Figure 3Binding of purified recombinant ATPIF1 - GFP to CV . Mitochondrial membranes were disrupted by freeze - thaw and exposed to ATPIF1 - GFP at different ratios of mitochondrial ( mt ) protein / ATPIF1 purified protein . Binding was assessed in BNGE by quantifying ATPIF1 - GFP fluorescence . ( A ) BNGE assessed for ATPIF1 - GFP binding ( top ) ; CV hydrolytic activity ( middle ) and protein loading ( bottom ) . Binding of purified recombinant ATPIF1 - GFP to CV . Mitochondrial membranes were disrupted by freeze - thaw and exposed to ATPIF1 - GFP at different ratios of mitochondrial ( mt ) protein / ATPIF1 purified protein . ( C - D ) Representative acidification rate profile showing the effects on ATP hydrolytic activity ( C ) and its quantification ( D ) ( n = 4 ) . For each biological replicate , technical replicates were averaged . Data represent average ± SEM . Binding of purified recombinant ATPIF1 - GFP to CV . Mitochondrial membranes were disrupted by freeze - thaw and exposed to ATPIF1 - GFP at different ratios of mitochondrial ( mt ) protein / ATPIF1 purified protein . Binding was assessed in BNGE by quantifying ATPIF1 - GFP fluorescence . Binding competition assay between ATPIF1 - GFP and EPI to CV . ( E ) BNGE assessed for ATPIF1 - GFP binding ( top ) ; CV hydrolytic activity ( middle ) and protein loading ( bottom ) .

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 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 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

Figure 3Binding of purified recombinant ATPIF1-GFP to CV. Mitochondrial membranes were disrupted by freeze-thaw and exposed to ATPIF1-GFP at different ratios of mitochondrial (mt) protein/ATPIF1 purified protein. Binding was assessed in BNGE by quantifying ATPIF1-GFP fluorescence. (A) BNGE assessed for ATPIF1-GFP binding (top); CV hydrolytic activity (middle) and protein loading (bottom).Binding of purified recombinant ATPIF1-GFP to CV. Mitochondrial membranes were disrupted by freeze-thaw and exposed to ATPIF1-GFP at different ratios of mitochondrial (mt) protein/ATPIF1 purified protein. (C-D) Representative acidification rate profile showing the effects on ATP hydrolytic activity (C) and its quantification (D) (n=4). For each biological replicate, technical replicates were averaged. Data represent average ± SEM.Binding of purified recombinant ATPIF1-GFP to CV. Mitochondrial membranes were disrupted by freeze-thaw and exposed to ATPIF1-GFP at different ratios of mitochondrial (mt) protein/ATPIF1 purified protein. Binding was assessed in BNGE by quantifying ATPIF1-GFP fluorescence. Binding competition assay between ATPIF1-GFP and EPI to CV. (E) BNGE assessed for ATPIF1-GFP binding (top); CV hydrolytic activity (middle) and protein loading (bottom).


Figure 4 ( A - B ) PLA of CV - bound ATPIF1 produces a red fluorescence signal where anti - ATPIF1 and anti - ATP5A1 antibodies are localized at a distance less than 10nm of each other . Anti - TOMM20 ( green ) was used to reveal mitochondrial architecture . ( A ) Representative high - resolution Airyscan confocal micrographs showing control fibroblasts treated with EPI for 24hrs . Maximum intensity projection is shown . Scale bars : 20 µm and 5 µm in zoomed image . Note that PLA reveals the spatial distribution of ATPIF1 binding along the mitochondrial length . ( B ) Analysis of ATPIF1 - CV complexes occurrence quantified as PLA dots per µm3of mitochondria . Effect of EPI is shown relative to untreated . ( n = 2 from > 50 replicates per n ) . In all cases , for each biological replicate , technical replicates were averaged . Data represent average ± SEM . Two - way ANOVA followed by Šídák ' s multiple comparisons test shows statistical differences depicted by p - value . ( E ) mtATP content measured in control and human mitochondrial disease fibroblasts treated for 30min with increasing concentrations of EPI as indicated . Analysis shows the mean intensity of mtATP probe in MTG area normalized per % of correspondent untreated cells ( n = 3 ) . In all cases , for each biological replicate , technical replicates were averaged . Data represent average ± SEM . Two - way ANOVA followed by Šídák ' s multiple comparisons test shows statistical differences depicted by p - value .

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

Figure 4(A-B) PLA of CV-bound ATPIF1 produces a red fluorescence signal where anti-ATPIF1 and anti-ATP5A1 antibodies are localized at a distance less than 10nm of each other. Anti-TOMM20 (green) was used to reveal mitochondrial architecture. (A) Representative high-resolution Airyscan confocal micrographs showing control fibroblasts treated with EPI for 24hrs. Maximum intensity projection is shown. Scale bars: 20 µm and 5 µm in zoomed image. Note that PLA reveals the spatial distribution of ATPIF1 binding along the mitochondrial length. (B) Analysis of ATPIF1-CV complexes occurrence quantified as PLA dots per µm3of mitochondria. Effect of EPI is shown relative to untreated. (n=2 from >50 replicates per n). In all cases, for each biological replicate, technical replicates were averaged. Data represent average ± SEM. Two-way ANOVA followed by Šídák's multiple comparisons test shows statistical differences depicted by p-value.(E) mtATP content measured in control and human mitochondrial disease fibroblasts treated for 30min with increasing concentrations of EPI as indicated. Analysis shows the mean intensity of mtATP probe in MTG area normalized per % of correspondent untreated cells (n=3). In all cases, for each biological replicate, technical replicates were averaged. Data represent average ± SEM. Two-way ANOVA followed by Šídák's multiple comparisons test shows statistical differences depicted by p-value.


Figure 5PLA of CV - bound ATPIF1 ( red ) . Anti - TOMM20 ( green ) was used to reveal mitochondrial architecture . ( A ) Representative confocal micrographs showing fibroblasts treated with EPI for 24hrs . Maximum intensity projection is shown . Scale bars : 20 µm and 5 µm zoomed images . ( B ) Analysis of ATPIF1 - CV complexes occurrence quantified as PLA dots per µm3of mitochondria in patient fibroblasts normalized in % of Ctrl untreated ( n = 2 from > 50 replicates per n ) . In all cases , data represent average ± SEM . For each biological replicate , technical replicates were averaged . Two - way ANOVA followed by Šídák ' s multiple comparisons test shows statistical differences depicted by p - value . PLA of CV - bound ATPIF1 ( red ) . Anti - TOMM20 ( green ) was used to reveal mitochondrial architecture . ( C ) Effect of EPI on ATPIF1 - CV complexes occurrence quantified as PLA dots per µm3 under the indicated conditions . Values are shown as relative to control untreated . In all cases , data represent average ± SEM . For each biological replicate , technical replicates were averaged . Two - way ANOVA followed by Šídák ' s multiple comparisons test shows statistical differences depicted by p - value . ( E - G ) Effect of EPI on CIII - deficient ( BCS1L ) cells stable - expressing ATPIF1WT or ATPIF1H49K . ( E ) CV ATP hydrolytic capacity measured by the acidification rate and normalized maximal respiration . Maximal respiration was determined using the OCR channel ( Max OCR on SR ) . Effect of EPI is shown as % of control cells , untreated with EPI . Cells were treated for 24hrs with 50nM EPI ( n ≥ 4 ) . ( F ) mtATP content measured as fluorescence intensity per mitochondria area and shown as % of control untreated ( n = 5 ) . Mitochondrial area was quantified using MTG staining . Cells were treated for 30min . ( G ) Total cellular ATP content shown as % of untreated cells . Cells were treated for 24hrs ( n ≥ 4 ) . In all cases , data represent average ± SEM . For each biological replicate , technical replicates were averaged . Two - way ANOVA followed by Šídák ' s multiple comparisons test shows statistical differences depicted by p - value .

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 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 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 O O O O O O O O O O O O O O O O O O O O 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 5PLA of CV-bound ATPIF1 (red). Anti-TOMM20 (green) was used to reveal mitochondrial architecture. (A) Representative confocal micrographs showing fibroblasts treated with EPI for 24hrs. Maximum intensity projection is shown. Scale bars: 20 µm and 5 µm zoomed images. (B) Analysis of ATPIF1-CV complexes occurrence quantified as PLA dots per µm3of mitochondria in patient fibroblasts normalized in % of Ctrl untreated (n=2 from >50 replicates per n). In all cases, data represent average ± SEM. For each biological replicate, technical replicates were averaged. Two-way ANOVA followed by Šídák's multiple comparisons test shows statistical differences depicted by p-value.PLA of CV-bound ATPIF1 (red). Anti-TOMM20 (green) was used to reveal mitochondrial architecture. (C) Effect of EPI on ATPIF1-CV complexes occurrence quantified as PLA dots per µm3 under the indicated conditions. Values are shown as relative to control untreated. In all cases, data represent average ± SEM. For each biological replicate, technical replicates were averaged. Two-way ANOVA followed by Šídák's multiple comparisons test shows statistical differences depicted by p-value.(E-G) Effect of EPI on CIII-deficient (BCS1L) cells stable-expressing ATPIF1WT or ATPIF1H49K. (E) CV ATP hydrolytic capacity measured by the acidification rate and normalized maximal respiration. Maximal respiration was determined using the OCR channel (Max OCR on SR). Effect of EPI is shown as % of control cells, untreated with EPI. Cells were treated for 24hrs with 50nM EPI (n≥4). (F) mtATP content measured as fluorescence intensity per mitochondria area and shown as % of control untreated (n=5). Mitochondrial area was quantified using MTG staining. Cells were treated for 30min. (G) Total cellular ATP content shown as % of untreated cells. Cells were treated for 24hrs (n≥4). In all cases, data represent average ± SEM. For each biological replicate, technical replicates were averaged. Two-way ANOVA followed by Šídák's multiple comparisons test shows statistical differences depicted by p-value.


Figure 6D2 . mdx mice , harboring the mdx mutation in a DBA / 2J genetic background ( D2 . mdx ) were subjected to exercise induced gastrocnemius injury by stimulating muscle contraction ( eccentric injury ) . Levels of CV , ATPIF1 and maximal CV ATP hydrolytic capacity were measured . ( A ) Representative western blot image ( left ) and quantification ( right ) showing CV ( ATP5A1 ) and CII ( SDHA ) levels in gastrocnemius homogenate in WT and mdx mice after 24hrs of eccentric injury ( inj ) . Vinculin was used as loading control . Note that only in mdx mice eccentric injury results in reduced CV content . n = 4 . For each biological replicate , technical replicates were averaged . Data represent average ± SEM . Two - way ANOVA followed by Šídák ' s multiple comparisons test shows statistical differences depicted by p - value . D2 . mdx mice , harboring the mdx mutation in a DBA / 2J genetic background ( D2 . mdx ) were subjected to exercise induced gastrocnemius injury by stimulating muscle contraction ( eccentric injury ) . Levels of CV , ATPIF1 and maximal CV ATP hydrolytic capacity were measured . ( C ) Analysis of levels of the cleaved OPA1 in gastrocnemius homogenate in WT and mdx mice 24hrs after eccentric injury . n = 4 . For each biological replicate , technical replicates were averaged . Data represent average ± SEM . Two - way ANOVA followed by Šídák ' s multiple comparisons test shows statistical differences depicted by p - value .

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 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

Figure 6D2.mdx mice, harboring the mdx mutation in a DBA/2J genetic background (D2.mdx) were subjected to exercise induced gastrocnemius injury by stimulating muscle contraction (eccentric injury). Levels of CV, ATPIF1 and maximal CV ATP hydrolytic capacity were measured. (A) Representative western blot image (left) and quantification (right) showing CV (ATP5A1) and CII (SDHA) levels in gastrocnemius homogenate in WT and mdx mice after 24hrs of eccentric injury (inj). Vinculin was used as loading control. Note that only in mdx mice eccentric injury results in reduced CV content. n=4. For each biological replicate, technical replicates were averaged. Data represent average ± SEM. Two-way ANOVA followed by Šídák's multiple comparisons test shows statistical differences depicted by p-value.D2.mdx mice, harboring the mdx mutation in a DBA/2J genetic background (D2.mdx) were subjected to exercise induced gastrocnemius injury by stimulating muscle contraction (eccentric injury). Levels of CV, ATPIF1 and maximal CV ATP hydrolytic capacity were measured. (C) Analysis of levels of the cleaved OPA1 in gastrocnemius homogenate in WT and mdx mice 24hrs after eccentric injury. n=4. For each biological replicate, technical replicates were averaged. Data represent average ± SEM. Two-way ANOVA followed by Šídák's multiple comparisons test shows statistical differences depicted by p-value.


Figure 7D2 . mdx mice were treated with EPI orally twice daily , with doses ranging from 0 . 5 to 15 mg / kg / day . After 13 days of treatment , mice were subjected to exercise induced gastrocnemius injury by stimulating muscle contraction and the effect of EPI on muscle force were measured . ( A ) Plantarflexor force quantification after eccentric injury . n = 8 . For each biological replicate , technical replicates were averaged . Data represent average ± SEM . unpaired t - tests ( A , shows statistical differences depicted by p - value . D2 . mdx mice were treated with EPI orally After 13 days of treatment , mice were subjected to exercise induced gastrocnemius injury by stimulating muscle contraction and OPA1 cleavage were measured . ( E ) Levels of cleaved OPA1 in gastrocnemius homogenate from mdx mice subjected to the indicated combinations of injury and EPI treatment . n = 8 . For each biological replicate , technical replicates were averaged . Data represent average ± SEM . Two - way ANOVA followed by Šídák ' s multiple comparisons testD2 . mdx mice were treated with EPI ( G ) Detection and quantification of myofiber membrane integrity or damage following injury using Evans Blue dye ( EBD ) in gastrocnemius muscle from vehicle and EPI treated mdx mice . Damaged fiber membranes allow dye entry that appears as red color . Yellow asterisks indicate damage fibers ( n ≥ 4 ) . unpaired t - tests G ) shows statistical differences depicted by p - value .

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 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 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

Figure 7D2.mdx mice were treated with EPI orally twice daily, with doses ranging from 0.5 to 15 mg/kg/day. After 13 days of treatment, mice were subjected to exercise induced gastrocnemius injury by stimulating muscle contraction and the effect of EPI on muscle force were measured. (A) Plantarflexor force quantification after eccentric injury. n=8. For each biological replicate, technical replicates were averaged. Data represent average ± SEM. unpaired t-tests (A, shows statistical differences depicted by p-value.D2.mdx mice were treated with EPI orally After 13 days of treatment, mice were subjected to exercise induced gastrocnemius injury by stimulating muscle contraction and OPA1 cleavage were measured. (E) Levels of cleaved OPA1 in gastrocnemius homogenate from mdx mice subjected to the indicated combinations of injury and EPI treatment. n=8. For each biological replicate, technical replicates were averaged. Data represent average ± SEM. Two-way ANOVA followed by Šídák's multiple comparisons testD2.mdx mice were treated with EPI (G) Detection and quantification of myofiber membrane integrity or damage following injury using Evans Blue dye (EBD) in gastrocnemius muscle from vehicle and EPI treated mdx mice. Damaged fiber membranes allow dye entry that appears as red color. Yellow asterisks indicate damage fibers (n≥4). unpaired t-tests G) shows statistical differences depicted by p-value.


Figure 1 ( B ) ParB clusters on F plasmid in vivo . Typical E . coli cells ( DLT3594 ) displays foci of ParBF - mVenus protein ( top ) expressed from the endogenous genetic locus of the F plasmid ( F1 - 10B - mVenus ) . The nucleoid is labelled with Hu - mCherry ( central ) . The overlay ( bottom ) combines the two fluorescent channels . Over 99 % of cells harbor ParBF foci . White bars : 1 µm ( C ) ParBF binding outside parSF on the F plasmid is compatible with a power - law decay . High resolution ChIP - seq performed on DLT3586 carrying the F plasmid ( F1 - 10B ) . The ParB density , normalized to 1 at the first bp downstream the last parSF binding repeat after background subtraction , is displayed over 14 - Kbp on the right side of parSF . Monte Carlo simulations and analytic formula are represented in red and dotted black lines , respectively . MC simulations were performed with a Freely - Jointed Chain of linear length L = 15 - Kbp and a cluster radius σ = 75nm . The two other parameters , the Kuhn length a = 10 - bp and the total number of proteins on the F plasmid Nt = 360 ( related to the normalization constant of the protein concentration κ = 0 . 41 ) were fitted from the ChIP - seq data ( see text and Fig . EV2 ) . As a benchmark for simulations , the analytics are obtained from Eq . ( 1 ) with the same parameters . Inset ; The ParBF binding profile ( black line ) is represented as the number of nucleotide reads over 80 - Kbp centered at parS . The number of reads in the Input sample ( grey line ) is normalized to the total number of reads in the IP sampleTypical E . coli cell displays foc expressed from the endogenous genetic locus of the F plasmid ( F1 - 10B - mVenus ) . The nucleoid is labelled with Hu - mCherry ( central ) . The overlay ( bottom ) combines the two fluorescent channels parSF inserted at the xylE locus on E . coli chromosome from DLT358ParBF binding outside parSF on the F plasmid is compatible with a power - law decay . High resolution ChIP - seq performed on DLT3586 carrying the F plasmid ( F1 - 10B ) . The ParB density , normalized to 1 at the first bp downstream the last parSF binding repeat after background subtraction , is displayed over 14 - Kbp on the right side of parSF . Monte Carlo simulations and analytic formula are represented in red and dotted black lines , respectively . MC simulations were performed with a Freely - Jointed Chain of linear length L = 15 - Kbp and a cluster radius σ = 75nm . The two other parameters , the Kuhn length a = 10 - bp and the total number of proteins on the F plasmid Nt = 360 ( related to the normalization constant of the protein concentration κ = 0 . 41 ) were fitted from the ChIP - seq dat parSF inserted at the xylE locus on E . coli chromosome fro

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 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 O O O O O 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 1(B) ParB clusters on F plasmid in vivo. Typical E. coli cells (DLT3594) displays foci of ParBF-mVenus protein (top) expressed from the endogenous genetic locus of the F plasmid (F1-10B-mVenus). The nucleoid is labelled with Hu-mCherry (central). The overlay (bottom) combines the two fluorescent channels. Over 99 % of cells harbor ParBF foci. White bars: 1 µm(C) ParBF binding outside parSF on the F plasmid is compatible with a power-law decay. High resolution ChIP-seq performed on DLT3586 carrying the F plasmid (F1-10B). The ParB density, normalized to 1 at the first bp downstream the last parSF binding repeat after background subtraction, is displayed over 14-Kbp on the right side of parSF. Monte Carlo simulations and analytic formula are represented in red and dotted black lines, respectively. MC simulations were performed with a Freely-Jointed Chain of linear length L=15-Kbp and a cluster radius σ=75nm. The two other parameters, the Kuhn length a=10-bp and the total number of proteins on the F plasmid Nt=360 (related to the normalization constant of the protein concentration κ=0.41) were fitted from the ChIP-seq data (see text and Fig. EV2). As a benchmark for simulations, the analytics are obtained from Eq.(1) with the same parameters. Inset; The ParBF binding profile (black line) is represented as the number of nucleotide reads over 80-Kbp centered at parS. The number of reads in the Input sample (grey line) is normalized to the total number of reads in the IP sampleTypical E. coli cell displays foc expressed from the endogenous genetic locus of the F plasmid (F1-10B-mVenus). The nucleoid is labelled with Hu-mCherry (central). The overlay (bottom) combines the two fluorescent channels parSF inserted at the xylE locus on E. coli chromosome from DLT358ParBF binding outside parSF on the F plasmid is compatible with a power-law decay. High resolution ChIP-seq performed on DLT3586 carrying the F plasmid (F1-10B). The ParB density, normalized to 1 at the first bp downstream the last parSF binding repeat after background subtraction, is displayed over 14-Kbp on the right side of parSF. Monte Carlo simulations and analytic formula are represented in red and dotted black lines, respectively. MC simulations were performed with a Freely-Jointed Chain of linear length L=15-Kbp and a cluster radius σ=75nm. The two other parameters, the Kuhn length a=10-bp and the total number of proteins on the F plasmid Nt=360 (related to the normalization constant of the protein concentration κ=0.41) were fitted from the ChIP-seq dat parSF inserted at the xylE locus on E. coli chromosome fro DLT207


Figure 2 ( A ) Normalized and rescaled ParBF binding profiles at different ParBF / parSF ratio . ChIP - seq density on the right side of parSF inserted at xylE were measured in DLT2075 induced ( 16 , 28 ) or not ( 0 . 4 ) with IPTG ( 100 and 500 µM ) , or carrying HCN plasmids pZC302 ( 0 . 04 ) or pJYB57 ( 0 . 016 ) , normalized as in Fig . 1C , E with the amplitudes of the curves rescaled by the indicated factors ( 1 . 2 , 10 or 50 ) to overlap with the curves of highest amplitude . The ParB / parS ratio is calculated relative to the one of F plasmid as determined from Western blot analyses ( Appendix Fig . S2B ) . Monte Carlo simulations and analytical formula are plotted with the same parameters as in Fig . 1E . Note ( i ) that the dips at ~ 9 - kbp are not visible for the low levels of available ParB since the signal is close to the basal level and ( ii ) that the ChIP - seq data at 100 µM IPTG induction ( 16 ) are the same as in Fig . 1E . Inset ; Same as in the main to display the density without rescaling . ( B ) ParBF are dispersed in the cell upon titration by HCN plasmids . ParBF - mVenus expressed from pJYB294 were imaged as in Fig . 1D in DLT3577 ( left ) and DLT3576 ( right ) carrying pZC302 and pJYB57 , respectively . The number of extra parSF per cell , indicated on top of each raw , are estimated from the copy number per cell of HCN plasmids carrying 10 specific binding sites . White bars : 1 µm . ( C ) The size of ParBF clusters is independent of the intracellular ParBF concentration . We considered two possible evolutions of the cluster size upon variations of ParB amount in the framework of " Nucleation & caging " with corresponding schematics drawn on the right . For direct comparison with ( A ) , all curves are displayed with a rescaling of the amplitude corresponding to the WT expression level . Top : constant ParB concentration ; supposing that clusters are compact , the cluster radius σ would depend on the number m of ParB like $ \ \ sigma = m ^ { \ frac { 1 } { 3 } } $ . Predictions profiles , plotted at different ratio of ParB / parS , vary within the range of the experimental levels tested . Bottom : constant cluster size ; ParB concentrations vary but the range of exploration remains the same resulting in

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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 2(A) Normalized and rescaled ParBF binding profiles at different ParBF/parSF ratio. ChIP-seq density on the right side of parSF inserted at xylE were measured in DLT2075 induced (16, 28) or not (0.4) with IPTG (100 and 500 µM), or carrying HCN plasmids pZC302 (0.04) or pJYB57 (0.016), normalized as in Fig. 1C, E with the amplitudes of the curves rescaled by the indicated factors (1.2, 10 or 50) to overlap with the curves of highest amplitude. The ParB/parS ratio is calculated relative to the one of F plasmid as determined from Western blot analyses (Appendix Fig. S2B). Monte Carlo simulations and analytical formula are plotted with the same parameters as in Fig. 1E. Note (i) that the dips at ~9-kbp are not visible for the low levels of available ParB since the signal is close to the basal level and (ii) that the ChIP-seq data at 100 µM IPTG induction (16) are the same as in Fig. 1E. Inset; Same as in the main to display the density without rescaling. (B) ParBF are dispersed in the cell upon titration by HCN plasmids. ParBF-mVenus expressed from pJYB294 were imaged as in Fig. 1D in DLT3577 (left) and DLT3576 (right) carrying pZC302 and pJYB57, respectively. The number of extra parSF per cell, indicated on top of each raw, are estimated from the copy number per cell of HCN plasmids carrying 10 specific binding sites. White bars: 1 µm. (C) The size of ParBF clusters is independent of the intracellular ParBF concentration. We considered two possible evolutions of the cluster size upon variations of ParB amount in the framework of "Nucleation & caging" with corresponding schematics drawn on the right. For direct comparison with (A), all curves are displayed with a rescaling of the amplitude corresponding to the WT expression level. Top: constant ParB concentration; supposing that clusters are compact, the cluster radius σ would depend on the number m of ParB like$\ \sigma = m^{\frac{1}{3}}$. Predictions profiles, plotted at different ratio of ParB/parS, vary within the range of the experimental levels tested. Bottom: constant cluster size; ParB concentrations vary but the range of exploration remains the same resulting in overlappin


Figure 3 ( A ) The formation of secondary ParBF - DNA complexes requires the box II motif . EMSA were performed with a 144 - bp 32P - labelled DNA fragments ( C144 ) carrying a single 16 - bp parS binding motif . Reaction mixtures containing 100 µg . ml - 1 sonicated salmon sperm DNA were incubated in the absence ( - ) or the presence of increasing concentrations ( grey triangle ; 10 , 30 , 100 , 300 , and 1000 nM ) of ParBF or ParBF - 3R * . Positions of free and bound probes are indicated on the left . B1 represents complexes involving the specific interaction on the 16 - bp binding site , while B " 2 and B " 3 complexes represent secondary complexes involving the parSF site with one or two additional nsDNA - binding interactions , respectively ( Sanchez et al , ( B ) ParBF cluster formation requires the box II motif . Epifluorescence microscopy of ParBF - 3R * - mVenus from DLT3566 is displayed as in Fig . 1D . White bars : 1 µm ( C ) ParBF in vivo DNA binding in the vicinity of parSF sites requires the box II motif . ChIP - seq was performed on DLT3726 carrying parSF in the xylE chromosomal locus and expressing ParBF - 3R * variant . ParBF - 3R * DNA binding profile displayed the number of nucleotide reads as a function of the E . coli genomic coordinates . The peak at parSF covered approximately 950 - bp , which corresponds to the 402 - bp between the 1st and 10th specific binding sites and ~ 280 - bp on each sides ( representing the average size of the DNA library ; see Appendix Fig . S3E ) . No ParBF - 3R * enrichment was found on parSF - flanking DNA and elsewhere on the chromosome . Inset , zoom in on the right side of parSF over 5 - Kbp with the ParB density , normalized to 1 at the first bp after the last parS binding repeat , plotted as a function of the distance from parSF . Note that a highly similar DNA binding pattern is obtained with ParBF - 3R * - mVenus ( strain DLT3566 ; Appendix Fig . S3C )

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 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 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 3(A) The formation of secondary ParBF-DNA complexes requires the box II motif. EMSA were performed with a 144-bp 32P-labelled DNA fragments (C144) carrying a single 16-bp parS binding motif. Reaction mixtures containing 100 µg.ml-1 sonicated salmon sperm DNA were incubated in the absence (-) or the presence of increasing concentrations (grey triangle; 10, 30, 100, 300, and 1000 nM) of ParBF or ParBF-3R*. Positions of free and bound probes are indicated on the left. B1 represents complexes involving the specific interaction on the 16-bp binding site, while B"2 and B"3 complexes represent secondary complexes involving the parSF site with one or two additional nsDNA-binding interactions, respectively (Sanchez et al,(B) ParBF cluster formation requires the box II motif. Epifluorescence microscopy of ParBF-3R*-mVenus from DLT3566 is displayed as in Fig. 1D. White bars: 1 µm(C) ParBF in vivo DNA binding in the vicinity of parSF sites requires the box II motif. ChIP-seq was performed on DLT3726 carrying parSF in the xylE chromosomal locus and expressing ParBF-3R* variant. ParBF-3R* DNA binding profile displayed the number of nucleotide reads as a function of the E. coli genomic coordinates. The peak at parSF covered approximately 950-bp, which corresponds to the 402-bp between the 1st and 10th specific binding sites and ~280-bp on each sides (representing the average size of the DNA library; see Appendix Fig. S3E). No ParBF-3R* enrichment was found on parSF-flanking DNA and elsewhere on the chromosome. Inset, zoom in on the right side of parSF over 5-Kbp with the ParB density, normalized to 1 at the first bp after the last parS binding repeat, plotted as a function of the distance from parSF. Note that a highly similar DNA binding pattern is obtained with ParBF -3R*-mVenus (strain DLT3566; Appendix Fig. S3C)


Figure 4 ( A ) Schematic representation of the genomic locus of the chromosome 1 of V . cholerae with the three parS sites , named parS1 - 3 . The rRNA operon ( blue rectangle ) spans the genomic coordinates 53823 to 59123 ( B ) ChIP - seq performed on strain N16961 is displayed as the number of nucleotide reads in function of the genomic coordinates . Correspondence to the parS1 - 3 location represented in ( A ) is indicated by grey dotted lines . The number of reads in the Input sample ( grey line ) is normalized to the total number of reads in the IP sample . ( C ) We modeled the ChIP - Seq data as in Fig . 1C - E by means of MC simulations with a Freely jointed chain of size N = 2000 monomers of size a = 16 - bp . Data are normalized after background subtraction to the read value at parS1 ( genomic coordinate 62438 ) . The best fit was achieved with σ = 25nm and an amplitude κ = 0 . 15 leading to Nt ~ 50 ParB on the chromosome . In the MC simulation , we accounted for the finite width of the distribution around parS sites by including the average fragment size of the DNA

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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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(A) Schematic representation of the genomic locus of the chromosome 1 of V. cholerae with the three parS sites, named parS1-3. The rRNA operon (blue rectangle) spans the genomic coordinates 53823 to 59123 (B) ChIP-seq performed on strain N16961 is displayed as the number of nucleotide reads in function of the genomic coordinates. Correspondence to the parS1-3 location represented in (A) is indicated by grey dotted lines. The number of reads in the Input sample (grey line) is normalized to the total number of reads in the IP sample. (C) We modeled the ChIP-Seq data as in Fig.1C-E by means of MC simulations with a Freely jointed chain of size N=2000 monomers of size a=16-bp. Data are normalized after background subtraction to the read value at parS1 (genomic coordinate 62438). The best fit was achieved with σ=25nm and an amplitude κ=0.15 leading to Nt~50 ParB on the chromosome. In the MC simulation, we accounted for the finite width of the distribution around parS sites by including the average fragment size of the DNA librar


Figure 5ChIP - sequencing assays were performed on DLT2075 ( xylE : : parSF ) expressing ParBF grown in exponential ( expo ) or stationary ( stat ) phases with addition of rifampicin when indicated ( + Rif ) . The assays have been performed in duplicate for the + Rif and once for the stationary phase experiments . ( A ) ParBF DNA binding around parSF is independent of active transcription . The color - coded ParBF profiles are represented over 50 - Kbp as the relative ParB density normalized to 1 at the first bp after the last parSF binding site . Loci A , C , E and F are defined in Appendix Fig . S1A . ( B ) The dips and peaks are highly similar in the three indicated conditions . Same as in ( A ) with zoom in on the right side of parSF up to 9 - Kpb and normalization to 1 at genomic coordinate 230 . The dotted line corresponds to the analytics description of " Nucleation and caging " ( see details in Fig . 1C - E ) . ( C ) ParBF binding profile upstream of the locus A . Same as in ( A ) with zoom in from - 6 . 5 to - 16 . 5 - Kbp by normalization to 1 at genomic coordinate - 6 . 5 - Kbp ( upstream of the dip at the locus A ) . The ParBF DNA binding profile remains compatible to a power - law , represented by the analytics description ( dotted line ) , upstream of the locus A in stationary phase ( black ) and in exponential phase ( blue ) . Also , the dips and peaks are highly similar in both conditions . These data are not in favor of the " 1D - spreading " or the " Spreading and bridging " models that predicts a basal uniform distribution or a linear decrease after a barrier , respectively ( Broedersz et al , 2014 ) . ( D ) The promoter region at locus A prevents ParBF DNA binding . Chip - seq assays were performed in isogenic xylE : : parSF strains ( DLT2075 ; black curve ) in which the locus A is replaced by a kanamycin gene ( DLT3651 ; red curve ) . The assay in the ∆ ( locus A ) genomic context has been performed once . The relative ParB density as a function of the distance from parSF

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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 5ChIP-sequencing assays were performed on DLT2075 (xylE::parSF) expressing ParBF grown in exponential (expo) or stationary (stat) phases with addition of rifampicin when indicated (+Rif). The assays have been performed in duplicate for the +Rif and once for the stationary phase experiments. (A) ParBF DNA binding around parSF is independent of active transcription. The color-coded ParBF profiles are represented over 50-Kbp as the relative ParB density normalized to 1 at the first bp after the last parSF binding site. Loci A, C, E and F are defined in Appendix Fig. S1A. (B) The dips and peaks are highly similar in the three indicated conditions. Same as in (A) with zoom in on the right side of parSF up to 9-Kpb and normalization to 1 at genomic coordinate 230. The dotted line corresponds to the analytics description of "Nucleation and caging" (see details in Fig. 1C-E). (C) ParBF binding profile upstream of the locus A. Same as in (A) with zoom in from -6.5 to -16.5-Kbp by normalization to 1 at genomic coordinate -6.5-Kbp (upstream of the dip at the locus A). The ParBF DNA binding profile remains compatible to a power-law, represented by the analytics description (dotted line), upstream of the locus A in stationary phase (black) and in exponential phase (blue). Also, the dips and peaks are highly similar in both conditions. These data are not in favor of the "1D-spreading" or the "Spreading and bridging" models that predicts a basal uniform distribution or a linear decrease after a barrier, respectively (Broedersz et al, 2014). (D) The promoter region at locus A prevents ParBF DNA binding. Chip-seq assays were performed in isogenic xylE::parSF strains (DLT2075; black curve) in which the locus A is replaced by a kanamycin gene (DLT3651; red curve). The assay in the ∆(locus A) genomic context has been performed once. The relative ParB density as a function of the distance from parSF is


Figure 6ParBF exchange between foci was measured by FRAP an from two - foci cell of DLT1215 carrying pJYB234 A - Representative images of a photobleached cell during a FRAP experiment . The 488 nm laser was pulsed ( Bleach ) on one of the two foci at ~ 2 . 4 sec ( black arrow ) . Red and blue arrows correspond to the bleached and unbleached focus , respectively . Time is indicated in second ( upper right ) . The cell outline is drawn in red . Scale bar : 1 μmParBF exchange between foci was measured by FRAP and FLIM ( fluorescence lifetime imaging microscopy ) from two - foci cell of DLT1215 carrying pJYB234 B - Quantification of ParBF - mVenus fluorescence intensity over time . The dynamics of fluorescence intensity is shown from averaging 18 independent measurements of the bleached ( FRAP , red line ) and unbleached ( FLIM , blue line ) foci . Foci fluorescence intensity in each experiment was normalized to the average intensity of each focus before

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 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

Figure 6ParBF exchange between foci was measured by FRAP an from two-foci cell of DLT1215 carrying pJYB234 A- Representative images of a photobleached cell during a FRAP experiment. The 488 nm laser was pulsed (Bleach) on one of the two foci at ~2.4 sec (black arrow). Red and blue arrows correspond to the bleached and unbleached focus, respectively. Time is indicated in second (upper right). The cell outline is drawn in red. Scale bar: 1 μmParBF exchange between foci was measured by FRAP and FLIM (fluorescence lifetime imaging microscopy) from two-foci cell of DLT1215 carrying pJYB234 B- Quantification of ParBF-mVenus fluorescence intensity over time. The dynamics of fluorescence intensity is shown from averaging 18 independent measurements of the bleached (FRAP, red line) and unbleached (FLIM, blue line) foci. Foci fluorescence intensity in each experiment was normalized to the average intensity of each focus before photobleaching


Figure 1A Immunoblot of endogenous BiP resolved by native - PAGE from lysates of CHO - K1 S21 wild - type ( wt ) or FICD - / - cells either transiently overexpressing wild - type FICD ( high expression level ; Hi ) or mutant FICDE234G ( E / G ) or stably expressing recombinant wild - type FICD ( low expression level ; Lo ) . The cells in lanes 1 - 4 were mock transfected . Where indicated cells were exposed to cycloheximide ( CHX ; 100 µg / mL ) for 3 h before lysis . Unmodified ( ' A ' ) and AMPylated ( ' B ' ) monomeric and oligomeric ( II and III ) forms of BiP are indicated . Immunoblots of the same samples resolved by SDS - PAGE report on FICD , total BiP and eIF2α ( loading control ) . Data representative of four independent experiments are shown . B Wild - type FICD forms homomeric complexes in vivo . Immunoblots of orthogonally - tagged wild - type and Leu258Asp mutant FICD in the input cell lysate and following recovery by pull - down with streptavidin ( recognising the AviTag ) or anti - FLAG antibody . Proteins were detected with fluorescently - labelled streptavidin ( StrepIR800 ) or FLAG antibody . Data representative of three independent experiments are shown . C Immunoblot of endogenous BiP from transfected CHO - K1 S21 FICD - / - cells ( as in A ) . Note that cells expressing monomeric FICDL258D accumulate AMPylated BiP . Data representative of three independent experiments are shown . D Size - exclusion chromatography ( SEC ) analysis of wild - type and mutant FICD proteins ( each at 20 µM ) . The elution times of protein standards are indicated as a reference . Note that the Leu258Asp mutation monomerises FICD , while Gly299Ser causes partial monomerisation . E Comparison of the signal - averaged sedimentation coefficients of wild - type ( red ) and monomeric mutant FICDL258D ( blue ) , as measured by analytical ultracentrifugation . A fit for monomer - dimer association ( solid red line ) , constrained using the average value for the monomeric protein ( dashed line , 2 . 82 S , Sw , 20 = 3 . 02 S ) , yielded a Kd of 1 . 2 nM with a 95 % confidence interval between 1 . 1 to 1 . 4 nM and a value of 4 . 08 S for the dimer ( Sw , 20 = 4 . 36 S ) . The fitted data points are from three independent experiments . F Autoradiograph of BiP , AMPylated in vitro by the indicated FICD derivatives , with [ α - 32P ] - ATP as a substrate and resolved by SDS - PAGE . Proteins in the gel were visualised by Coomassie staining . A representative result of three independent experiments is shown . The graph shows the quantified relative BiP - AMP signals and SD bars generated by wild - type ( wt ) FICD and the indicated monomeric mutants .

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 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 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 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 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

Figure 1A Immunoblot of endogenous BiP resolved by native-PAGE from lysates of CHO-K1 S21 wild-type (wt) or FICD-/- cells either transiently overexpressing wild-type FICD (high expression level; Hi) or mutant FICDE234G (E/G) or stably expressing recombinant wild-type FICD (low expression level; Lo). The cells in lanes 1-4 were mock transfected. Where indicated cells were exposed to cycloheximide (CHX; 100 µg/mL) for 3 h before lysis. Unmodified ('A') and AMPylated ('B') monomeric and oligomeric (II and III) forms of BiP are indicated. Immunoblots of the same samples resolved by SDS-PAGE report on FICD, total BiP and eIF2α (loading control). Data representative of four independent experiments are shown.B Wild-type FICD forms homomeric complexes in vivo. Immunoblots of orthogonally-tagged wild-type and Leu258Asp mutant FICD in the input cell lysate and following recovery by pull-down with streptavidin (recognising the AviTag) or anti-FLAG antibody. Proteins were detected with fluorescently-labelled streptavidin (StrepIR800) or FLAG antibody. Data representative of three independent experiments are shown.C Immunoblot of endogenous BiP from transfected CHO-K1 S21 FICD-/- cells (as in A). Note that cells expressing monomeric FICDL258D accumulate AMPylated BiP. Data representative of three independent experiments are shown.D Size-exclusion chromatography (SEC) analysis of wild-type and mutant FICD proteins (each at 20 µM). The elution times of protein standards are indicated as a reference. Note that the Leu258Asp mutation monomerises FICD, while Gly299Ser causes partial monomerisation.E Comparison of the signal-averaged sedimentation coefficients of wild-type (red) and monomeric mutant FICDL258D (blue), as measured by analytical ultracentrifugation. A fit for monomer-dimer association (solid red line), constrained using the average value for the monomeric protein (dashed line, 2.82 S, Sw,20 = 3.02 S), yielded a Kd of 1.2 nM with a 95% confidence interval between 1.1 to 1.4 nM and a value of 4.08 S for the dimer (Sw,20 = 4.36 S). The fitted data points are from three independent experiments.F Autoradiograph of BiP, AMPylated in vitro by the indicated FICD derivatives, with [α-32P]-ATP as a substrate and resolved by SDS-PAGE. Proteins in the gel were visualised by Coomassie staining. A representative result of three independent experiments is shown. The graph shows the quantified relative BiP-AMP signals and SD bars generated by wild-type (wt) FICD and the indicated monomeric mutants.


Figure 2A Monomerising FICD mutations inhibit deAMPylation . Shown is a representative plot of data points and fit curves of the time - dependent deAMPylation of a fluorescent BiPV461F - AMPFAM by the indicated FICD proteins ( at 7 . 5 µM ) as detected by a change in fluorescence polarisation ( FP ) . DeAMPylation rates calculated from independent experimentsB - C Dimer interface mutants both AMPylate and deAMPylate BiP . Shown are representative autoradiographs of thin layer chromatography ( TLC ) plates revealing AMP produced from reactions containing [ α - 32P ] - ATP and the indicated FICD enzymes in the presence or absence of the co - substrate BiP ( arrow indicates direction of nucleotide migration ) . The radioactive signals were quantified and the AMP signals were normalised to the total nucleotide signal in each sample . Plotted below are mean values ± SD from at least three independent experiments . Unpaired t - tests were performed . E Detection of the time - dependent accumulation of AMPylated BiPT229A - V461F in radioactive reactions , containing [ α - 32P ] - ATP and the indicated FICD proteins , in the presence of excess trap . At the specified time - points samples were taken and analysed by SDS - PAGE . The autoradiograph ( 32P ) illustrates the radioactive signals , which represent AMPylated BiP . Proteins were visualised by Coomassie staining . The radioactive signals were quantified and presented in the graph below . Mean values ± SD of three independent experiments are 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 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 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

Figure 2A Monomerising FICD mutations inhibit deAMPylation. Shown is a representative plot of data points and fit curves of the time-dependent deAMPylation of a fluorescent BiPV461F-AMPFAM by the indicated FICD proteins (at 7.5 µM) as detected by a change in fluorescence polarisation (FP). DeAMPylation rates calculated from independent experimentsB-C Dimer interface mutants both AMPylate and deAMPylate BiP. Shown are representative autoradiographs of thin layer chromatography (TLC) plates revealing AMP produced from reactions containing [α-32P]-ATP and the indicated FICD enzymes in the presence or absence of the co-substrate BiP (arrow indicates direction of nucleotide migration). The radioactive signals were quantified and the AMP signals were normalised to the total nucleotide signal in each sample. Plotted below are mean values ± SD from at least three independent experiments. Unpaired t-tests were performed.E Detection of the time-dependent accumulation of AMPylated BiPT229A-V461F in radioactive reactions, containing [α-32P]-ATP and the indicated FICD proteins, in the presence of excess trap. At the specified time-points samples were taken and analysed by SDS-PAGE. The autoradiograph (32P) illustrates the radioactive signals, which represent AMPylated BiP. Proteins were visualised by Coomassie staining. The radioactive signals were quantified and presented in the graph below. Mean values ± SD of three independent experiments are shown.


Figure 3A Autoradiographs of in vitro reactions containing varying concentration of wild - type FICD protein and fixed concentrations of BiPT229A - V461F and [ α - 32P ] - ATP as co - substrates , resolved by SDS - PAGE after the indicated incubation times . The proteins were visualised by Coomassie staining of the gel ( bottom ) . The reactions shown on the right were performed in the presence of an excess of S - SFICDA252C - H363A - C421S ( trap ) to delay de - modification of BiP . Representative gels are shown , and similar results were observed in three independent experiments . B As in ( A ) but with 0 . 2 µM of the indicated FICD variant . The radioactive signals were detected by autoradiography , quantified , and normalised to the signal in lane 6 . The mean radioactive signals ± SD from three independent experiments are given . The proteins were visualised by staining with Coomassie . C As in ( A ) but with dilutions of FICDC421S or covalently linked S - SFICDA252C - C421S . Reactions were preceded by a 16 h incubation of FICD in presence or absence of the reducing agent ( DTT ) . Representative gels are shown of three independent experiments . D Forced dimerisation does not significantly alter deAMPylation rates . Time - dependent deAMPylation of fluorescent BiPV461F - AMPFAM by the indicated FICD proteins ( at 7 . 5 µM ) assayed by fluorescence polarisation A representative experiment ( data points and fit curves ) is shownE Representative autoradiograph of thin layer chromatography ( TLC ) plates revealing AMP produced from reactions containing [ α - 32P ] - ATP and the indicated FICD enzymes in the presence of the co - substrate BiP . AMP signals were normalised to the total nucleotide signal in each sample and the graph below plots mean values ± SD from at least three independent experiments .

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

Figure 3A Autoradiographs of in vitro reactions containing varying concentration of wild-type FICD protein and fixed concentrations of BiPT229A-V461F and [α-32P]-ATP as co-substrates, resolved by SDS-PAGE after the indicated incubation times. The proteins were visualised by Coomassie staining of the gel (bottom). The reactions shown on the right were performed in the presence of an excess of S-SFICDA252C-H363A-C421S (trap) to delay de-modification of BiP. Representative gels are shown, and similar results were observed in three independent experiments. B As in (A) but with 0.2 µM of the indicated FICD variant. The radioactive signals were detected by autoradiography, quantified, and normalised to the signal in lane 6. The mean radioactive signals ± SD from three independent experiments are given. The proteins were visualised by staining with Coomassie. C As in (A) but with dilutions of FICDC421S or covalently linked S-SFICDA252C-C421S. Reactions were preceded by a 16 h incubation of FICD in presence or absence of the reducing agent (DTT). Representative gels are shown of three independent experiments.D Forced dimerisation does not significantly alter deAMPylation rates. Time-dependent deAMPylation of fluorescent BiPV461F-AMPFAM by the indicated FICD proteins (at 7.5 µM) assayed by fluorescence polarisation A representative experiment (data points and fit curves) is shownE Representative autoradiograph of thin layer chromatography (TLC) plates revealing AMP produced from reactions containing [α-32P]-ATP and the indicated FICD enzymes in the presence of the co-substrate BiP. AMP signals were normalised to the total nucleotide signal in each sample and the graph below plots mean values ± SD from at least three independent experiments.


Figure 4B Size - exclusion chromatography elution profile of wild - type and mutant FICD proteins ( each at 20 µM ) . Protein absorbance at 280 nm is plotted against elution time . The elution times of protein standards are indicated as a reference . C Radioactive in vitro AMPylation reactions containing the indicated FICD proteins , [ α - 32P ] - ATP , and BiPT229A - V461F were analysed by SDS - PAGE . The radioactive BiP - AMP signals were detected by autoradiography and proteins were visualised by Coomassie staining of the gel . D Representative autoradiograph of thin layer chromatography ( TLC ) plates revealing AMP produced from reactions containing [ α - 32P ] - ATP and the indicated FICD enzymes in the presence of the co - substrate BiP . The radioactive signals were quantified and the AMP signals were normalised to the total nucleotide signal in each sample . The graph shows mean AMP values ± SD from three independent experiments . E Melting temperatures ( Tm ) of the indicated FICD mutants ( at 2 µM ) were measured by differential scanning fluorimetry ( DSF ) . Shown is the mean Tm ± SD of three independent experiments . The inset shows melt curves with their negative first derivatives from a representative experiment . F A plot of the melting temperature of the indicated FICD proteins in absence ( Apo ) or presence of nucleotides . Shown are the mean Tm values ± SD of three independent DSF experiments . Monomeric FICDL258D ( mFICD ) and FICDL258D - E234G ( mFICDE / G ) as well as dimeric wild - type FICD ( dFICD ) and FICDE234G ( dFICDE / G ) were tested . ADP and ATP concentrations in mM are given in parentheses .

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 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 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 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

Figure 4B Size-exclusion chromatography elution profile of wild-type and mutant FICD proteins (each at 20 µM). Protein absorbance at 280 nm is plotted against elution time. The elution times of protein standards are indicated as a reference.C Radioactive in vitro AMPylation reactions containing the indicated FICD proteins, [α-32P]-ATP, and BiPT229A-V461F were analysed by SDS-PAGE. The radioactive BiP-AMP signals were detected by autoradiography and proteins were visualised by Coomassie staining of the gel.D Representative autoradiograph of thin layer chromatography (TLC) plates revealing AMP produced from reactions containing [α-32P]-ATP and the indicated FICD enzymes in the presence of the co-substrate BiP. The radioactive signals were quantified and the AMP signals were normalised to the total nucleotide signal in each sample. The graph shows mean AMP values ± SD from three independent experiments.E Melting temperatures (Tm) of the indicated FICD mutants (at 2 µM) were measured by differential scanning fluorimetry (DSF). Shown is the mean Tm ± SD of three independent experiments. The inset shows melt curves with their negative first derivatives from a representative experiment.F A plot of the melting temperature of the indicated FICD proteins in absence (Apo) or presence of nucleotides. Shown are the mean Tm values ± SD of three independent DSF experiments. Monomeric FICDL258D (mFICD) and FICDL258D-E234G (mFICDE/G) as well as dimeric wild-type FICD (dFICD) and FICDE234G (dFICDE/G) were tested. ADP and ATP concentrations in mM are given in parentheses.


Figure 6A BioLayer interferometry ( BLI ) derived association and dissociation traces of monomeric FICDL258D - H363A ( mFICDH363A ) or dimeric FICDH363A ( dFICDH363A ) from immobilised biotinylated BiPT229A - V461F in absence or presence of nucleotides . Unless indicated ( * ) BiP was saturated with ATP before exposure to FICD variants . A representative experiment of three independent repetitions is shown . B BLI dissociation traces of proteins as in ( A ) . At t = 0 a pre - assembled complex of immobilised , ATP - saturated BiP and the indicated FICD proteins ( associated without ATP ) were transferred into a solution without or with ATP , as indicated . A representative experiment is shown and the biphasic dissociation kinetics are quantified in ( C ) and ( D ) . C Graph of the slow dissociation rates ( koff , slow ) of monomeric FICD from BiP : ATP as shown in ( B ) . Bars represent mean values ± SD of three independent experiments . D The ATP - induced fold change in the percentage of the dissociation phase attributed to a fast dissociation ( % Fast ) , koff , fast , and koff , slow derived from the data represented in ( B ) . Bars represent mean values ± SD of three independent experiments . E ( i ) BLI assay workflow used to derive FICD dimer off rate data presented in ( ii ) ( ii ) Observed dimer off rates under different nucleotide conditions ( 5 mM , where applicable ) . ATP , but no ADP , significantly increases the dimer dissociation rate [ n . s . : not significant by Tukey test ] . Data shown is the mean ± SD of four independent experiments . F Validation of the homo - FRET dimerisation probe . The dimerisation sensitive TMR fluorescence of the labelled FRET dimer ( 2 . 5 nM ) is de - quenched specifically by equilibration with excess unlabelled dimerisation - competent FICDH363A but not monomeric FICDL258D - H363A . Mean ± SD of three independent experiments . G Homo - FRET measurement of nucleotide dependent modulation of the FICD monomer - dimer equilibrium . ATP increases and ADP decreases the proportion of monomeric FICD . * Plateaus were constrained to a shared best fit value . Data shown is the mean ± SD of four independent experiments .

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

Figure 6A BioLayer interferometry (BLI) derived association and dissociation traces of monomeric FICDL258D-H363A (mFICDH363A) or dimeric FICDH363A (dFICDH363A) from immobilised biotinylated BiPT229A-V461F in absence or presence of nucleotides. Unless indicated (*) BiP was saturated with ATP before exposure to FICD variants. A representative experiment of three independent repetitions is shown. B BLI dissociation traces of proteins as in (A). At t = 0 a pre-assembled complex of immobilised, ATP-saturated BiP and the indicated FICD proteins (associated without ATP) were transferred into a solution without or with ATP, as indicated. A representative experiment is shown and the biphasic dissociation kinetics are quantified in (C) and (D). C Graph of the slow dissociation rates (koff,slow) of monomeric FICD from BiP:ATP as shown in (B). Bars represent mean values ± SD of three independent experiments. D The ATP-induced fold change in the percentage of the dissociation phase attributed to a fast dissociation (%Fast), koff,fast, and koff,slow derived from the data represented in (B). Bars represent mean values ± SD of three independent experiments.E (i) BLI assay workflow used to derive FICD dimer off rate data presented in (ii) (ii) Observed dimer off rates under different nucleotide conditions (5 mM, where applicable). ATP, but no ADP, significantly increases the dimer dissociation rate [n.s.: not significant by Tukey test]. Data shown is the mean ± SD of four independent experiments.F Validation of the homo-FRET dimerisation probe. The dimerisation sensitive TMR fluorescence of the labelled FRET dimer (2.5 nM) is de-quenched specifically by equilibration with excess unlabelled dimerisation-competent FICDH363A but not monomeric FICDL258D-H363A. Mean ± SD of three independent experiments.G Homo-FRET measurement of nucleotide dependent modulation of the FICD monomer-dimer equilibrium. ATP increases and ADP decreases the proportion of monomeric FICD. * Plateaus were constrained to a shared best fit value. Data shown is the mean ± SD of four independent experiments.


Figure 1 ( E ) Representative immunoblot for key lipolytic enzymes HSL and ATGL protein expression and quantification ( n = 5 - 6 / group ) . Data are represented as mean ± s . e . m . E , Unpaired Student ' s t - test . ( F ) Immunoblot analysis of autophagic flux in mWAT ( upper panel ) and gWAT ( lower panel ) adipose tissue stimulated ex vivo with lysosomal inhibitors 100nM Bafilomycin A1 and 20mM NH4Cl for 4 hours or DMSO ( Vehicle ) ( n = 3 - 4 / group ) . Data are represented as mean ± s . e . m . Unpaired Student ' s t - test . ( G ) Representative transmission electron microscopy images from mesenteric adipose tissue 7 days post DSS - induced colitis induction . Lower panel is showing magnification of selected area . White arrows show autophagosomal structures . ( J ) Representative immunoblot for LC3 - I / - II and ACTIN protein expression and quantification of autophagic flux in creeping fat tissues ( CrF ) and adjacent mesenteric adipose tissues ( Ad . MAT ) of Crohn ' s disease patients ( n = 3 / group ) . Additionally , autophagic flux was determined in the mesenteric adipose tissue ( MAT ) of a colorectal cancer patient as control ( dotted line ) . Data are represented as mean ± s . e . m . ( J ) Paired Student ' s t - test .

O O 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 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 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

Figure 1(E) Representative immunoblot for key lipolytic enzymes HSL and ATGL protein expression and quantification (n = 5-6/group). Data are represented as mean ± s.e.m. E, Unpaired Student's t-test.(F) Immunoblot analysis of autophagic flux in mWAT (upper panel) and gWAT (lower panel) adipose tissue stimulated ex vivo with lysosomal inhibitors 100nM Bafilomycin A1 and 20mM NH4Cl for 4 hours or DMSO (Vehicle) (n = 3-4/group). Data are represented as mean ± s.e.m. Unpaired Student's t-test.(G) Representative transmission electron microscopy images from mesenteric adipose tissue 7 days post DSS-induced colitis induction. Lower panel is showing magnification of selected area. White arrows show autophagosomal structures.(J) Representative immunoblot for LC3-I/-II and ACTIN protein expression and quantification of autophagic flux in creeping fat tissues (CrF) and adjacent mesenteric adipose tissues (Ad. MAT) of Crohn's disease patients (n = 3/group). Additionally, autophagic flux was determined in the mesenteric adipose tissue (MAT) of a colorectal cancer patient as control (dotted line). Data are represented as mean ± s.e.m. (J) Paired Student's t-test.


Figure 2 ( C ) Representative immunoblot for LC3 - I and LC3 - II protein expression and quantification of LC3 conversion ratio ( LC3 - II / LC3 - I ) ( n = 3 / group ) . Data are represented as mean ± s . e . m . C , Unpaired Student ' s t - test . ( F ) Colon length after two weeks post - deletion ( steady state ; n = 14 / group ) and after DSS at day 7 ( n = 18 - 22 / group ) . Data are represented as mean ± s . e . m . , F , Unpaired Student ' s t - test . ( G ) Representative H & E staining images ( 10x magnification ) of colon sections and quantification of histological score at steady state ( n = 9 / group ) and DSS colitis ( n = 18 - 22 / group ) . Data are represented as mean ± s . e . m . G , Unpaired Student ' s t - test . ( I ) Absolute number CD45 + immune cells from colons at steady state ( n = 13 - 14 / group ) or at 7 days post - DSS induction ( n = 18 - 22 / group ) . ( J ) Frequency of myeloid cell population in colon at day 7 post - DSS induction ( n = 18 - 25 / group ) . ( K ) Absolute number of Ly6C + monocytes discriminated by the absence or presence of MHCII for infiltrating and inflammatory monocytes respectively ( n = 18 - 25 / group ) . Data are represented as mean ± s . e . m . I ) Unpaired Student ' s t - test . ( J , K ) Two - Way ANOVA .

O O 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 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 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 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

Figure 2(C) Representative immunoblot for LC3-I and LC3-II protein expression and quantification of LC3 conversion ratio (LC3-II/LC3-I) (n = 3/group). Data are represented as mean ± s.e.m. C, Unpaired Student's t-test.(F) Colon length after two weeks post-deletion (steady state; n = 14/group) and after DSS at day 7 (n = 18-22/group). Data are represented as mean ± s.e.m. , F, Unpaired Student's t-test.(G) Representative H&E staining images (10x magnification) of colon sections and quantification of histological score at steady state (n = 9/group) and DSS colitis (n = 18-22/group). Data are represented as mean ± s.e.m. G, Unpaired Student's t-test.(I) Absolute number CD45+ immune cells from colons at steady state (n = 13-14/group) or at 7 days post-DSS induction (n = 18-22/group). (J) Frequency of myeloid cell population in colon at day 7 post-DSS induction (n = 18-25/group). (K) Absolute number of Ly6C+ monocytes discriminated by the absence or presence of MHCII for infiltrating and inflammatory monocytes respectively (n = 18-25/group). Data are represented as mean ± s.e.m. I) Unpaired Student's t-test. (J, K) Two-Way ANOVA.


Figure 3 ( A ) Ex vivo lipolysis measured by released free fatty acid ( left , n = 4 - 5 / group ) and glycerol ( right , n = 7 - 8 / group ) in culture supernatant of adipose tissue explants simulated with isoproterenol ( 10µM ) for 1 - 2h . ( B ) Ex vivo lipolysis measured by released free fatty acid ( left , n = 4 / group ) and glycerol ( right , n = 7 / group ) adipose tissue explants simulated with TNFα ( 100ng / mL ) for 24h before replacing with fresh medium in the absence of TNFα for 3h . Data are represented as mean ± s . e . m . ( A , B , Two - Way ANOVA . ( B , Unpaired Student ' s t - test . ( E ) Concentration of individual FFA species in serum in water - treated and DSS - treated mice as measured by FID - GC ( n = 12 - 14 / group ) . Data are represented as mean ± s . e . m . E ) Two - Way ANOVA .

O O 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

Figure 3(A) Ex vivo lipolysis measured by released free fatty acid (left, n = 4-5/group) and glycerol (right, n = 7-8/group) in culture supernatant of adipose tissue explants simulated with isoproterenol (10µM) for 1-2h. (B) Ex vivo lipolysis measured by released free fatty acid (left, n = 4/group) and glycerol (right, n = 7/group) adipose tissue explants simulated with TNFα (100ng/mL) for 24h before replacing with fresh medium in the absence of TNFα for 3h. Data are represented as mean ± s.e.m. (A, B, Two-Way ANOVA. (B, Unpaired Student's t-test.(E) Concentration of individual FFA species in serum in water-treated and DSS-treated mice as measured by FID-GC (n = 12-14/group). Data are represented as mean ± s.e.m. E) Two-Way ANOVA.


Figure 4 ( C ) Ex vivo lipolysis assays on Atg7 - deficient adipose tissue explants simulated with isoproterenol ( 10µM ) for 1 - 2h ( n = 5 - 6 / group ) . Data are represented as mean ± s . e . m . ( C , Two - Way ANOVA . ( H ) Expression of pro - inflammatory cytokines in colon tissues at 7 days post - DSS induction ( n = 8 / group ) . Dotted line represents non - inflamed controls . Data are represented as mean ± s . e . m . Unpaired Student ' s t - test . ( K ) Absolute number of Ly6C + monocytes discriminated by the absence or presence of MHCII for infiltrating and inflammatory monocytes respectively ( n = 8 / group ) . Data are represented as mean ± s . e . m . K ) Unpaired Student ' s t - test .

O O 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 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 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

Figure 4(C) Ex vivo lipolysis assays on Atg7-deficient adipose tissue explants simulated with isoproterenol (10µM) for 1-2h (n = 5-6/group). Data are represented as mean ± s.e.m. (C, Two-Way ANOVA.(H) Expression of pro-inflammatory cytokines in colon tissues at 7 days post-DSS induction (n = 8/group). Dotted line represents non-inflamed controls. Data are represented as mean ± s.e.m. Unpaired Student's t-test.(K) Absolute number of Ly6C+ monocytes discriminated by the absence or presence of MHCII for infiltrating and inflammatory monocytes respectively (n = 8/group). Data are represented as mean ± s.e.m. K) Unpaired Student's t-test.


Figure 5 ( E ) Fold change expression of NRF2 - target genes in primary visceral adipocytes at day 7 after DSS induction from normalized counts of RNAseq dataset ( n = 6 / group ) . Data are represented as mean ± s . e . m . Unpaired Student ' s t - test . ( H ) Representative immunoblot of EPHX1 and EPHX2 in gonadal adipose tissues at day 7 after DSS induction . Asterix indicating non - specific band ( n = 14 - 18 / group ) . Data are represented as mean ± s . e . m . Unpaired Student ' s t - test .

O O 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 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

Figure 5(E) Fold change expression of NRF2-target genes in primary visceral adipocytes at day 7 after DSS induction from normalized counts of RNAseq dataset (n = 6/group). Data are represented as mean ± s.e.m. Unpaired Student's t-test.(H) Representative immunoblot of EPHX1 and EPHX2 in gonadal adipose tissues at day 7 after DSS induction. Asterix indicating non-specific band (n = 14-18/group). Data are represented as mean ± s.e.m. Unpaired Student's t-test.


Figure 6 ( B ) Colitis was induced in mice for 7 days and adipose tissues were extracted and cultured for 6 hours in serum - starved medium . Secretion of IL - 10 and from mesenteric ( left panel ) and gonadal adipose tissues ( right panel ) was measured by ELISA . Shapes identify individual experiments ( n = 5 - 15 / group ) . Data are represented as mean ± s . e . m . ( B ) Two - Way ANOVA with regression for experiment . ( F ) Serum cytokines upon DSS - induced colitis at day 7 post - induction ( n = 17 - 23 / group ) . Data are represented as mean ± s . e . m . Two - Way ANOVA .

O O 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 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

Figure 6(B) Colitis was induced in mice for 7 days and adipose tissues were extracted and cultured for 6 hours in serum-starved medium. Secretion of IL-10 and from mesenteric (left panel) and gonadal adipose tissues (right panel) was measured by ELISA. Shapes identify individual experiments (n = 5-15/group). Data are represented as mean ± s.e.m. (B) Two-Way ANOVA with regression for experiment.(F) Serum cytokines upon DSS-induced colitis at day 7 post-induction (n = 17-23/group). Data are represented as mean ± s.e.m. Two-Way ANOVA.


Figure 7 ( A ) Quantification of Il10 transcript levels in RAW264 . 7 upon stimulation to epoxy fatty acids ( n = 3 / group ) . ( B ) Quantification of IL - 10 protein levels in the supernatant of RAW264 . 7 upon stimulation to epoxy fatty acids ( n = 3 / group ) . Data are represented as mean ± s . e . m . ( A , B ) One - Way ANOVA .

O O 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

Figure 7(A) Quantification of Il10 transcript levels in RAW264.7 upon stimulation to epoxy fatty acids (n = 3/group). (B) Quantification of IL-10 protein levels in the supernatant of RAW264.7 upon stimulation to epoxy fatty acids (n = 3/group). Data are represented as mean ± s.e.m. (A,B) One-Way ANOVA.


Figure 1Transgenic ESZscan4 _ Emerald cells were sorted and total RNAs were extracted and subjected to RT - qPCR . The graph shows the mRNA expression level of Arg2 in ES subpopulations . ( B ) Transgenic ESZscan4 _ Emerald cells were sorted and total proteins were extracted and subjected to immunoblotting . The image is a representative of three independent biological western blotting analysis on arg2 expression in sorted ESZscan4 _ Emerald and control cells . Transcript ( left panel ) and protein expression ( right panel ) levels of Arg2 in ESZscan4 _ LNGFR cells after transfection with a non - silencing siRNA ( siCtrl ) or siRNA against Arg2 ( siArg2 ) . Imaging flow cytometry analysis on Zscan4 + siArg2 and Zscan4 + siCtrl cells . Samples were stained by labelling histone H3 protein . The intensity of H3 fluorescent signal was measured in the nuclear region by creating a mask defined by DAPI staining . The texture feature of fluorescent signal was analysed by using the modulation method ( for details see Materials and Methods ) . Two cell populations ( R1 and R2 ) correspond to two different patterns of fluorescent signal : homogenous and clustered distribution , respectively .

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 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 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 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

Figure 1Transgenic ESZscan4_Emerald cells were sorted and total RNAs were extracted and subjected to RT-qPCR. The graph shows the mRNA expression level of Arg2 in ES subpopulations.(B)Transgenic ESZscan4_Emerald cells were sorted and total proteins were extracted and subjected to immunoblotting. The image is a representative of three independent biological western blotting analysis on arg2 expression in sorted ESZscan4_Emerald and control cells.Transcript (left panel) and protein expression (right panel) levels of Arg2 in ESZscan4_LNGFR cells after transfection with a non-silencing siRNA (siCtrl) or siRNA against Arg2 (siArg2).Imaging flow cytometry analysis on Zscan4+ siArg2 and Zscan4+ siCtrl cells. Samples were stained by labelling histone H3 protein. The intensity of H3 fluorescent signal was measured in the nuclear region by creating a mask defined by DAPI staining. The texture feature of fluorescent signal was analysed by using the modulation method (for details see Materials and Methods). Two cell populations (R1 and R2) correspond to two different patterns of fluorescent signal: homogenous and clustered distribution, respectively.


Figure 2representative SeaHorse time courses of OCR and ECAR respectively ; where are indicated the following compounds added : oligomycin ( olig . ) , carbonyl cyanide 4 - ( trifluoromethoxy ) phenylhydrazone ( FCCP ) , rotenone plus antimycin A ( R / A ) , glucose ( Glu ) , 2 - deoxyglucose ( 2DG ) . Each point in the OCR and ECAR time - courses is the average ± S . D . of four technical replicates . OCR / ECAR ratio ; ( 4 technical replicates each ) ; Basal , ( resting OCR ) / ( ECAR + Glu ) ; Max , ( OCR + FCCP ) / ( ECAR + Olig . ) Cellular NAD + and NADH content and NAD + / NADH . Confocal microscopy analysis of the mitochondrial membrane potential DYm . Pictures on the right , representative imaging of cells loaded with tetramethylrhodamine , ethyl ester ( TMRE ) ; histogram on the left , averaged fluorescence intensity / cell ± SD of n = 3 independent biological replicates ; * , P < 0 . 05 .

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 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 B-PANEL_START 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

Figure 2representative SeaHorse time courses of OCR and ECAR respectively; where are indicated the following compounds added: oligomycin (olig.), carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP), rotenone plus antimycin A (R/A), glucose (Glu), 2-deoxyglucose (2DG). Each point in the OCR and ECAR time-courses is the average ± S.D. of four technical replicates.OCR/ECAR ratio; (4 technical replicates each); Basal, (resting OCR)/(ECAR +Glu); Max, (OCR+FCCP)/(ECAR+Olig.)Cellular NAD+ and NADH content and NAD+/NADH.Confocal microscopy analysis of the mitochondrial membrane potential DYm. Pictures on the right, representative imaging of cells loaded with tetramethylrhodamine, ethyl ester (TMRE); histogram on the left, averaged fluorescence intensity/cell ± SD of n=3 independent biological replicates; *, P < 0.05.


Figure 3Representative confocal microscopy analysis of cells loaded with MitoTracker Green ( pictures on the left ) ; digital enlargements and false colors rendering of details shown as white contoured squares ( pictures on the right ) . Electron microscopy images of sorted ESZscan4 _ Emerald and control cells . Scale bar 1μmRepresentative confocal microscopy analysis of cells loaded with dichlorofluorescein - diacetate converted in dichlorofluorescein ( DCF ) ( pictures on the left ) ; digital enlargements and false - color rendering of details shown as white contoured squares ( pictures on the right ) ; histogram on the left , averaged fluorescence intensity / cell ± SEM of n = 3 independent biological replicates .

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 B-PANEL_START 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 3Representative confocal microscopy analysis of cells loaded with MitoTracker Green (pictures on the left); digital enlargements and false colors rendering of details shown as white contoured squares (pictures on the right).Electron microscopy images of sorted ESZscan4_Emerald and control cells. Scale bar 1μmRepresentative confocal microscopy analysis of cells loaded with dichlorofluorescein-diacetate converted in dichlorofluorescein (DCF) (pictures on the left); digital enlargements and false-color rendering of details shown as white contoured squares (pictures on the right); histogram on the left, averaged fluorescence intensity/cell ± SEM of n=3 independent biological replicates.


Figure 4Basal R / A - sensitive OCR measured as in Fig . 2A in the absence and in the presence of a cocktail of inhibitors ( BPTES + etomoxir + UK5099 ) ; End . Sub . , OCR measured with endogenous substrates ; Exo . Sub . , OCR measured with glutamine ( Gln ) , fatty acids ( FA ) and glucose ( Glc ) supplemented in the assay medium . The superimposed bars are means ± SEM of n = 3 independent biological replicates ( 3 technical replicates each ) ; Measurement of dependency and flexibility of the OCR to either of Gln , FA , Glc ; the assay was carried out according to the MitoFuel protocol ( see Material and Methods ) ; the superimposed bars ( whose sum corresponds to the capacity ) are means ± SEM of n = 3 independent biological replicates ( 3 technical replicates each ) ; LC - MSMS analysis of the cellular content of free aminoacids ; the symbols of aminoacids are indicated in x - axis ; the amount expressed as μmol / μg protein is indicated in y - axis . The inset shows the content of ornithine , citrulline and arginine . ( D ) LC - MSMS analysis of the cellular content of acylcarnitines ; the length of the acyl moiety is indicated by the number of carbon atoms in abscissa ; bars values are means ± SEM of n = 3 independent biological replicates ; The inset shows the content of carnitine ( C0 ) and acetylcarnitine ( C2 ) .

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 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 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

Figure 4Basal R/A-sensitive OCR measured as in Fig. 2A in the absence and in the presence of a cocktail of inhibitors (BPTES + etomoxir + UK5099); End. Sub., OCR measured with endogenous substrates; Exo. Sub., OCR measured with glutamine (Gln), fatty acids (FA) and glucose (Glc) supplemented in the assay medium. The superimposed bars are means ± SEM of n=3 independent biological replicates (3 technical replicates each);Measurement of dependency and flexibility of the OCR to either of Gln, FA, Glc; the assay was carried out according to the MitoFuel protocol (see Material and Methods); the superimposed bars (whose sum corresponds to the capacity) are means ± SEM of n=3 independent biological replicates (3 technical replicates each);LC-MSMS analysis of the cellular content of free aminoacids; the symbols of aminoacids are indicated in x-axis; the amount expressed as μmol/μg protein is indicated in y-axis. The inset shows the content of ornithine, citrulline and arginine.(D)LC-MSMS analysis of the cellular content of acylcarnitines; the length of the acyl moiety is indicated by the number of carbon atoms in abscissa; bars values are means ± SEM of n=3 independent biological replicates; The inset shows the content of carnitine (C0) and acetylcarnitine (C2).


Figure 1Electron micrograph of fibrils formed by a synthetic peptide encompassing the M45 RHIM . Representative of three samples examined . Scale bar 100 nmHT29 cell survival 18h after treatment with TNF + BV - 6 + z - VAD - fmk to induce necroptosis . Cells expressed lentivirus constructs encoding 1 - 90 or 1 - 277 fragments of M45 and were cultured + / - the addition of coumermycin A1 to induce dimerisation . N = 2 for M45 - 90 gyrase + / - coumermycin A1 and 4 for M45 - 277 gyrase and for empty vector , + / - coumermycin

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 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

Figure 1Electron micrograph of fibrils formed by a synthetic peptide encompassing the M45 RHIM. Representative of three samples examined. Scale bar 100 nmHT29 cell survival 18h after treatment with TNF+BV-6+z-VAD-fmk to induce necroptosis. Cells expressed lentivirus constructs encoding 1-90 or 1-277 fragments of M45 and were cultured +/- the addition of coumermycin A1 to induce dimerisation. N=2 for M45-90 gyrase +/- coumermycin A1 and 4 for M45-277 gyrase and for empty vector, +/- coumermycin A1


Figure 2Thioflavin T fluorescence as a function of time , following dilution of WT or AAAA His ‑ Ub ‑ M451 ‑ 90 , His - Ub - RIPK1497 ‑ 583 , and His - Ub - RIPK3387 ‑ 518 from 8 M to 200 mM urea - containing buffer . Buffer sample contains ThT but no protein . Samples were tested in triplicate and solid line is the average of the triplicates and the error bars ( in grey ) represent + / - s . dAbsorbance spectra from solutions containing Congo red with WT ( black lines ) or AAAA mutant ( dark grey lines ) His ‑ Ub ‑ M451 ‑ 90 , His - Ub - RIPK1497 ‑ 583 , and His - Ub - RIPK3387 ‑ 518 that have been dialysed against Assembly Buffer . Congo red sample alone with no protein ( light grey line ) served as a control for the experiment . Experiment conducted onceElectron micrograph of material eluted in peak 1 from the three MBP fusion proteins , as labelled . These are representative micrographs of the material observed . Scale bar 100 nm in all panelsRepresentative micrographs of insoluble material generated after treatment with thrombin removed the MBP . Experiment repeated three times . Scale bar 100 nm in all

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 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 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 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

Figure 2Thioflavin T fluorescence as a function of time, following dilution of WT or AAAA His‑Ub‑M451‑90, His-Ub-RIPK1497‑583, and His-Ub-RIPK3387‑518 from 8 M to 200 mM urea-containing buffer. Buffer sample contains ThT but no protein. Samples were tested in triplicate and solid line is the average of the triplicates and the error bars (in grey) represent +/- s.dAbsorbance spectra from solutions containing Congo red with WT (black lines) or AAAA mutant (dark grey lines) His‑Ub‑M451‑90, His-Ub-RIPK1497‑583, and His-Ub-RIPK3387‑518 that have been dialysed against Assembly Buffer. Congo red sample alone with no protein (light grey line) served as a control for the experiment. Experiment conducted onceElectron micrograph of material eluted in peak 1 from the three MBP fusion proteins, as labelled. These are representative micrographs of the material observed. Scale bar 100 nm in all panelsRepresentative micrographs of insoluble material generated after treatment with thrombin removed the MBP. Experiment repeated three times. Scale bar 100 nm in all panel


Figure 3Fluorescence polarisation profiles from incorporation of FITC - M451 ‑ 90 into excess unlabeled M451 ‑ 90 ( green ) , RIPK1497 ‑ 583 ( red ) , RIPK3387 ‑ 518 ( blue ) or ZBP1170 - 355 ( purple ) . Average polarisation profile from smoothed triplicate samples plotted , with s . d . indicated . Experiment performed twice and representative results shown hereRepresentative fluorescence intensity trace showing homomeric assembly over 40 s in mixtures of ( C ) mCHERRY - RIPK1497 ‑ 583 and YPet - RIPK1497 ‑ 583 and ( D ) mCHERRY - RIPK3387 ‑ 518 and YPet - RIPK3387 ‑ 518 . E , F Representative fluorescence intensity traces showing heteromeric assembly over 40 s in mixtures of ( E ) mCHERRY - RIPK1497 ‑ 583 and YPet - RIPK3387 ‑ 518 ( inset is expansion of region indicated by * ) and ( F ) mCHERRY - RIPK1497 ‑ 583 - AAAA and YPet - RIPK3387 ‑ 518 . All mixtures examined in at least three different experiments on three separate

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 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

Figure 3Fluorescence polarisation profiles from incorporation of FITC-M451‑90 into excess unlabeled M451‑90 (green), RIPK1497‑583 (red), RIPK3387‑518 (blue) or ZBP1170-355 (purple). Average polarisation profile from smoothed triplicate samples plotted, with s.d. indicated. Experiment performed twice and representative results shown hereRepresentative fluorescence intensity trace showing homomeric assembly over 40 s in mixtures of (C) mCHERRY-RIPK1497‑583 and YPet-RIPK1497‑583 and (D) mCHERRY-RIPK3387‑518 and YPet-RIPK3387‑518. E, F Representative fluorescence intensity traces showing heteromeric assembly over 40 s in mixtures of (E) mCHERRY-RIPK1497‑583 and YPet-RIPK3387‑518 (inset is expansion of region indicated by *) and (F) mCHERRY-RIPK1497‑583-AAAA and YPet-RIPK3387‑518. All mixtures examined in at least three different experiments on three separate occasions


Figure 4Representative fluorescence intensity traces collected over 50 s for mixtures of ( A ) mCHERRY - M451 - 90 and YPet ‑ only ; ( B ) mCHERRY - M451 - 90 and YPet ‑ RIPK1497 ‑ 583 ; ( C ) mCHERRY - M451 - 90 and YPet - RIPK3387 ‑ 518 ; ( D ) mCHERRY - M451 - 90 and YPet - ZBP1170 - 355 ; ( E ) mCHERRY - M451 - 90AAAA and YPet ‑ only ; ( F ) mCHERRY - M451 - 90AAAA and YPet - RIPK1497 - 583 ; ( G ) mCHERRY - M451 - 90AAAA and YPet - RIPK3387 ‑ 518 and ( H ) mCHERRY - M451 - 90 and YPet - ZBP1170 - 355 . Independent replicates performed at least

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

Figure 4Representative fluorescence intensity traces collected over 50 s for mixtures of (A) mCHERRY-M451-90 and YPet‑only; (B) mCHERRY-M451-90 and YPet‑RIPK1497‑583; (C) mCHERRY-M451-90 and YPet-RIPK3387‑518; (D) mCHERRY-M451-90 and YPet-ZBP1170-355; (E) mCHERRY-M451-90AAAA and YPet‑only; (F) mCHERRY-M451-90AAAA and YPet-RIPK1497-583; (G) mCHERRY-M451-90AAAA and YPet-RIPK3387‑518 and (H) mCHERRY-M451-90 and YPet-ZBP1170-355. Independent replicates performed at least twice