2 biological replicates ) . ( B and C ) Growth and viability of wild type , phm8 and pnp1 / urh1 strains in starvation . ( D ) Growth of wild type , phm8 and pnp1 / urh1 strains in the switch from glucose to glycerol + ethanol . The x axis represents hours after starvation and the logarithmic y axis represents optical density ( A600 ) in ( B and D ) and percentage of live cells calculated by dividing the number of colonies formed after starvation by the number of colonies formed before starvation in ( C ) ( mean ± range of N = 2 biological replicates ) .

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Figure 7(A) Level of hallmark metabolites and energy charge in wild type, phm8 and pnp1/urh1 strains in starvation. The x axis represents hours after starvation, and the logarithmic y axis represents energy charge ([ATP]+0.5[ADP]/([ATP]+[ADP]+[AMP]) or absolute intracellular concentration (mean±range of N=2 biological replicates).(B and C) Growth and viability of wild type, phm8 and pnp1/urh1 strains in starvation.(D) Growth of wild type, phm8 and pnp1/urh1 strains in the switch from glucose to glycerol+ethanol. The x axis represents hours after starvation and the logarithmic y axis represents optical density (A600) in (B and D) and percentage of live cells calculated by dividing the number of colonies formed after starvation by the number of colonies formed before starvation in (C) (mean±range of N=2 biological replicates).


Figure 8 ( C and D ) DHAP , S7P , G6P , and glutathione levels and NADPH / NADP + ratio in wild type , phm8 and pnp1 / urh1 strains in the experiment shown in ( A ) The x axis represents minutes after oxidative stress , and the y axis represents absolute intracellular concentration or ratio of intracellular concentration ( mean ± range of N = 2 biological replicates ) . ( E ) Viability of wild type , phm8 and pnp1 / urh1 strains in oxidative stress . The y axis represents percentage of survived cells calculated by dividing the number of colonies formed after oxidative stress by the number of colonies formed under the same starvation condition but without H2O2 treatment ( mean ± range of N = 2 biological replicates ) .

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Figure 8(C and D) DHAP, S7P, G6P, and glutathione levels and NADPH/NADP+ ratio in wild type, phm8 and pnp1/urh1 strains in the experiment shown in (A) The x axis represents minutes after oxidative stress, and the y axis represents absolute intracellular concentration or ratio of intracellular concentration (mean±range of N=2 biological replicates).(E) Viability of wild type, phm8 and pnp1/urh1 strains in oxidative stress. The y axis represents percentage of survived cells calculated by dividing the number of colonies formed after oxidative stress by the number of colonies formed under the same starvation condition but without H2O2 treatment (mean±range of N=2 biological replicates).


Figure 2 Fluorescence intensity of the FRET donor ( GFP ) is shown before and after photobleaching of the FRET acceptor ( mCherry ) in ImageJ Smart pseudocolor scheme ( for intensity values see bars on the right ) . FRET is observed as an increase of donor fluorescence after acceptor photobleaching . The indicated GFP - and mCherry - tagged endocytic proteins localize at the endocytic patches on the plasma membrane of live yeast cells . The enrichment of individual endocytic patches ( diameter of 50 - 80 nm ) on the plasma membrane , caused by the latA - induced block of their subsequent invagination and / or their higher abundance at incipient buds , resulted in their apparent clustering on the diffraction - limited fluorescence images ( pixel size , 178 nm ) . All raw acquisitions of GFP fluorescence intensities before and after photobleaching are shown

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Figure 2 Fluorescence intensity of the FRET donor (GFP) is shown before and after photobleaching of the FRET acceptor (mCherry) in ImageJ Smart pseudocolor scheme (for intensity values see bars on the right). FRET is observed as an increase of donor fluorescence after acceptor photobleaching. The indicated GFP- and mCherry-tagged endocytic proteins localize at the endocytic patches on the plasma membrane of live yeast cells. The enrichment of individual endocytic patches (diameter of 50-80 nm) on the plasma membrane, caused by the latA-induced block of their subsequent invagination and/or their higher abundance at incipient buds, resulted in their apparent clustering on the diffraction-limited fluorescence images (pixel size, 178 nm). All raw acquisitions of GFP fluorescence intensities before and after photobleaching are shown


Figure 4 Partial loss of FRET between Ent1 - mTurquoise2 and Sla1 - mNeonGreen during endocytic membrane invagination . FRET values ( in % ) of individual endocytic patches ( n = 74 , 75 ; 69 , 73 for ( B ) and ( C ) , respectively ) sorted according to the absence / presence of Abp1 protein are shown as box plots . Center , top and bottom lines of box plots show the medians , the 25th and 75th percentiles of individual datasets , respectively . Whiskers extend 1 . 5 times the interquartile range from the 25th and 75th percentiles . Notches indicate 95 % confidence intervals of the medians . presence of FRET between Ent1 - mTurquoise2 and Sla2 - mNeonGreen during endocytic membrane invagination . FRET values ( in % ) of individual endocytic patches ( n = 74 , 75 ; 69 , 73 for ( B ) and ( C ) , respectively ) sorted according to the absence / presence of Abp1 protein are shown as box plots . Center , top and bottom lines of box plots show the medians , the 25th and 75th percentiles of individual datasets , respectively . Whiskers extend 1 . 5 times the interquartile range from the 25th and 75th percentiles . Notches indicate 95 % confidence intervals of the medians .

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

Figure 4 Partial loss of FRET between Ent1-mTurquoise2 and Sla1-mNeonGreen during endocytic membrane invagination. FRET values (in %) of individual endocytic patches (n=74, 75; 69, 73 for (B) and (C), respectively) sorted according to the absence/presence of Abp1 protein are shown as box plots. Center, top and bottom lines of box plots show the medians, the 25th and 75th percentiles of individual datasets, respectively. Whiskers extend 1.5 times the interquartile range from the 25th and 75th percentiles. Notches indicate 95% confidence intervals of the medians.  presence of FRET between Ent1-mTurquoise2 and Sla2-mNeonGreen during endocytic membrane invagination. FRET values (in %) of individual endocytic patches (n=74, 75; 69, 73 for (B) and (C), respectively) sorted according to the absence/presence of Abp1 protein are shown as box plots. Center, top and bottom lines of box plots show the medians, the 25th and 75th percentiles of individual datasets, respectively. Whiskers extend 1.5 times the interquartile range from the 25th and 75th percentiles. Notches indicate 95% confidence intervals of the medians.


Figure 5 Ede1 , Apl1 , Yap1801 , Gts1 , Bzz1 , Lsb3 , Las17 , and Vrp1 proteins tagged with mNeonGreen were photobleached in individual endocytic sites of LatA - treated cells and fluorescence recovery was followed every 0 . 5 s for 60 s ( 40 s in case of the highly dynamic Bzz1 protein ) . The curves represent means ± 95 % confidence intervals ( n = 9 - 17 ) .

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Figure 5 Ede1, Apl1, Yap1801, Gts1, Bzz1, Lsb3, Las17, and Vrp1 proteins tagged with mNeonGreen were photobleached in individual endocytic sites of LatA-treated cells and fluorescence recovery was followed every 0.5 s for 60 s (40 s in case of the highly dynamic Bzz1 protein). The curves represent means ± 95% confidence intervals (n = 9-17).


Figure 1C ) RNA binding - deficient TDP - 43 mutants generated by site - directed substitutions in RRM1 ( F2L ) or in both RRM domains ( F4L ) and substitution of RRM1 and RRM2 with monomeric GFP ( N - GFP - C ) . Phase separation of the mutants without added RNA ( control ) or in the presence A ( GU ) 6 ( 8 μM ) , A ( GU ) 18 ( 2 . 6 μM ) , or A ( CA ) 6 ( 8 μM ) at 150 mM NaCl ( 4 µM protein ) .

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Figure 1C) RNA binding-deficient TDP-43 mutants generated by site-directed substitutions in RRM1 (F2L) or in both RRM domains (F4L) and substitution of RRM1 and RRM2 with monomeric GFP (N-GFP-C). Phase separation of the mutants without added RNA (control) or in the presence A(GU)6 (8 μM), A(GU)18 (2.6 μM), or A(CA)6 (8 μM) at 150 mM NaCl (4 µM protein).


Figure 2B ) TDP - 43 condensates in the presence of the different RNA sequences . TDP - 43 and RNA oligonucleotide concentration is 4 μM and 3 . 9 μM , respectively . Representative images for 3 biological replicates using 2 different protein preparations . Scale bars , 10 μm . C ) LLPS measured by turbidity in the same experimental conditions as panel B . Mean and SD from 3 biological replicates with 2 different protein preparations . D ) Lysate from HEK 293 cells expressing a GFP - tagged copy of either wild - type ( WT ) or F147 / 149 / 229 / 231L ( F4L ) TDP - 43 was added to a mixture of recombinant WT or F4L TDP - 43 ( 5 . 3 μM , 10 % Cy3 - labeled protein ) and A ( GU ) 6 ( 22 . 8 μM ) , A ( CA ) 18 ( 7 . 6 μM ) , A ( GU ) 18 ( 7 . 6 μM ) , or no RNA control at 250 mM NaCl . Droplets were imaged by brightfield and fluorescence microscopy . Representative images for 3 biological replicates using 2 protein preparations . Scale bars , 10 μm . E ) LLPS measured by turbidity in the same experimental conditions as panel D . Mean and SD of 4 biological replicates using 2 recombinant protein preparations . Analyzed by one - way ANOVA ( F ( 7 , 24 ) = 69 . 30 , P < 0 . 0001 ) . Sidak ' s multiple comparisons test was used to compare selected groups . * P < 0 . 05 , * * * * P < 0 . 0001 .

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Figure 2B) TDP-43 condensates in the presence of the different RNA sequences. TDP-43 and RNA oligonucleotide concentration is 4 μM and 3.9 μM, respectively. Representative images for 3 biological replicates using 2 different protein preparations. Scale bars, 10 μm. C) LLPS measured by turbidity in the same experimental conditions as panel B. Mean and SD from 3 biological replicates with 2 different protein preparations. D) Lysate from HEK 293 cells expressing a GFP-tagged copy of either wild-type (WT) or F147/149/229/231L (F4L) TDP-43 was added to a mixture of recombinant WT or F4L TDP-43 (5.3 μM, 10% Cy3-labeled protein) and A(GU)6 (22.8 μM), A(CA)18 (7.6 μM), A(GU)18 (7.6 μM), or no RNA control at 250 mM NaCl. Droplets were imaged by brightfield and fluorescence microscopy. Representative images for 3 biological replicates using 2 protein preparations. Scale bars, 10 μm. E) LLPS measured by turbidity in the same experimental conditions as panel D. Mean and SD of 4 biological replicates using 2 recombinant protein preparations. Analyzed by one-way ANOVA (F(7,24)=69.30, P<0.0001). Sidak's multiple comparisons test was used to compare selected groups. *P<0.05, ****P<0.0001.


Figure 3A ) Liquid droplets observed by brightfield and fluorescence microscopy when Oregon green labeled TDP - 43 ( 4 μM , 10 % labeled protein ) was mixed with no RNA control or increasing A ( GU ) 18 RNA concentration at 250 mM salt . Representative images from 3 biological replicates using 2 different protein preparations . Scale bars , 10 μm . D ) Phase separation of TDP - 43 ( 4 μM ) at increasing salt concentration without RNA or in the presence of increasing A ( GU ) 18 concentration . Scale bars , 10 μm . Inlay added to top right panel for increased visibility , scale bar 1 μm . E ) Phase diagram derived from panel D , denoting either no droplets or droplet formation for each condition .

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Figure 3A) Liquid droplets observed by brightfield and fluorescence microscopy when Oregon green labeled TDP-43 (4 μM, 10% labeled protein) was mixed with no RNA control or increasing A(GU)18 RNA concentration at 250 mM salt. Representative images from 3 biological replicates using 2 different protein preparations. Scale bars, 10 μm.D) Phase separation of TDP-43 (4 μM) at increasing salt concentration without RNA or in the presence of increasing A(GU)18 concentration. Scale bars, 10 μm. Inlay added to top right panel for increased visibility, scale bar 1 μm. E) Phase diagram derived from panel D, denoting either no droplets or droplet formation for each condition.


Figure 4A ) TDP - 43 droplets observed by brightfield and fluorescence microscopy when Oregon green labeled TDP - 43 ( 4 μM , 10 % labeled protein ) was mixed with no RNA control or GU - repeat RNA oligonucleotides of increasing length at 250 mM salt . As indicated , RNA concentration varied to maintain a constant total number of TDP - 43 binding sites . Scale bars , 10 μm . B ) The plot shows droplet area quantified as a function of GU - repeat length . Mean and SD of > 300 droplets from 3 biological replicates using 3 different protein preparations . C ) TDP - 43 concentration in the light phase ( Cout ) as a function of GU ( blue ) or CA ( red ) - repeat length quantified by Bradford protein assay . Mean and SD from > 5 biological replicates using 2 different protein preparations . ( 250 mM NaCl , 4 µM TDP - 43 )

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Figure 4A) TDP-43 droplets observed by brightfield and fluorescence microscopy when Oregon green labeled TDP-43 (4 μM, 10% labeled protein) was mixed with no RNA control or GU-repeat RNA oligonucleotides of increasing length at 250 mM salt. As indicated, RNA concentration varied to maintain a constant total number of TDP-43 binding sites. Scale bars, 10 μm. B) The plot shows droplet area quantified as a function of GU-repeat length. Mean and SD of >300 droplets from 3 biological replicates using 3 different protein preparations. C) TDP-43 concentration in the light phase (Cout) as a function of GU (blue) or CA (red)-repeat length quantified by Bradford protein assay. Mean and SD from >5 biological replicates using 2 different protein preparations. (250 mM NaCl, 4 µM TDP-43)


Figure 5B ) Phase separation of full - length and TDP - 43 variants ( 4 μM ) mixed with A ( GU ) 30 RNA ( 0 . 8 μM ) or no RNA control at 250 mM NaCl observed by brightfield microscopy . Representative images for 3 biological replicates using 2 different protein preparations . Scale bars , 10 μm .

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Figure 5B) Phase separation of full-length and TDP-43 variants (4 μM) mixed with A(GU)30 RNA (0.8 μM) or no RNA control at 250 mM NaCl observed by brightfield microscopy. Representative images for 3 biological replicates using 2 different protein preparations. Scale bars, 10 μm.


Figure 6A ) TDP - 43 ALS - linked amino acid substitutions K181E , A315T / E , A321G , Q331K , M337V , and A382T . Boxed in the C - terminal domain is the α - helical structure within the conserved region ( a . a . 320 - 343 ) . Droplets formed by TDP - 43 wild - type ( WT ) and the different mutants ( 4 μM ) observed by brightfield microscopy in the presence of no RNA control or A ( GU ) 18 RNA ( 3 . 9 μM ) at 250 mM NaCl . Mutations A321G and M337V , showing decreased liquidity of the condensates in the presence of A ( GU ) 18 , are boxed in red . Representative images for 3 biological replicates using 3 different protein preparations of WT and M337V , 2 preparations of A321G , Q331K , K181E and one preparation of A315T / E and A382T . Scale bars , 10 μm . B ) Phase separation of WT ( 4 μM , 10 % Oregon green - labeled protein ) and M337V ( 4 μM , 10 % Cy - 3 - labeled protein ) observed by brightfield and fluorescence microscopy in the presence of A ( GU ) 18 ( 3 . 9 μM ) at 250 mM NaCl . The middle panel shows mixing of these WT ( 2 μM ) and M337V ( 2 μM ) samples . Representative images for 3 biological replicates using 2 protein preparations . Scale bars , 10 μm .

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Figure 6A) TDP-43 ALS-linked amino acid substitutions K181E, A315T/E, A321G, Q331K, M337V, and A382T. Boxed in the C-terminal domain is the α-helical structure within the conserved region (a.a. 320-343). Droplets formed by TDP-43 wild-type (WT) and the different mutants (4 μM) observed by brightfield microscopy in the presence of no RNA control or A(GU)18 RNA (3.9 μM) at 250 mM NaCl. Mutations A321G and M337V, showing decreased liquidity of the condensates in the presence of A(GU)18, are boxed in red. Representative images for 3 biological replicates using 3 different protein preparations of WT and M337V, 2 preparations of A321G, Q331K, K181E and one preparation of A315T/E and A382T. Scale bars, 10 μm.B) Phase separation of WT (4 μM, 10% Oregon green-labeled protein) and M337V (4 μM, 10% Cy-3-labeled protein) observed by brightfield and fluorescence microscopy in the presence of A(GU)18 (3.9 μM) at 250 mM NaCl. The middle panel shows mixing of these WT (2 μM) and M337V (2 μM) samples. Representative images for 3 biological replicates using 2 protein preparations. Scale bars, 10 μm.


Figure 7A ) Binding of TDP - 43 to GU - rich RNA promotes liquid properties of TDP - 43 condensates , as shown by the fusion of small condensates into larger droplets . This fluidity is impaired by the loss of RNA binding affinity or disease - linked mutations that alter phase separation , such as M337V and A321G , which could explain increased TDP - 43 aggregation .

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Figure 7A) Binding of TDP-43 to GU-rich RNA promotes liquid properties of TDP-43 condensates, as shown by the fusion of small condensates into larger droplets. This fluidity is impaired by the loss of RNA binding affinity or disease-linked mutations that alter phase separation, such as M337V and A321G, which could explain increased TDP-43 aggregation.


Figure 1stoichiometry ' s for B56α measured by ITC . Global direct fitting shown for one experiment ( reverse ) . Each dot is the integrated heat per injection and the error bars represent uncertainty with this integrated value . The experiment was done in both direct ( B56 in cell ) and reverse ( B56 in syringe ) with similar results . C ) Time from Nuclear envelope breakdown ( NEBD ) to mitotic exit of cells expressing the indicated B56 inhibitors with each circle representing a single cell . Only cells with similar expression levels of the various B56 inhibitor constructs were analysed . Median time is indicated by red line . A representative result from at least three independent experiments is shown . At least 25 cells were counted per condition in the experiment shown . A Mann - Whitney U ‐ test was used for statistical analysis ( ns : non ‐ significant , * * * P ≤ 0 . 001 ) . D ) Volcano plot representing mass spectrometry identified proteins co - purifying with B56 inhibitor versus control inhibitor from HeLa cells . PP2A - B56 subunits co - purifying with the B56 inhibitor are indicated . E ) and F ) Competition assay in HeLa cells stably expressing RFP - tagged B56 inhibitor ( LxxIxE ) or control inhibitor ( AxxAxA ) . YFP - B56α was transfected into and subsequently purified from these cell lines . Loss of binding of indicated proteins determined by either mass spectrometry ( pink - B56 SLIM containing protein and known B56 interactor ; blue - known B56 interactor , green - B56 SLIM containing protein ) ( E ) or Western blotting ( F ) .

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Figure 1stoichiometry's for B56α measured by ITC. Global direct fitting shown for one experiment (reverse). Each dot is the integrated heat per injection and the error bars represent uncertainty with this integrated value. The experiment was done in both direct (B56 in cell) and reverse (B56 in syringe) with similar results.C) Time from Nuclear envelope breakdown (NEBD) to mitotic exit of cells expressing the indicated B56 inhibitors with each circle representing a single cell. Only cells with similar expression levels of the various B56 inhibitor constructs were analysed. Median time is indicated by red line. A representative result from at least three independent experiments is shown. At least 25 cells were counted per condition in the experiment shown. A Mann-Whitney U‐test was used for statistical analysis (ns: non‐significant, ***P ≤ 0.001).D) Volcano plot representing mass spectrometry identified proteins co-purifying with B56 inhibitor versus control inhibitor from HeLa cells. PP2A-B56 subunits co-purifying with the B56 inhibitor are indicated.E) and F) Competition assay in HeLa cells stably expressing RFP-tagged B56 inhibitor (LxxIxE) or control inhibitor (AxxAxA). YFP-B56α was transfected into and subsequently purified from these cell lines. Loss of binding of indicated proteins determined by either mass spectrometry (pink - B56 SLIM containing protein and known B56 interactor; blue - known B56 interactor, green - B56 SLIM containing protein) (E) or Western blotting (F).


Figure 2A - B ) Schematic of synchronisation protocol for G1 / S ( A ) or mitotic ( B ) arrested cells and accompanying volcano plot of phosphorylation sites quantified . The phosphorylation sites showing an increase in the presence of the B56 inhibitor are shown in light grey . Phosphorylation sites in a protein containing an LxxIxE motif are coloured pink .

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Figure 2A-B) Schematic of synchronisation protocol for G1/S (A) or mitotic (B) arrested cells and accompanying volcano plot of phosphorylation sites quantified. The phosphorylation sites showing an increase in the presence of the B56 inhibitor are shown in light grey. Phosphorylation sites in a protein containing an LxxIxE motif are coloured pink.


Figure 3A ) Michaelis - Menten kinetic parameters of purified PP2A - B56α and PP2A - B55α holoenzymes were determined against the indicated phospho - peptides . Mean and standard deviation shown in plots as black bars ( n = 3 independent experiments ) B - D ) In vitro dephosphorylation by the PP2A - B55α and PP2A - B56α holoenzymes of panels of phosphorylated peptides as indicated . Mean and standard deviation shown in plots as black bars ( n = 3 independent experiments )

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Figure 3A) Michaelis-Menten kinetic parameters of purified PP2A-B56α and PP2A-B55α holoenzymes were determined against the indicated phospho-peptides. Mean and standard deviation shown in plots as black bars (n=3 independent experiments)B-D) In vitro dephosphorylation by the PP2A-B55α and PP2A-B56α holoenzymes of panels of phosphorylated peptides as indicated. Mean and standard deviation shown in plots as black bars (n=3 independent experiments)


Figure 4B ) The indicated YFP - FoxO3 constructs were transfected into HeLa cells , purified using YFP resin and PP2A - B56 binding determined by Western blotting . PP2A - A ; scaffold subunit . C ) - D ) YFP - FoxO3 constructs were transfected into HeLa cells and lysates ( pS413 ) or YFP purifications ( pS253 ) were subjected to Western Blotting and probed with the indicated phosphoantibodies . Quantifications arise from five ( pS413 ) or three ( pS253 ) independent experiments . Mean and standard deviation shown in plots as black bars . E ) Steady - state localization of the indicated YFP - FOXO3 variants in HeLa cells . Each data point represents quantification from a single cell . A representative result from at least three independent experiments is shown . At least 25 cells were counted per condition in the experiment shown . Scale bar is 10 μm . Mann - Whitney test significance values ; ∗ p < 0 . 02 , ∗ ∗ ∗ p < 0 . 0002 . F ) Dephosphorylation by the PP2A - B56α holoenzyme complex of substrates with increasing length between phosphorylation sites and binding motifs as depicted . Engineered substrates containing three TP sites were phosphorylated with radioactive ATP using Cdk1 and incubated with the PP2A - B56α holoenzyme . Removal of radioactive phosphate was monitored over time . Mean and standard deviation from 3 experiments is shown .

O B-PANEL_START O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4B) The indicated YFP-FoxO3 constructs were transfected into HeLa cells, purified using YFP resin and PP2A-B56 binding determined by Western blotting. PP2A-A; scaffold subunit.C) - D) YFP-FoxO3 constructs were transfected into HeLa cells and lysates (pS413) or YFP purifications (pS253) were subjected to Western Blotting and probed with the indicated phosphoantibodies. Quantifications arise from five (pS413) or three (pS253) independent experiments. Mean and standard deviation shown in plots as black bars.E) Steady-state localization of the indicated YFP-FOXO3 variants in HeLa cells. Each data point represents quantification from a single cell. A representative result from at least three independent experiments is shown. At least 25 cells were counted per condition in the experiment shown. Scale bar is 10 μm. Mann-Whitney test significance values; ∗p < 0.02, ∗∗∗p < 0.0002.F) Dephosphorylation by the PP2A-B56α holoenzyme complex of substrates with increasing length between phosphorylation sites and binding motifs as depicted. Engineered substrates containing three TP sites were phosphorylated with radioactive ATP using Cdk1 and incubated with the PP2A-B56α holoenzyme. Removal of radioactive phosphate was monitored over time. Mean and standard deviation from 3 experiments is shown.


Figure 5B ) KD values obtained by ITC measurements with full - length recombinant B56α and indicated ADAM17 variant peptides . C ) The indicated full - length murine Myc - ADAM17 derivatives were transfected into HeLa cells stably expressing YFP - B56α . YFP - B56α was purified ( IP ) and ADAM17 binding determined by Western blotting . PP2A - C ; catalytic subunit , PP2A - A ; scaffold subunit . D ) Dephosphorylation by the PP2A - B56α holoenzyme complex of the indicated phosphorylated GST - ADAM17 ( V724 - C827 ) fragments . The GST - ADAM17 ( V724 - C827 ) substrates was phosphorylated with radioactive ATP using Protein Kinase A and incubated with the PP2A - B56α holoenzyme . Removal of radioactive phosphate was monitored over time . The mean and Standard deviation of 4 independent experiments are shown . E ) Protein expression of ADAM17 variants in the DLD - 1 Adam17 - / - cell line , determined by Western blot . GAPDH was used as an internal loading control . F ) Amphiregulin ( AREG ) shedding measured by ELISA of conditioned media from untreated , H2O2 treated or irradiated with x - ray DLD - 1 Adam17 - / - cells ( clone # 1 ) expressing full - length ADAM17 variants ( wt , I762A or LEE ) . Two sided , unpaired Students t - test test was applied to test for significant differences * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 . Mean and standard deviation indicated from at least three independent experiments . G ) Exogenous wt and I762A full length ADAM17 was immuno - purified from Adam17 - / - cells treated with H2O2 and subjected to label - free LC - MS / MS to determine differential phosphorylation status of T735 and S808 ( S811 in murine ADAM17 ) .

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Figure 5B) KD values obtained by ITC measurements with full-length recombinant B56α and indicated ADAM17 variant peptides.C) The indicated full-length murine Myc-ADAM17 derivatives were transfected into HeLa cells stably expressing YFP-B56α. YFP-B56α was purified (IP) and ADAM17 binding determined by Western blotting. PP2A-C; catalytic subunit, PP2A-A; scaffold subunit.D) Dephosphorylation by the PP2A-B56α holoenzyme complex of the indicated phosphorylated GST-ADAM17 (V724-C827) fragments. The GST-ADAM17 (V724-C827) substrates was phosphorylated with radioactive ATP using Protein Kinase A and incubated with the PP2A-B56α holoenzyme. Removal of radioactive phosphate was monitored over time. The mean and Standard deviation of 4 independent experiments are shown.E) Protein expression of ADAM17 variants in the DLD-1 Adam17-/- cell line, determined by Western blot. GAPDH was used as an internal loading control.F) Amphiregulin (AREG) shedding measured by ELISA of conditioned media from untreated, H2O2 treated or irradiated with x-ray DLD-1 Adam17-/- cells (clone #1) expressing full-length ADAM17 variants (wt, I762A or LEE). Two sided, unpaired Students t-test test was applied to test for significant differences *p<0.05, **p<0.01, ***p<0.001. Mean and standard deviation indicated from at least three independent experiments.G) Exogenous wt and I762A full length ADAM17 was immuno-purified from Adam17-/- cells treated with H2O2 and subjected to label-free LC-MS/MS to determine differential phosphorylation status of T735 and S808 (S811 in murine ADAM17).


Figure 6A ) EGFR activation in the ADAM17 variant expressing DLD - 1 Adam17 - / - cells upon H2O2 treatment , determined by Western blot and quantified below as the ratio of EGFR autophosphorylated at Tyr1068 to total EGFR . Two sided , unpaired Students t - test test was applied to test for significant differences * p < 0 . 05 , * * p < 0 . 01 . Mean and standard deviation indicated from three independent experiments . ( B , C ) B ) Cell proliferation and C ) Matrigel invasion of the ADAM17 variant expressing DLD - 1 Adam17 - / - cells . Two sided , unpaired Students t - test test was applied to test for significant differences * p < 0 . 05 , * * p < 0 . 01 . Mean and standard deviation indicated from three independent experiments . D ) Protein expression of the ADAM17 variants in the mouse breast cancer cell line 4T1 Adam17 - / - , determined by Western blot . GAPDH was used as an internal loading control . E ) Average tumor volume ± SEM of 4T1 Adam17 - / - cells expressing the ADAM17 variants injected into the mammary fat pad of Balb / c mice ( n = 10 for each group ) . Indicated significances are between the I762A and LEE tumors . Two sided , unpaired Students t - test test was applied to test for significant differences * p < 0 . 05 . Mean and standard error of the mean indicated . F ) Survival curve of the tumor bearing mice injected with 4T1 Adam17 - / - cells expressing the ADAM17 variants ( n = 10 for each group ) . The indicated significances are between the LEE and wt tumor bearing mice , and the LEE and I762A tumor bearing mice . Log - rank test was applied to test for significant differences . * p < 0 . 05 .

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Figure 6A) EGFR activation in the ADAM17 variant expressing DLD-1 Adam17-/- cells upon H2O2 treatment, determined by Western blot and quantified below as the ratio of EGFR autophosphorylated at Tyr1068 to total EGFR. Two sided, unpaired Students t-test test was applied to test for significant differences *p<0.05, **p<0.01. Mean and standard deviation indicated from three independent experiments.(B, C) B) Cell proliferation and C) Matrigel invasion of the ADAM17 variant expressing DLD-1 Adam17-/- cells. Two sided, unpaired Students t-test test was applied to test for significant differences *p<0.05, **p<0.01. Mean and standard deviation indicated from three independent experiments.D) Protein expression of the ADAM17 variants in the mouse breast cancer cell line 4T1 Adam17-/-, determined by Western blot. GAPDH was used as an internal loading control.E) Average tumor volume ± SEM of 4T1 Adam17-/- cells expressing the ADAM17 variants injected into the mammary fat pad of Balb/c mice (n=10 for each group). Indicated significances are between the I762A and LEE tumors. Two sided, unpaired Students t-test test was applied to test for significant differences *p<0.05. Mean and standard error of the mean indicated.F) Survival curve of the tumor bearing mice injected with 4T1 Adam17-/- cells expressing the ADAM17 variants (n=10 for each group). The indicated significances are between the LEE and wt tumor bearing mice, and the LEE and I762A tumor bearing mice. Log-rank test was applied to test for significant differences. *p<0.05.


Figure 1A Western blot analysis of lysates from WT and MIC10 KO or MIC60 KO HAP1 cells . MIC10 KO cells show a drastic reduction of MIC13 , MIC26 and MIC27 protein levels while protein levels of other MICOS components remain unchanged . MIC60 KO cells show a drastic reduction of protein levels of all MICOS components . B Representative electron micrographs of WT , MIC10 KO and MIC60 KO HAP1 cells . Scale bar 500 nm . C Quantification of CJs per mitochondrial section from different mitochondria in WT , MIC10 and MIC60 KO HAP1 cells using EM represented as boxplots . Boxplots show median and interquartile range from 25 to 75 percentile and whiskers represent minimum and maximum value . Data from n = 55 - 69 mitochondria ( from 2 independent experiments ) are shown as data points in the boxplots . * * * * P < 0 . 0001 ( using unpaired Student ' s t - test ) . D Quantification of cristae per mitochondrial section from different mitochondria in WT , MIC10 and MIC60 KO HAP1 cells using EM represented as boxplots . Boxplots show median and interquartile range from 25 to 75 percentile and whiskers represent minimum and maximum value . Data from n = 55 - 69 mitochondria ( from 2 independent experiments ) are shown as data points in the boxplots . * * * * P < 0 . 0001 ( using unpaired Student ' s t - test ) . E Comparison of oxygen consumption rates ( pmol O2 / s , normalized for cell numbers by Hoechst staining ) of basal , proton leak , maximum and non - mitochondrial respiration in WT and MIC10 KO cells obtained from 3 independent experiments . The data is normalized to basal respiration from HAP1 WT . Bar and error bar represent mean ± SEM from 3 independent experiments . * P = 0 . 034 for basal ( using one sample t - test ) and * P = 0 . 018 for maximum respiration ( using unpaired Student ' s t - test ) . F Comparison of oxygen consumption rates ( pmol O2 / s , normalized for cell numbers by Hoechst staining ) of basal , proton leak , maximum and non - mitochondrial respiration in WT and MIC60 KO cells obtained from 3 independent experiments . The data is normalized to basal respiration from HAP1 WT . Bar and error bar represent mean ± SEM from 3 independent experiments . * P = 0 . 01 for basal ( using one sample t - test ) , * * * P = 0 . 0004 for maximum respiration and * P = 0 . 012 for non - mitochondrial respiration ( using unpaired Student ' s t - test ) .

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Figure 1A Western blot analysis of lysates from WT and MIC10 KO or MIC60 KO HAP1 cells. MIC10 KO cells show a drastic reduction of MIC13, MIC26 and MIC27 protein levels while protein levels of other MICOS components remain unchanged. MIC60 KO cells show a drastic reduction of protein levels of all MICOS components.B Representative electron micrographs of WT, MIC10 KO and MIC60 KO HAP1 cells. Scale bar 500 nm.C Quantification of CJs per mitochondrial section from different mitochondria in WT, MIC10 and MIC60 KO HAP1 cells using EM represented as boxplots. Boxplots show median and interquartile range from 25 to 75 percentile and whiskers represent minimum and maximum value. Data from n = 55-69 mitochondria (from 2 independent experiments) are shown as data points in the boxplots. ****P < 0.0001 (using unpaired Student's t-test).D Quantification of cristae per mitochondrial section from different mitochondria in WT, MIC10 and MIC60 KO HAP1 cells using EM represented as boxplots. Boxplots show median and interquartile range from 25 to 75 percentile and whiskers represent minimum and maximum value. Data from n = 55-69 mitochondria (from 2 independent experiments) are shown as data points in the boxplots. ****P < 0.0001 (using unpaired Student's t-test).E Comparison of oxygen consumption rates (pmol O2/s, normalized for cell numbers by Hoechst staining) of basal, proton leak, maximum and non-mitochondrial respiration in WT and MIC10 KO cells obtained from 3 independent experiments. The data is normalized to basal respiration from HAP1 WT. Bar and error bar represent mean ± SEM from 3 independent experiments. *P = 0.034 for basal (using one sample t-test) and *P = 0.018 for maximum respiration (using unpaired Student's t-test).F Comparison of oxygen consumption rates (pmol O2/s, normalized for cell numbers by Hoechst staining) of basal, proton leak, maximum and non-mitochondrial respiration in WT and MIC60 KO cells obtained from 3 independent experiments. The data is normalized to basal respiration from HAP1 WT. Bar and error bar represent mean ± SEM from 3 independent experiments. *P = 0.01 for basal (using one sample t-test), ***P = 0.0004 for maximum respiration and *P = 0.012 for non-mitochondrial respiration (using unpaired Student's t-test).


Figure 2A Representative STED super - resolution images of WT and MIC10 KO HAP1 cells immunostained with MIC60 or MIC10 antibodies ( top panel ) and TOMM70 ( middle panel ) . Bottom panel shows merged images . Arrowheads show colocalization of MIC60 and TOMM70 punctae . Scale bar 500 nm . B Quantification of median interpunctae distance ( IPD ) per mitochondrion between MIC60 or MIC10 punctae in indicated cell lines represented by boxplots . Boxplots show median and interquartile range from 25 to 75 percentile and whiskers represent minimum and maximum value . Median IPD from n = 10 - 16 mitochondria ( from 2 independent experiments ) are shown as data points in the boxplots . ns , not significant , unpaired Student ' s t - test .

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Figure 2A Representative STED super-resolution images of WT and MIC10 KO HAP1 cells immunostained with MIC60 or MIC10 antibodies (top panel) and TOMM70 (middle panel). Bottom panel shows merged images. Arrowheads show colocalization of MIC60 and TOMM70 punctae. Scale bar 500 nm.B Quantification of median interpunctae distance (IPD) per mitochondrion between MIC60 or MIC10 punctae in indicated cell lines represented by boxplots. Boxplots show median and interquartile range from 25 to 75 percentile and whiskers represent minimum and maximum value. Median IPD from n = 10-16 mitochondria (from 2 independent experiments) are shown as data points in the boxplots. ns, not significant, unpaired Student's t-test.


Figure 3FRAP curves ( curve fitted ) of WT Hela cells expressing GFP - tagged fusion proteins of MIC60 ( CJs ) , MIC10 ( CJs ) , TOMM20 ( OM ) , TIMM23 ( IBM ) and ATP5I ( CM ) . Average FRAP curves ( intensities in arbitrary units ) for each marker were obtained ( 3 independent experiments , 6 - 10 mitochondria for each experiment ) . Error bars for each time point representing SEM are shown . C FRAP curves ( curve fitted ) of MIC13 KO Hela cells expressing GFP - tagged fusion proteins of MIC60 ( CJs ) , MIC10 ( CJs ) , TOMM20 ( OM ) , TIMM23 ( IBM ) and ATP5I ( CM ) . Average FRAP curves ( intensities in arbitrary units ) for each marker were obtained ( 3 independent experiments , 6 - 10 mitochondria for each experiment ) . Error bars for each time point representing SEM are shown . D Histogram showing average percentage of mobile fractions , obtained from curve fits of all mitochondria expressing GFP - tagged versions of MIC60 ( CJs ) , MIC10 ( CJs ) , TOMM20 ( OM ) , TIMM23 ( IBM ) and ATP5I ( CM ) in WT and MIC13 KO Hela cells ( data calculated from FRAP curves shown in Figure 3B and 3C that are obtained from 3 independent experiments , 6 - 10 mitochondria from each experiment ) .

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Figure 3FRAP curves (curve fitted) of WT Hela cells expressing GFP-tagged fusion proteins of MIC60 (CJs), MIC10 (CJs), TOMM20 (OM), TIMM23 (IBM) and ATP5I (CM). Average FRAP curves (intensities in arbitrary units) for each marker were obtained (3 independent experiments, 6-10 mitochondria for each experiment). Error bars for each time point representing SEM are shown.C FRAP curves (curve fitted) of MIC13 KO Hela cells expressing GFP-tagged fusion proteins of MIC60 (CJs), MIC10 (CJs), TOMM20 (OM), TIMM23 (IBM) and ATP5I (CM). Average FRAP curves (intensities in arbitrary units) for each marker were obtained (3 independent experiments, 6-10 mitochondria for each experiment). Error bars for each time point representing SEM are shown.D Histogram showing average percentage of mobile fractions, obtained from curve fits of all mitochondria expressing GFP-tagged versions of MIC60 (CJs), MIC10 (CJs), TOMM20 (OM), TIMM23 (IBM) and ATP5I (CM) in WT and MIC13 KO Hela cells (data calculated from FRAP curves shown in Figure 3B and 3C that are obtained from 3 independent experiments, 6-10 mitochondria from each experiment).


Figure 4A - D Representative single particle tracks of cells expressing MIC60 - ( A ) and MIC10 - SNAP ( B ) in WT Hela cells and MIC60 - ( C ) and MIC10 - SNAP ( D ) in MIC13 KO Hela cells , stained with silicone - rhodamine dye , and imaged at a rate of 33 ms / frame . Single tracks were colour - coded according to temporal appearance . Scale Bar 5 µm . Insets in each image show zoomed - in images of few representative tracks . E Cumulative frequency of tracks having corresponding values of instantaneous diffusion coefficients ( insD ) in WT and MIC13 KO Hela cells expressing MIC60 - and MIC10 - SNAP stained with silicone - rhodamine . Number of tracks analysed ( obtained from 2 independent experiments ) : n = 2541 , 2540 , 3560 and 1441 in WT Hela cells expressing MIC60 - SNAP and MIC10 - SNAP , MIC13 KO Hela cells expressing MIC60 - SNAP and MIC10 - SNAP , respectively . ( * * * * P ≤ 0 . 0001 for all possible comparisons , unpaired Student ' s t - test ) . F Percentage of subtracks having directed motion in WT and MIC13 KO Hela cells expressing MIC60 - and MIC10 - SNAP and in WT and MIC60 KO HAP1 cells expressing MIC10 - SNAP . Number of corresponding subtracks analysed ( obtained from 2 independent experiments ) : n = 2561 , 2443 , 3402 , 1360 , 561 and 1110 . Data are mean ± SEM . * P ≤ 0 . 05 , * * * P ≤ 0 . 001 , * * * * P ≤ 0 . 0001 , unpaired Student ' s t - test .

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Figure 4A-D Representative single particle tracks of cells expressing MIC60- (A) and MIC10-SNAP (B) in WT Hela cells and MIC60- (C) and MIC10-SNAP (D) in MIC13 KO Hela cells, stained with silicone-rhodamine dye, and imaged at a rate of 33 ms/frame. Single tracks were colour-coded according to temporal appearance. Scale Bar 5 µm. Insets in each image show zoomed-in images of few representative tracks.E Cumulative frequency of tracks having corresponding values of instantaneous diffusion coefficients (insD) in WT and MIC13 KO Hela cells expressing MIC60- and MIC10-SNAP stained with silicone-rhodamine. Number of tracks analysed (obtained from 2 independent experiments): n = 2541, 2540, 3560 and 1441 in WT Hela cells expressing MIC60-SNAP and MIC10-SNAP, MIC13 KO Hela cells expressing MIC60-SNAP and MIC10-SNAP, respectively. (****P ≤ 0.0001 for all possible comparisons, unpaired Student's t-test).F Percentage of subtracks having directed motion in WT and MIC13 KO Hela cells expressing MIC60- and MIC10-SNAP and in WT and MIC60 KO HAP1 cells expressing MIC10-SNAP. Number of corresponding subtracks analysed (obtained from 2 independent experiments): n = 2561, 2443, 3402, 1360, 561 and 1110. Data are mean ± SEM. *P ≤ 0.05, ***P ≤ 0.001, ****P ≤ 0.0001, unpaired Student's t-test.


Figure 5A , C Representative live - cell STED super - resolution images ( t = 0 s ) showing WT ( A ) and MIC13 KO ( C ) Hela cells expressing MIC10 - SNAP stained with silicone - rhodamine . Box in ( A and C ) mark selection shown as a zoom in panel ( B and D ) respectively . Scale bar 500 nm . E Blind quantification of merging and splitting events of CJs in WT and MIC13 KO Hela cells expressing MIC10 - SNAP ( 3 independent experiments , 5 - 8 mitochondria for each experiment ) represented as boxplots . Boxplots show median and interquartile range from 25 to 75 percentile and whiskers represent minimum and maximum value . * P = 0 . 026 ( merging and splitting events in WT cells ) and * * * * P < 0 . 0001 ( merging and splitting events of WT vs . MIC13 KO ) , unpaired Student ' s t - test .

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Figure 5A, C Representative live-cell STED super-resolution images (t=0 s) showing WT (A) and MIC13 KO (C) Hela cells expressing MIC10-SNAP stained with silicone-rhodamine. Box in (A and C) mark selection shown as a zoom in panel (B and D) respectively. Scale bar 500 nm.E Blind quantification of merging and splitting events of CJs in WT and MIC13 KO Hela cells expressing MIC10-SNAP (3 independent experiments, 5-8 mitochondria for each experiment) represented as boxplots. Boxplots show median and interquartile range from 25 to 75 percentile and whiskers represent minimum and maximum value. *P = 0.026 (merging and splitting events in WT cells) and ****P < 0.0001 (merging and splitting events of WT vs. MIC13 KO), unpaired Student's t-test.