Figure 2AdV - GFP was incubated with 20 ug / ml h9C12 antibody or 20 mg / ml IVIg in PBS . These AdV - Ab complexes were then diluted 1 : 2 to give final doses of viral particles being added to WT THP - 1 cells ( with antibody complexed ) as indicated . A single experiment representative of 3 independent experiments is shown . THP - 1s were stimulated with a constant dose of AdV - GFP ( 50 000 pp / cell ) , and complexed with decreasing antibody concentrations as indicated . A single experiment representative of 2 independent experiments is shown . HMDM were stimulated directly ( TNF ) or primed ( IL - 1β , LDH ) with AdV - Ab complexes as in ( B ) . Data for each individual donor ( 2 total ) is shown . THP - 1 were stimulated with AdV ( 50 000 pp / cell ) complexed with h9C12 ( 20 ug / ml ) or IVIg ( 20 or 0 . 8 mg / ml ) for 16h and cell death was measured by LDH releaseTHP - 1 were stimulated with AdV ( 50 000 pp / cell ) complexed with h9C12 ( 20 ug / ml ) or IVIg ( 20 or 0 . 8 mg / ml ) for 16h and cell viability measured by PrestoBlue assayHMDM were primed , or not , and stimulated with AdV ( 50 000 pp / cell ) complexed with h9C12 ( 20 ug / ml ) or IVIg ( 20 or 0 . 8 mg / ml ) for 16h and cell death was measured by LDH releaseHMDM were primed , or not , and stimulated with AdV ( 50 000 pp / cell ) complexed with h9C12 ( 20 ug / ml ) or IVIg ( 20 or 0 . 8 mg / ml ) for 16h and cell viability measured by PrestoBlue

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Figure 2AdV-GFP was incubated with 20 ug/ml h9C12 antibody or 20 mg/ml IVIg in PBS. These AdV-Ab complexes were then diluted 1:2 to give final doses of viral particles being added to WT THP-1 cells (with antibody complexed) as indicated. A single experiment representative of 3 independent experiments is shown.THP-1s were stimulated with a constant dose of AdV-GFP (50 000 pp/cell), and complexed with decreasing antibody concentrations as indicated. A single experiment representative of 2 independent experiments is shown.HMDM were stimulated directly (TNF) or primed (IL-1β, LDH) with AdV-Ab complexes as in (B). Data for each individual donor (2 total) is shown.THP-1 were stimulated with AdV (50 000 pp/cell) complexed with h9C12 (20 ug/ml) or IVIg (20 or 0.8 mg/ml) for 16h and cell death was measured by LDH releaseTHP-1 were stimulated with AdV (50 000 pp/cell) complexed with h9C12 (20 ug/ml) or IVIg (20 or 0.8 mg/ml) for 16h and cell viability measured by PrestoBlue assayHMDM were primed, or not, and stimulated with AdV (50 000 pp/cell) complexed with h9C12 (20 ug/ml) or IVIg (20 or 0.8 mg/ml) for 16h and cell death was measured by LDH releaseHMDM were primed, or not, and stimulated with AdV (50 000 pp/cell) complexed with h9C12 (20 ug/ml) or IVIg (20 or 0.8 mg/ml) for 16h and cell viability measured by PrestoBlue assay


Figure 3HMDM were stimulated with AdV ( 50 000 pp / cell ) complexed with antibody at indicated doses for 2h and viral genomes were measured by qPCR . ( n = 4 , mean ± s . e . m ) AdV - mCherry was added to AdV - GFP at a ratio of 1 : 200 and 50 000 pp / cell of this mix was complexed with h9C12 and IVIg and indicated doses . HMDM were stimulated with these complexes for 16h and infection measured as mCherry fluorescence by flow cytometry . In virus alone the percentage of mCherry positive cells was ~ 20 % , the relative infection with antibodies was normalised to virus alone as 100 % . HMDM were stimulated for 3 h with AdV - Ab complexes as in ( A , B ) or with LeuLeu at 10μM , and lysosomal damage measured by loss of acridine orange red fluorescence using flow cytometry . HMDM were stimulated as in ( C ) and cell death was assessed by an increase in e780 fluorescence . The mean of 3 independent donors is shown . ( E ) The MFI of the AO - red channel ( 685nM ) for the populations determined as e780 positive ( dying ) or e780 negative ( live ) for the condition of AdV + IVIg 20 was measured and plotted as mean + SEM of the three donors shown in ( D ) .

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Figure 3HMDM were stimulated with AdV (50 000 pp/cell) complexed with antibody at indicated doses for 2h and viral genomes were measured by qPCR. (n = 4, mean ± s.e.m)AdV-mCherry was added to AdV-GFP at a ratio of 1:200 and 50 000 pp/cell of this mix was complexed with h9C12 and IVIg and indicated doses. HMDM were stimulated with these complexes for 16h and infection measured as mCherry fluorescence by flow cytometry. In virus alone the percentage of mCherry positive cells was ~ 20%, the relative infection with antibodies was normalised to virus alone as 100%.HMDM were stimulated for 3 h with AdV-Ab complexes as in (A,B) or with LeuLeu at 10μM, and lysosomal damage measured by loss of acridine orange red fluorescence using flow cytometry.HMDM were stimulated as in (C) and cell death was assessed by an increase in e780 fluorescence. The mean of 3 independent donors is shown.(E)The MFI of the AO- red channel (685nM) for the populations determined as e780 positive (dying) or e780 negative (live) for the condition of AdV + IVIg 20 was measured and plotted as mean + SEM of the three donors shown in (D).


Figure 4Primed or ( C ) un - primed HMDM were stimulated with AdV ( 50 000 pp / cell ) + / - h9C12 ( 20 μg / ml ) for 16h . Ctrl or Trim21 - deficient THP - 1s were stimulated for 4 h with 1000 U / ml IFNα and TRIM21 expression measured by immunoblot . WT or TRIM21 - deficient THP - 1s were stimulated with AdV ( 50 000 pp / cell ) and 20 μg / ml h9C12 or 20 mg / ml IVIg or with 200 ng / well HT - DNA or 10 μM Nigericin for 16h , and IL - 1β measured in the supernatant by ELISA . WT or TRIM21 - deficient THP - 1s were stimulated with AdV ( 50 000 pp / cell ) and 20 μg / ml h9C12 or 20 mg / ml IVIg or with 200 ng / well HT - DNA or 10 μM Nigericin for 16h , and TNF measured in the supernatant by ELISA . WT mice were injected i . v . with 2 . 5ug WT or H433A h9C12 antibody , then the next day injected i . v . with 2 . 5 x 1011 pp AdV - GFP . 4h later spleens were harvested and neutrophil influx measured by flow cytometry . HMDM were stimulated with AdV - GFP ( 250 pp / cell ) in the presence of h9C12 antibodies at indicated doses . Infection was measured after 24h by flow cytometry . Infection relative to AdV alone is shown graphed on the left ( n = 4 mean ± s . e . m ) . Representative plots and a comparison of the different h9C12 mutants at 0 . 2 ug / ml are also shown in the graph on the right ( n = 4 , mean ± s . e . m . ) .

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Figure 4Primed or (C) un- primed HMDM were stimulated with AdV (50 000 pp/cell) +/- h9C12 (20 μg/ml) for 16h.Ctrl or Trim21- deficient THP-1s were stimulated for 4 h with 1000 U/ml IFNα and TRIM21 expression measured by immunoblot.WT or TRIM21- deficient THP-1s were stimulated with AdV (50 000 pp/cell) and 20 μg/ml h9C12 or 20 mg/ml IVIg or with 200 ng/well HT-DNA or 10 μM Nigericin for 16h, and IL-1β measured in the supernatant by ELISA.WT or TRIM21- deficient THP-1s were stimulated with AdV (50 000 pp/cell) and 20 μg/ml h9C12 or 20 mg/ml IVIg or with 200 ng/well HT-DNA or 10 μM Nigericin for 16h, and TNF measured in the supernatant by ELISA.WT mice were injected i.v. with 2.5ug WT or H433A h9C12 antibody, then the next day injected i.v. with 2.5 x 1011 pp AdV-GFP. 4h later spleens were harvested and neutrophil influx measured by flow cytometry.HMDM were stimulated with AdV-GFP (250 pp/cell) in the presence of h9C12 antibodies at indicated doses. Infection was measured after 24h by flow cytometry. Infection relative to AdV alone is shown graphed on the left (n = 4 mean ± s.e.m). Representative plots and a comparison of the different h9C12 mutants at 0.2 ug/ml are also shown in the graph on the right(n = 4,mean ± s.e.m.).


Figure 5HMDM were stimulated with AdV ( 50 000 pp / cell ) and antibody ( h9C12 ; 20 ug / ml , IVIg : 20 mg / ml ) or 10 μg / ml pI : C for 3h and gene expression was measured by qPCR ( n = 4 mean ± s . e . m ) . HMDM were stimulated as in ( A ) , or with 10 ng / ml LPS for 6 h and pro - IL - 1β levels in the cytosol measured by western blot . Blot is representative of three independent donors . HMDM were stimulated as in ( A ) , or with 10 ng / ml LPS for 3 h and NLRP3 mRNA expression measured by qPCR . Data shows average ± s . d . of 2 independent donorsHMDM were stimulated as in ( B ) and NLRP3 levels in the cytosol measured by western blot . Blot is representative of two independent donors . TLR9 mRNA levels from PBMCs , CD19 + ve B cells , CD14 + ve monocytes or HMDM derived from CD14 + ve monocytes were assessed by qPCR and copy number was determined relative to actin copy number ( n = 5 mean ± s . e . m ) . THP - 1s expressing ASC - GFP were stimulated for 6 h with virus and antibody or 200 ng / well HT - DNA or 10 μM Nigericin , in the presence of the pan - caspase inhibitor zVAD - fmk . A representative image ( scale bar 100 μm ) and quantification of number of cells with ASC specks from three independent experiments ( mean ± s . e . m , * P - value ≤ 0 . 05 , paired , two - tailed t - test ) is shown .

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Figure 5HMDM were stimulated with AdV (50 000 pp/cell) and antibody (h9C12; 20 ug/ml, IVIg: 20 mg/ml) or 10 μg/ml pI:C for 3h and gene expression was measured by qPCR (n = 4 mean ± s.e.m).HMDM were stimulated as in (A), or with 10 ng/ml LPS for 6 h and pro-IL-1β levels in the cytosol measured by western blot. Blot is representative of three independent donors.HMDM were stimulated as in (A), or with 10 ng/ml LPS for 3 h and NLRP3 mRNA expression measured by qPCR. Data shows average ± s.d. of 2 independent donorsHMDM were stimulated as in (B) and NLRP3 levels in the cytosol measured by western blot. Blot is representative of two independent donors.TLR9 mRNA levels from PBMCs, CD19+ve B cells, CD14+ve monocytes or HMDM derived from CD14+ve monocytes were assessed by qPCR and copy number was determined relative to actin copy number (n = 5 mean ± s.e.m).THP-1s expressing ASC-GFP were stimulated for 6 h with virus and antibody or 200 ng/well HT-DNA or 10 μM Nigericin, in the presence of the pan-caspase inhibitor zVAD-fmk. A representative image (scale bar 100 μm) and quantification of number of cells with ASC specks from three independent experiments (mean ± s.e.m, *P-value ≤ 0.05, paired, two-tailed t-test) is shown.


Figure 6Primed HMDM were treated with MCC950 ( 1 μM ) for 30 mins before stimulation overnight with AdV ( 50 000 pp / cell ) and h9C12 ( 20 μg / ml ) or IVIg ( 20 mg / ml ) or 10 μM Nigericin . IL - 1β ( A ) or TNF ( B ) or LDH release ( C ) were measured in cell supernatants . Data shows average ± s . d . of two independent donors and is representative of three independent experiments . WT , NLRP3 , ASC or Caspase - 1 - deficient THP - 1s were stimulated as in ( A ) and IL - 1β in the supernatant measured by ELISA . Data is representative of two independent experiments . HMDM were primed with 10 ug / ml pI : C for 2h , treated with 100 μM VX - 765 for 30 mins before stimulation for 16 h as in ( A ) . IL - 1β or LDH release was measured in cell supernatants . Data shows average ± s . d . of two independent donorsPrimed HMDM were treated with KCl as indicated for 1h , before being stimulated for a further 6h as in ( A ) . IL - 1β and TNF were measured in cell supernatants by ELISA . Data is representative of three independent donors . Primed HMDM were stimulated with AdV and 20 μg / ml h9C12 or 10 μM Nigericin for 3h , and then immunostained for ASC or intracellular antibody . Co - localisation was assessed by confocal microscopy . Data is representative of two independent donors .

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Figure 6Primed HMDM were treated with MCC950 (1 μM) for 30 mins before stimulation overnight with AdV (50 000 pp/cell) and h9C12 (20 μg/ml) or IVIg (20 mg/ml) or 10 μM Nigericin. IL-1β (A) or TNF (B) or LDH release (C) were measured in cell supernatants. Data shows average ± s.d. of two independent donors and is representative of three independent experiments.WT, NLRP3, ASC or Caspase-1 - deficient THP-1s were stimulated as in (A) and IL-1β in the supernatant measured by ELISA. Data is representative of two independent experiments.HMDM were primed with 10 ug/ml pI:C for 2h, treated with 100 μM VX-765 for 30 mins before stimulation for 16 h as in (A). IL-1β or LDH release was measured in cell supernatants. Data shows average ± s.d. of two independent donorsPrimed HMDM were treated with KCl as indicated for 1h, before being stimulated for a further 6h as in (A). IL-1β and TNF were measured in cell supernatants by ELISA. Data is representative of three independent donors.Primed HMDM were stimulated with AdV and 20 μg/ml h9C12 or 10 μM Nigericin for 3h, and then immunostained for ASC or intracellular antibody. Co-localisation was assessed by confocal microscopy. Data is representative of two independent donors.


Figure 7AIM2 mRNA levels from PBMCs , CD19 + ve B cells , CD14 + ve Monocytes or HMDM derived from CD14 + ve monocytes . were assessed by qPCR and copy number was determined relative to Actin copy number ( n = 5 mean ± s . e . m ) . HMDM were stimulated with AdV ( 50 000 pp / cell ) and 20 μg / ml h9C12 for 1h , washed 2x with SFM , then whole cell lysates harvested after a further 5h . Viral hexon and transgene ( GFP ) expression in the cytosol was assessed by western blot . Data is representative of two independent experiments . THP - 1s expressing either a control guide RNA or targeting cGAS and STING were generated and stimulated with 1000 U / ml IFNα for 4 h and protein levels assessed by western blot . THP - 1s deficient in cGAS and STING were stimulated with AdV ( 50 000 pp / cell ) and 20 μg / ml h9C12 , 200 ng / well HT - DNA , 10 μM Nigericin or 10 ng / ml LPS for 16 h . Data shows combined data ( mean ± s . e . m ) of 3 experiments with absolute protein values ( upper panel ) or as % cytokine output of KO cells relative to Ctrl treated cells ( lower panel ) , * P - value ≤ 0 . 05 , * * * P - value ≤ 0 . 001 unpaired , two - tailed t - test ) . WT THP - 1s were treated with 5 μM H151 for 30 mins before stimulation as in ( D ) . Data in upper panel is representative of 3 independent experiments . Data in lower panel shows combined data of these 3 experiments ( mean ± s . e . m ) showing H151 treated cells relative to media treated cells ( * P - value ≤ 0 . 05 , * * * P - value ≤ 0 . 001 unpaired , two - tailed t - test ) . ASC - GFP THP - 1s were treated with 5 μM H151 for 30 mins before stimulation with AdV - mCherry ( 50 000 pp / cell ) + 20 μg / ml h9C12 or 20 mg / ml IVIg or 200 ng / well HT - DNA for 8h . A representative image ( scale bar 50 μm ) and quantification of number of cells with ASC specks from one representative experiment of three is shown .

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Figure 7AIM2 mRNA levels from PBMCs, CD19+ve B cells, CD14+ve Monocytes or HMDM derived from CD14+ve monocytes. were assessed by qPCR and copy number was determined relative to Actin copy number (n = 5 mean ± s.e.m).HMDM were stimulated with AdV (50 000 pp/cell) and 20 μg/ml h9C12 for 1h, washed 2x with SFM, then whole cell lysates harvested after a further 5h. Viral hexon and transgene (GFP) expression in the cytosol was assessed by western blot. Data is representative of two independent experiments.THP-1s expressing either a control guide RNA or targeting cGAS and STING were generated and stimulated with 1000 U/ml IFNα for 4 h and protein levels assessed by western blot.THP-1s deficient in cGAS and STING were stimulated with AdV (50 000 pp/cell) and 20 μg/ml h9C12, 200 ng/ well HT-DNA, 10 μM Nigericin or 10 ng/ml LPS for 16 h. Data shows combined data (mean ± s.e.m) of 3 experiments with absolute protein values (upper panel) or as % cytokine output of KO cells relative to Ctrl treated cells (lower panel), *P-value ≤ 0.05, ***P-value ≤ 0.001 unpaired, two-tailed t-test).WT THP-1s were treated with 5 μM H151 for 30 mins before stimulation as in (D). Data in upper panel is representative of 3 independent experiments. Data in lower panel shows combined data of these 3 experiments (mean ± s.e.m) showing H151 treated cells relative to media treated cells (*P-value ≤ 0.05, ***P-value ≤ 0.001 unpaired, two-tailed t-test).ASC-GFP THP-1s were treated with 5 μM H151 for 30 mins before stimulation with AdV-mCherry (50 000 pp/cell) + 20 μg/ml h9C12 or 20 mg/ml IVIg or 200 ng/ well HT-DNA for 8h. A representative image (scale bar 50 μm) and quantification of number of cells with ASC specks from one representative experiment of three is shown.


Figure 1Morphology of wild - type ( Tefm + / + ) and Tefm homozygous knockout ( Tefm - / - ) embryos at embryonic day 8 . 5 ( analyzed embryos , n = 43 ) . Scale bar , 500 μm . Western blot analyses of the TEFM protein level in heart mitochondrial extracts from 8 - week - old control ( L / L ) and tissue - specific Tefm knockout ( Tefm knockout ; L / L , cre ) mice ( n = 12 mice for each group ) . VDAC was used as loading control . Survival curve for control ( L / L ; n = 60 ) and Tefm knockout ( L / L , cre ; n = 22 ) mice . Heart weight to body weight ratio in 4 - and 8 - week - old control ( L / L ) and Tefm knockout mice ( L / L , cre ) . At 4 weeks : L / L n = 22 , L / L , cre n = 15 ; 8 weeks : L / L n = 41 , L / L , cre n = 39 .

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Figure 1Morphology of wild-type (Tefm+/+) and Tefm homozygous knockout (Tefm-/-) embryos at embryonic day 8.5 (analyzed embryos, n= 43). Scale bar, 500 μm.Western blot analyses of the TEFM protein level in heart mitochondrial extracts from 8-week-old control (L/L) and tissue-specific Tefm knockout (Tefm knockout; L/L, cre) mice (n = 12 mice for each group). VDAC was used as loading control.Survival curve for control (L/L; n= 60) and Tefm knockout (L/L, cre; n= 22) mice.Heart weight to body weight ratio in 4- and 8-week-old control (L/L) and Tefm knockout mice (L/L, cre). At 4 weeks: L/L n= 22, L/L, cre n= 15; 8 weeks: L/L n= 41, L/L, cre n= 39.


Figure 2Transmission electron micrographs of heart tissue in 8 - week - old control and Tefm knockout mice ( n = 5 mice for each group ) . Scale bar , 2 μm ( upper panel ) and 1 μm ( lower panel ) . Southern blot analyses of mtDNA levels in control and Tefm knockout hearts at the age of 4 and 8 weeks ( Upper 2 blots ; n = 6 mice at each time - point for each group ) . 18S rDNA was used as loading control . DNA isolated from Lrpprc and Mterf4 knockout hearts used as controls . Western blot analyses of VDAC and Histone H3 protein levels in control and Tefm knockout hearts at the age of 4 and 8 weeks ( Bottom 2 blots ; ; n = 6 mice at each time - point for each group ) . VDAC represents mitochondria loading and Histone H3 was used as a loading control for nucleus DNA . Respiratory chain complex activities were measured spectrophotometrically and normalized to citrate synthase activity in heart mitochondria from 8 - week - old control and Tefm knockout mice ( n = 5 mice for each group ) . The analyzed enzyme activities are NADH coenzyme Q reductase ( complex I ) , NADH cytochrome c reductase ( complex I / III ) , succinate dehydrogenase ( complex II ) and cytochrome c oxidase ( complex IV ) . Barplot of the significantly changed proteins ( Benjamini - Hochberg adjusted P < 0 . 05 ) in Tefm knockout mice compared to control mice ( n = 5 mice for each group ) , organized by individual OXPHOS complexes . Proteins are organized in alphabetic order based on gene name . Bars represent mean log2 [ L / L , cre / L / L ] ± 95 % confidence interval . Boxplots of the protein intensity ( LFQ ) of TEFM ( top ) and POLRMT ( bottom ) . Red dot : mean ; blue dots : intensity value for each biological replicate ( n = 5 mice for each group ) . Western blot analyses of POLRMT protein levels in control and Tefm knockout heart mitochondria at the age of 8 weeks ( n = 12 mice for each group ) . VDAC was used as loading control .

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Figure 2Transmission electron micrographs of heart tissue in 8-week-old control and Tefm knockout mice (n = 5 mice for each group). Scale bar, 2 μm (upper panel) and 1 μm (lower panel).Southern blot analyses of mtDNA levels in control and Tefm knockout hearts at the age of 4 and 8 weeks (Upper 2 blots; n= 6 mice at each time-point for each group). 18S rDNA was used as loading control. DNA isolated from Lrpprc and Mterf4 knockout hearts used as controls. Western blot analyses of VDAC and Histone H3 protein levels in control and Tefm knockout hearts at the age of 4 and 8 weeks (Bottom 2 blots; ; n= 6 mice at each time-point for each group). VDAC represents mitochondria loading and Histone H3 was used as a loading control for nucleus DNA.Respiratory chain complex activities were measured spectrophotometrically and normalized to citrate synthase activity in heart mitochondria from 8-week-old control and Tefm knockout mice (n= 5 mice for each group). The analyzed enzyme activities are NADH coenzyme Q reductase (complex I), NADH cytochrome c reductase (complex I/III), succinate dehydrogenase (complex II) and cytochrome c oxidase (complex IV).Barplot of the significantly changed proteins (Benjamini-Hochberg adjusted P <0.05) in Tefm knockout mice compared to control mice (n= 5 mice for each group), organized by individual OXPHOS complexes. Proteins are organized in alphabetic order based on gene name. Bars represent mean log2 [L/L, cre / L/L] ± 95% confidence interval.Boxplots of the protein intensity (LFQ) of TEFM (top) and POLRMT (bottom). Red dot: mean; blue dots: intensity value for each biological replicate (n= 5 mice for each group).Western blot analyses of POLRMT protein levels in control and Tefm knockout heart mitochondria at the age of 8 weeks (n= 12 mice for each group). VDAC was used as loading control.


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 O O O O O O O O O O O O O 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 3Northern blot analyses and relative quantification of mtRNA transcripts in control and Tefm knockout mouse hearts at the age of 4 and 8 weeks (n= 12 mice at each time-point for each group). 18S rRNA and 5.8S rRNA were used as loading controls, respectively. HSP transcripts: 12S rRNA 4 weeks P= 0.005966044, 8 weeks P= 0.0806819; 16S rRNA 4 weeks P= 0.000223004, 8 weeks P= 0.00243443; Nd1 4 weeks P= 0.0352088, 8 weeks P= 0.000344158; Cox1 4 weeks P= 0.000353895, 8 weeks P= 0.00145773; Cox2 4 weeks P= 0.0145193, 8 weeks P= 0.00578484; Cox3 4 weeks P= 0.00834317, 8 weeks P= 0.000392364; Nd5/Cyb 4 weeks P= 0.0010185, 8 weeks P= 0.00153049; Nd5 4 weeks P= 0.024621, 8 weeks P= 7.38074E-05; Cyb 4 weeks P= 3.46175E-06, 8 weeks P= 0.000991942; LSP transcript: Nd6 4 weeks P= 0.000162704, 8 weeks P= 0.000187855. (E) HSP transcripts: Tf 4 weeks P= 0.0297685, 8 weeks P= 0. 000212544; Tv 4 weeks P= 0.842023, 8 weeks P= 0.269231; Tl1 4 weeks P= 0.00921072, 8 weeks P= 0.000430068; Tm 4 weeks P= 0.0084742, 8 weeks P= 0.000142524; Tk 4 weeks P= 0.00297015, 8 weeks P= 0.00010425; Ts2 4 weeks P= 0.0128829, 8 weeks P= 2.27321E-05; Tl2 4 weeks P= 0.00701324, 8 weeks P= 0.000411549; Tt 4 weeks P= 0.00572118, 8 weeks P= 0.000807943; LSP transcript: Tp 4 weeks P= 0.0427191, 8 weeks P= 0.0483328; Te 4 weeks P= 0.464031, 8 weeks P= 0.13412; Ts1 4 weeks P= 0.0382215, 8 weeks P= 0.000478237; Tc 4 weeks P= 0.00418795, 8 weeks P= 0.000287525; Tn 4 weeks P= 0.00531668, 8 weeks P= 0.000374367; Ta 4 weeks P= 0.00247257, 8 weeks P= 0.000564597; Tq 4 weeks P= 0.00385524, 8 weeks P= 0.000727981.


Figure 4In organello transcription analyses using heart mitochondria isolated from 8 - week - old control and Tefm knockout mice ( n = 5 mice for each group ) . The steady - state levels of 12S rRNA and tRNAV were used as endogenous controls , due to their unchanged levels . VDAC was used as loading control for the equal input of mitochondrial extracts . This experiment has performed five times with either two or three replicates . Northern blot analyses of 7S RNA level in 4 - and 8 - week - old control and Tefm knockout mouse hearts ( n = 12 mice at each time - point for each group ) . 18S rRNA was used as loading control . RNA from tissue - specific Mterf4 knockout mouse hearts and tissue - specific Polrmt knockout mouse hearts were loaded as positive and negative control , respectively . Southern blot analyses of mtDNA to obtain 7S DNA in 8 - week - old control and Tefm knockout mice . Heart mitochondria from 3 control or knockout mice were pooled for preparation of mtDNA . Equal amounts of mtDNA shown on the top of the gel , were loaded as the endogenous control . This experiment has performed three times . In organello replication assays were performed on heart mitochondria isolated from 8 - week - old control and Tefm knockout mice ( n = 5 mice for each group ) . VDAC was used as loading control . Northern blot analyses of the 7S RNA levels in 4 - and 8 - week - old control and Tefm knockout mouse hearts ( n = 12 mice for each group ) . 5S rRNA was used as loading control . RNA from tissue - specific Mterf4 knockout mouse hearts and tissue - specific Polrmt knockout mouse hearts were loaded as positive and negative control , 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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

Figure 4In organello transcription analyses using heart mitochondria isolated from 8-week-old control and Tefm knockout mice (n= 5 mice for each group). The steady-state levels of 12S rRNA and tRNAV were used as endogenous controls, due to their unchanged levels. VDAC was used as loading control for the equal input of mitochondrial extracts. This experiment has performed five times with either two or three replicates.Northern blot analyses of 7S RNA level in 4- and 8-week-old control and Tefm knockout mouse hearts (n= 12 mice at each time-point for each group). 18S rRNA was used as loading control. RNA from tissue-specific Mterf4 knockout mouse hearts and tissue-specific Polrmt knockout mouse hearts were loaded as positive and negative control, respectively.Southern blot analyses of mtDNA to obtain 7S DNA in 8-week-old control and Tefm knockout mice. Heart mitochondria from 3 control or knockout mice were pooled for preparation of mtDNA. Equal amounts of mtDNA shown on the top of the gel, were loaded as the endogenous control. This experiment has performed three times.In organello replication assays were performed on heart mitochondria isolated from 8-week-old control and Tefm knockout mice (n= 5 mice for each group). VDAC was used as loading control.Northern blot analyses of the 7S RNA levels in 4- and 8-week-old control and Tefm knockout mouse hearts (n= 12 mice for each group). 5S rRNA was used as loading control. RNA from tissue-specific Mterf4 knockout mouse hearts and tissue-specific Polrmt knockout mouse hearts were loaded as positive and negative control, respectively.


Figure 5Strand - specific RT - qPCR detection of the mitochondrial precursor transcripts containing the antisense of mt - tRNAA and antisense of mt - tRNAC in control and Tefm knockout mice ( n = 12 mice for each group ) . The mt - mRNAs are shown in light blue and mt - tRNAs in grey . Arrows show the binding position of RT - qPCR primers . Actin transcript was used as endogenous control . Strand - specific RT - qPCR detection of the mitochondrial precursor transcripts spanning the junction of Nd5 and antisense of Nd6 in control and Tefm knockout mice ( n = 25 mice for each group ) . The mt - mRNAs are shown in light blue . Arrows show the binding position of RT - qPCR primers . Actin transcript was used as endogenous control . Northern blot analyses of the precursor transcripts containing the antisense of mt - tRNAA in 4 - and 8 - week - old control and Tefm knockout mouse hearts ( n = 6 mice for each group ) .

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 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

Figure 5Strand-specific RT-qPCR detection of the mitochondrial precursor transcripts containing the antisense of mt-tRNAA and antisense of mt-tRNAC in control and Tefm knockout mice (n= 12 mice for each group). The mt-mRNAs are shown in light blue and mt-tRNAs in grey. Arrows show the binding position of RT-qPCR primers. Actin transcript was used as endogenous control.Strand-specific RT-qPCR detection of the mitochondrial precursor transcripts spanning the junction of Nd5 and antisense of Nd6 in control and Tefm knockout mice (n= 25 mice for each group). The mt-mRNAs are shown in light blue. Arrows show the binding position of RT-qPCR primers. Actin transcript was used as endogenous control.Northern blot analyses of the precursor transcripts containing the antisense of mt-tRNAA in 4- and 8-week-old control and Tefm knockout mouse hearts (n= 6 mice for each group).


Figure 6Volcano plot showing TEFM - BioID . TEFM - BirA * - HA was stably expressed in HEK 293 cells , followed by streptavidin affinity purification and mass spectrometry analysis to detect the associated proteins . Mitochondrial targeted BirA * ( MTS - BirA * ) was used as a control . The significantly enriched proteins are presented above the horizontal dashed line with 5 % false discovery rate ( FDR ) significance . The blue dots highlight proteins involved in mtDNA transcription and the green dots represent proteins involved in RNA metabolism . All other proteins are in grey .

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Figure 6Volcano plot showing TEFM-BioID. TEFM-BirA*-HA was stably expressed in HEK 293 cells, followed by streptavidin affinity purification and mass spectrometry analysis to detect the associated proteins. Mitochondrial targeted BirA* (MTS-BirA*) was used as a control. The significantly enriched proteins are presented above the horizontal dashed line with 5% false discovery rate (FDR) significance. The blue dots highlight proteins involved in mtDNA transcription and the green dots represent proteins involved in RNA metabolism. All other proteins are in grey.


Figure 1B Elution volumes ( peak positions ) of the Munc18 - 1 mutants in the absence ( top ) and presence ( bottom ) of Syx1 using size - exclusion chromatography on Superdex - 200 column . C Effects of the domain 3a mutations on MUN - catalyzed transition detected by native - PAGE assay . Components addition was indicated on the top of every lane . D Quantitative analysis of the integrated densities of Munc18 - 1 / Syx1 bands in C . Effects of the QKAA , KKEE and L348R mutations on MUN - catalyzed transition detected by FRET assay . Schematic diagram is shown on the top of the chart . FRET between BODIPY FL - labeled Syb2 ( donor ) and TMR - labeled SN25 ( acceptor ) was monitored . F Effects of the QKAA , KKEE and L348R mutations on lipid mixing between liposomes with Munc18 - 1 / Syx1 and liposomes containing Syb2 in the presence of Munc13 ( M13 ) , SN25 and Syt1 / Ca2 + . Illustration of the reactions is shown on the top of the chart .

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Figure 1B Elution volumes (peak positions) of the Munc18-1 mutants in the absence (top) and presence (bottom) of Syx1 using size-exclusion chromatography on Superdex-200 column.C Effects of the domain 3a mutations on MUN-catalyzed transition detected by native-PAGE assay. Components addition was indicated on the top of every lane. D Quantitative analysis of the integrated densities of Munc18-1/Syx1 bands in C.  Effects of the QKAA, KKEE and L348R mutations on MUN-catalyzed transition detected by FRET assay. Schematic diagram is shown on the top of the chart. FRET between BODIPY FL-labeled Syb2 (donor) and TMR-labeled SN25 (acceptor) was monitored.  F Effects of the QKAA, KKEE and L348R mutations on lipid mixing between liposomes with Munc18-1/Syx1 and liposomes containing Syb2 in the presence of Munc13 (M13), SN25 and Syt1/Ca2+. Illustration of the reactions is shown on the top of the chart.


Figure 2 A Representative traces ( left ) , summary graphs of normalized frequency ( middle ) and normalized amplitude ( right ) of mIPSCs recorded from cultured cortical neurons that were infected with lentivirus expressing Munc18 - 1 WT or mutants , with or without Munc18 - 1 shRNAs ( Control , n = 25 ; None , n = 17 ; WT , n = 17 ; QKAA , n = 18 ; KKEE , n = 16 ; P335A , n = 23 ; L348R , n = 21 ) . B Sample traces ( left ) , summary graphs of normalized amplitude ( middle ) and normalized charge transfer ( right ) of evoked IPSCs recorded from neurons as described in panel A ( Control , n = 19 ; None , n = 19 ; WT , n = 19 ; QKAA , n = 13 ; KKEE , n = 18 ; P335A , n = 17 ; L348R , n = 21 ) . C Sample traces ( left ) and quantification of the normalized charge transfer evoked by hypertonic sucrose ( right ) recorded from the neurons described in panel A ( Control , n = 17 ; None , n = 13 ; WT , n = 17 ; QKAA , n = 17 ; KKEE , n = 18 ; P335A , n = 15 ; L348R , n = 14 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Representative traces (left), summary graphs of normalized frequency (middle) and normalized amplitude (right) of mIPSCs recorded from cultured cortical neurons that were infected with lentivirus expressing Munc18-1 WT or mutants, with or without Munc18-1 shRNAs (Control, n = 25; None, n = 17; WT, n = 17; QKAA, n = 18; KKEE, n = 16; P335A, n = 23; L348R, n = 21). B Sample traces (left), summary graphs of normalized amplitude (middle) and normalized charge transfer (right) of evoked IPSCs recorded from neurons as described in panel A (Control, n = 19; None, n = 19; WT, n = 19; QKAA, n = 13; KKEE, n = 18; P335A, n = 17; L348R, n = 21). C Sample traces (left) and quantification of the normalized charge transfer evoked by hypertonic sucrose (right) recorded from the neurons described in panel A (Control, n = 17; None, n = 13; WT, n = 17; QKAA, n = 17; KKEE, n = 18; P335A, n = 15; L348R, n = 14).


Figure 3 A Representative images of HEK293T cells transfected with mCherry2 - Syx1 or EGFP - Munc18 - 1 . Lipophilic dye DiD ( 1 , 1 - Dioctadecyl - 3 , 3 , 3 , 3 - tetramethylindodicarbocyanine ) was used as the plasma membrane marker . B Representative images of HEK293T cells co - transfected with mCherry2 - Syx1 and EGFP labeled WT or mutants of Munc18 - 1 . C , D Summary graphs of the membrane distribution of Syx1 ( C ) and Munc18 - 1 WT or mutants ( D ) quantified by the ratio of fluorescence at the plasma membrane ( PM ) to the total fluorescence in the whole cell ( total ) . DiD is the PM marker . Numbers of pictures analyzed are shown as follows : no Syx1 , n = 14 ; no M18 , n = 15 ; WT , n = 24 ; QKAA , n = 23 ; KKEE , n = 12 ; L348R , n = 22 . E Munc18 - 1 mutants retain interaction with Syx1 shown by GST pull - down experiment . A representative gel displayed is from one of three independent replicates .

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Figure 3 A Representative images of HEK293T cells transfected with mCherry2-Syx1 or EGFP-Munc18-1. Lipophilic dye DiD (1,1-Dioctadecyl-3,3,3,3-tetramethylindodicarbocyanine) was used as the plasma membrane marker. B Representative images of HEK293T cells co-transfected with mCherry2-Syx1 and EGFP labeled WT or mutants of Munc18-1.  C,D Summary graphs of the membrane distribution of Syx1 (C) and Munc18-1 WT or mutants (D) quantified by the ratio of fluorescence at the plasma membrane (PM) to the total fluorescence in the whole cell (total). DiD is the PM marker. Numbers of pictures analyzed are shown as follows: no Syx1, n = 14; no M18, n = 15; WT, n = 24; QKAA, n = 23; KKEE, n = 12; L348R, n = 22. E Munc18-1 mutants retain interaction with Syx1 shown by GST pull-down experiment. A representative gel displayed is from one of three independent replicates.


Figure 4B Example histograms of smFRET efficiency of Syx1 in complex with Munc18 - 1 WT in the absence and presence of the MUN domain , and within the SNARE complex . C − E Example histograms of smFRET efficiency of Syx1 bound to Munc18 - 1 QKAA ( C ) , KKEE ( D ) , and L348R ( E ) in the absence or presence of the MUN domain . The QKAA mutation strongly impaired the shift of smFRET efficiency in the presence of the MUN domain . F Quantitative results of the Gaussian peak center positions of the FRET efficiency distributions in C − E . G Effects of QKAA , KKEE , and L348R mutations on the interaction between the Munc18 - 1 / Syx1 complex and the MUN domain by GST pull - down assay combined with immunoblotting . Results were shown with 6 × His - tag antibody and GST - tag antibody .

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Figure 4B Example histograms of smFRET efficiency of Syx1 in complex with Munc18-1 WT in the absence and presence of the MUN domain, and within the SNARE complex. C−E Example histograms of smFRET efficiency of Syx1 bound to Munc18-1 QKAA (C), KKEE (D), and L348R (E) in the absence or presence of the MUN domain. The QKAA mutation strongly impaired the shift of smFRET efficiency in the presence of the MUN domain. F Quantitative results of the Gaussian peak center positions of the FRET efficiency distributions in C−E. G Effects of QKAA, KKEE, and L348R mutations on the interaction between the Munc18-1/Syx1 complex and the MUN domain by GST pull-down assay combined with immunoblotting. Results were shown with 6×His-tag antibody and GST-tag antibody.


Figure 5 B Effects of the QKAA , KKEE , and L348R mutations on binding of Syb2 to Munc18 - 1 / Syx1 complex in the presence and absence of MUN domain detected by GST pull - down assay . Bands of Munc18 - 1 are indicated with red asterisks . C Quantification of the normalized binding of Syb2 to Munc18 - 1 / Syx1 complex . Integrated intensities of Munc18 - 1 bands were quantified . QKAA and L348R , but not KKEE , disrupted Syb2 binding to the Munc18 - 1 / Syx1 complex .

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Figure 5 B Effects of the QKAA, KKEE, and L348R mutations on binding of Syb2 to Munc18-1/Syx1 complex in the presence and absence of MUN domain detected by GST pull-down assay. Bands of Munc18-1 are indicated with red asterisks. C Quantification of the normalized binding of Syb2 to Munc18-1/Syx1 complex. Integrated intensities of Munc18-1 bands were quantified. QKAA and L348R, but not KKEE, disrupted Syb2 binding to the Munc18-1/Syx1 complex.


Figure 6 A Ensemble FRET assay showing the different effects of the Munc18 - 1 P335A mutation and the Syx1 LEAA mutation in the transition from the Munc18 - 1 / Syx1 complex to the SNARE complex with and without the MUN domain . FRET signal between BODIPY FL - labeled Syb2 ( donor ) and TMR - labeled SN25 ( acceptor ) was monitored . B Quantification of the FRET results in A . C Single - molecule FRET assay showing the MUN domain drives the open of the Syx1 linker region independent of the conformational change of domain 3a . Example histograms of smFRET efficiency of Syx1 bound to Munc18 - 1 P335A mutant in the absence and presence of the MUN domain . D Quantitative results of the Gaussian peak center positions of the FRET efficiency distributions in C .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 A Ensemble FRET assay showing the different effects of the Munc18-1 P335A mutation and the Syx1 LEAA mutation in the transition from the Munc18-1/Syx1 complex to the SNARE complex with and without the MUN domain. FRET signal between BODIPY FL-labeled Syb2 (donor) and TMR-labeled SN25 (acceptor) was monitored. B Quantification of the FRET results in A.  C Single-molecule FRET assay showing the MUN domain drives the open of the Syx1 linker region independent of the conformational change of domain 3a. Example histograms of smFRET efficiency of Syx1 bound to Munc18-1 P335A mutant in the absence and presence of the MUN domain. D Quantitative results of the Gaussian peak center positions of the FRET efficiency distributions in C.


Figure 8 B Munc18 - 1 , Habc and H3 construct a tertiary complex . C The tertiary complex globally resembles the Munc18 - 1 / Syx1 complex . D Formation of the tertiary complex with Munc18 - 1 QKAA , KKEE , and L348R mutants . KKEE strongly impaired formation of the tertiary complex . E Munc18 - 1 displays interaction with Syx1 - Habc . F QKAA , KKEE , and L348R all retained interaction with Syx1 - Habc .

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Figure 8 B Munc18-1, Habc and H3 construct a tertiary complex. C The tertiary complex globally resembles the Munc18-1/Syx1 complex. D Formation of the tertiary complex with Munc18-1 QKAA, KKEE, and L348R mutants. KKEE strongly impaired formation of the tertiary complex. E Munc18-1 displays interaction with Syx1-Habc.F QKAA, KKEE, and L348R all retained interaction with Syx1-Habc.


Figure 1C Overabundance of MIAA in LAM plasma . Top panel shows each control and patient group ( number ( n ) of samples are indicated ) ; bottom panel shows aggregation of related pulmonary diseases . The asterisks indicate significant differences based on two - sided Mann - Whitney tests ( * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , and * * * * P < 0 . 0001 ; top panel , control - LAM P = 0 . 048 , LAM - Langerhans P = 1x10 - 4 , LAM - Sjögren P = 1x10 - 4 , LAM - lupus P = 1x10 - 3 , LAM - emphysema P = 0 . 041 ; bottom panel , LAM - Langerhans P = 7x10 - 6 and control - other P = 0 . 031 ) . Average values are indicated with lilac - colored lines . Comparison of the three groups : Kruskal - Wallis test P = 6x10 - 4 ( top panel ) and P = 3x10 - 5 ( bottom panel ) . E Overabundance of MIAA in LAM patients not treated with rapamycin relative to rapamycin - treated ( number ( n ) of samples are indicated ) . The asterisk indicates significant difference based on two - sided Mann - Whitney test ( P = 0 . 043 ) . Average values are indicated with lilac - colored lines . G Plasma levels of three other metabolic products derived from monoamine metabolism and showing significant differences from controls and / or between LAM and related pulmonary diseases ( individual samples indicated in panel C ) . The asterisks indicate significant differences based on two - sided Mann - Whitney tests ( * P < 0 . 05 , * * P < 0 . 01 , and * * * * P < 0 . 0001 ; 4 - HPA , control - LAM P = 0 . 024 and LAM - other P = 0 . 089 ; HVA , control - LAM P = 0 . 008 and LAM - other P = 0 . 013 ; and VMA , control - LAM P = 1x10 - 4 and LAM - other P = 0 . 10 ) . Average values are indicated with lilac - colored lines . Comparison of the three groups : Kruskal - Wallis test ; 4 - HPA P = 0 . 095 ; HVA P = 0 . 013 ; and VMA P = 0 . 005 .

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

Figure 1C Overabundance of MIAA in LAM plasma. Top panel shows each control and patient group (number (n) of samples are indicated); bottom panel shows aggregation of related pulmonary diseases. The asterisks indicate significant differences based on two-sided Mann-Whitney tests (*P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001; top panel, control-LAM P = 0.048, LAM-Langerhans P = 1x10-4, LAM-Sjögren P = 1x10-4, LAM-lupus P = 1x10-3, LAM-emphysema P = 0.041; bottom panel, LAM-Langerhans P = 7x10-6 and control-other P = 0.031). Average values are indicated with lilac-colored lines. Comparison of the three groups: Kruskal-Wallis test P = 6x10-4 (top panel) and P = 3x10-5 (bottom panel).E Overabundance of MIAA in LAM patients not treated with rapamycin relative to rapamycin-treated (number (n) of samples are indicated). The asterisk indicates significant difference based on two-sided Mann-Whitney test (P = 0.043). Average values are indicated with lilac-colored lines.G Plasma levels of three other metabolic products derived from monoamine metabolism and showing significant differences from controls and/or between LAM and related pulmonary diseases (individual samples indicated in panel C). The asterisks indicate significant differences based on two-sided Mann-Whitney tests (*P < 0.05, **P < 0.01, and ****P < 0.0001; 4-HPA, control-LAM P = 0.024 and LAM-other P = 0.089; HVA, control-LAM P = 0.008 and LAM-other P = 0.013; and VMA, control-LAM P = 1x10-4 and LAM-other P = 0.10). Average values are indicated with lilac-colored lines. Comparison of the three groups: Kruskal-Wallis test; 4-HPA P = 0.095; HVA P = 0.013; and VMA P = 0.005.


Figure 2A Overabundance of MIAA in LAM plasma ( UK cohort not treated with rapamycin ) relative to healthy women . The number ( n ) of individuals in each group is indicated ; one of 20 healthy controls did not pass the quality controls for LC - MS / MS . Asterisk indicates significance using two - sided Mann - Whitney test ( P = 0 . 018 ) . Average values are indicated with lilac - colored lines . B Overabundance of MIAA in plasma of LAM patients with AMLs . Asterisk indicates significance using two - sided Mann - Whitney test ( P = 0 . 042 ) . The number ( n ) of individuals in each group is indicated . Average values are indicated with lilac - colored lines .

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Figure 2A Overabundance of MIAA in LAM plasma (UK cohort not treated with rapamycin) relative to healthy women. The number (n) of individuals in each group is indicated; one of 20 healthy controls did not pass the quality controls for LC-MS/MS. Asterisk indicates significance using two-sided Mann-Whitney test (P = 0.018). Average values are indicated with lilac-colored lines. B Overabundance of MIAA in plasma of LAM patients with AMLs. Asterisk indicates significance using two-sided Mann-Whitney test (P = 0.042). The number (n) of individuals in each group is indicated. Average values are indicated with lilac-colored lines.


Figure 3A Representative images of immunohistochemical positivity of MAO - A / B in LAM lung lesions ( top panels , two patients ) and lung tissue from healthy ( non - LAM ) individuals ( bottom panels ) . In total , seven LAM patients and three healthy controls were analyzed . Brown - stained cells , counter - stained with hematoxylin , are considered positive . Scale bars are shown . B Representative images of immunofluorescence detection of MAO - A / B and αSMA in LAM lung lesions , nuclei stained with DAPI ( merged ) . C Graph showing gene expression differences ( log10 - fold changes ) of defined genes ( X - axis ) in Tsc2 - deficient relative to wild - type MEFs grown in DMEM 10 % FBS . The asterisks indicate significant differences with two - sided t - test ( * * P < 0 . 01 , * * * P < 0 . 001 , and * * * * P < 0 . 0001 ; Aldh1a3 P = 0 . 006 , Aldh2 P = 2x10 - 5 , Aldh3a1 P = 0 . 004 ; Aldh3b1 P = 0 . 006 ; Aldh3b2 , P = 0 . 17 , Maoa P = 7x10 - 5 , and Maob P = 7x10 - 4 ; replicates / condition n = 3 , assays n = 4 ) . The bars indicate mean ± SD . Dotted horizontal lines indicate 2 - fold ( top ) and 0 . 5 - fold ( bottom ) . D Western blot results ( independent experiments n = 4 ) from Tsc2 - deficient and wild - type MEFs exposed to DMSO or treated with 20 nM everolimus for 16 hours in DMEM 10 % FBS . Loading controls are shown . The inferred protein expression level is indicated by the ratio between the corresponding signal and loading control , standardized to the basal

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 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 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 3A Representative images of immunohistochemical positivity of MAO-A/B in LAM lung lesions (top panels, two patients) and lung tissue from healthy (non-LAM) individuals (bottom panels). In total, seven LAM patients and three healthy controls were analyzed. Brown-stained cells, counter-stained with hematoxylin, are considered positive. Scale bars are shown.B Representative images of immunofluorescence detection of MAO-A/B and αSMA in LAM lung lesions, nuclei stained with DAPI (merged).C Graph showing gene expression differences (log10-fold changes) of defined genes (X-axis) in Tsc2-deficient relative to wild-type MEFs grown in DMEM 10% FBS. The asterisks indicate significant differences with two-sided t-test (**P < 0.01, ***P < 0.001, and ****P < 0.0001; Aldh1a3 P = 0.006, Aldh2 P = 2x10-5, Aldh3a1 P = 0.004; Aldh3b1 P = 0.006; Aldh3b2, P = 0.17, Maoa P = 7x10-5, and Maob P = 7x10-4; replicates/condition n = 3, assays n = 4). The bars indicate mean ± SD. Dotted horizontal lines indicate 2-fold (top) and 0.5-fold (bottom).D Western blot results (independent experiments n = 4) from Tsc2-deficient and wild-type MEFs exposed to DMSO or treated with 20 nM everolimus for 16 hours in DMEM 10% FBS. Loading controls are shown. The inferred protein expression level is indicated by the ratio between the corresponding signal and loading control, standardized to the basal setting


Figure 4A No differences of Vdac1 / VDAC1 expression between MEF cell lines ( replicates / condition n = 3 and independent experiments n = 2 ) . The graph shows fold change expression ( mean ± SEM ) in Tsc2 - deficient relative to wild - type MEFs . B Flow cytometry results ( independent experiments n = 3 ) showing a higher percentage of MitoSOX red - positive cells in Tsc2 - deficient MEFs . C Flow cytometry results ( independent experiments n = 3 ) showing higher intensity ( X - axis , single channel intensity FL3 670 nm ) of MitoTracker red - positive cells in Tsc2 - deficient MEFs . D Higher basal cell respiration ( as measured by oxygen consumption , Y - axis ) in Tsc2 - deficient MEFs . The asterisks indicate significant difference with two - sided t - test ( P = 5x10 - 4 ; replicates / condition n = 5 , and independent experiments n = 3 ) . F Left panel , western blot results showing overexpression of catalase ( CAT ) in Tsc2 - deficient MEFs , and unaffected by exposure to everolimus ( independent experiments n = 2 ) . The CAT expression level is indicated by the ratio of the corresponding signal relative to loading control and basal setting ( noted as 1 ( ref ) ) . Right panel , Cat overexpression ( log10 - fold change ) in Tsc2 - deficient MEFs . The asterisks indicate significant difference with two - sided t - test ( P = 1x10 - 2 ; replicates / condition n = 3 , and independent experiments n = 2 ) . The bars indicate mean ± SD . Dotted horizontal lines indicate 2 - fold ( top ) and 0 . 5 - fold ( bottom ) . G Representative images of immunohistochemical detection of acrolein in LAM lung lesions .

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

Figure 4A No differences of Vdac1/VDAC1 expression between MEF cell lines (replicates/condition n = 3 and independent experiments n = 2). The graph shows fold change expression (mean ± SEM) in Tsc2-deficient relative to wild-type MEFs.B Flow cytometry results (independent experiments n = 3) showing a higher percentage of MitoSOX red-positive cells in Tsc2-deficient MEFs. C Flow cytometry results (independent experiments n = 3) showing higher intensity (X-axis, single channel intensity FL3 670 nm) of MitoTracker red-positive cells in Tsc2-deficient MEFs. D Higher basal cell respiration (as measured by oxygen consumption, Y-axis) in Tsc2-deficient MEFs. The asterisks indicate significant difference with two-sided t-test (P = 5x10-4; replicates/condition n = 5, and independent experiments n = 3).F Left panel, western blot results showing overexpression of catalase (CAT) in Tsc2-deficient MEFs, and unaffected by exposure to everolimus (independent experiments n = 2). The CAT expression level is indicated by the ratio of the corresponding signal relative to loading control and basal setting (noted as 1(ref)). Right panel, Cat overexpression (log10-fold change) in Tsc2-deficient MEFs. The asterisks indicate significant difference with two-sided t-test (P = 1x10-2; replicates/condition n = 3, and independent experiments n = 2). The bars indicate mean ± SD. Dotted horizontal lines indicate 2-fold (top) and 0.5-fold (bottom).G Representative images of immunohistochemical detection of acrolein in LAM lung lesions.


Figure 5A Overabundance of histamine in Tsc2 wild - type relative to Tsc2 - deficient MEFs , both growth in DMEM 10 % FBS . The asterisks indicate significant difference with two - sided t - test ( P = 0 . 017 ) ; replicates / condition n = 5 , and independent experiments n = 2 ) . Average values are indicated with lilac - colored lines . D Western blot results of HRH1 , SLC22A3 , and loading control from Tsc2 - deficient and wild - type MEFs exposed to DMSO or treated with 20 nM everolimus for 16 hours in DMEM 10 % or 0 . 5 % FBS ( independent experiments n = 2 ) . The expression levels are indicated by the ratio of the corresponding signal relative to loading control and basal settingE Representative images of immunohistochemical detection of HRH1 in LAM lung lesions . F Evaluation of cell viability inhibition by loratadine alone ( concentrations shown on X - axis , from 0 to 100 μM ) or combined with everolimus ( fixed to 20 nM ) in Tsc2 - deficient and wild - type MEF cultures grown in DMEM 10 % FBS for 72 hours . The synergistic combination index ( CIx < 1 ) is shown ( replicates / condition n = 3 , independent experiments n = 3 ) . Each data point represents the mean and SD . H Percentages of viability of Tsc2 - dificient and wild - type MEFs exposed to DMEM 10 % or 0 . 5 % FBS with or without L - histidine for 72 hours . The asterisks indicate significant differences with one - sided t - test ( 10 % FBS , P = 0 . 017 ; 0 . 5 % FBS , P = 5x10 - 4 ; replicates / condition n = 4 - 6 , independent experiments n = 2 ) . The bars indicate mean ± SD .

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

Figure 5A Overabundance of histamine in Tsc2 wild-type relative to Tsc2-deficient MEFs, both growth in DMEM 10% FBS. The asterisks indicate significant difference with two-sided t-test (P = 0.017); replicates/condition n = 5, and independent experiments n = 2). Average values are indicated with lilac-colored lines.D Western blot results of HRH1, SLC22A3, and loading control from Tsc2-deficient and wild-type MEFs exposed to DMSO or treated with 20 nM everolimus for 16 hours in DMEM 10% or 0.5% FBS (independent experiments n = 2). The expression levels are indicated by the ratio of the corresponding signal relative to loading control and basal settingE Representative images of immunohistochemical detection of HRH1 in LAM lung lesions.F Evaluation of cell viability inhibition by loratadine alone (concentrations shown on X-axis, from 0 to 100 μM) or combined with everolimus (fixed to 20 nM) in Tsc2-deficient and wild-type MEF cultures grown in DMEM 10% FBS for 72 hours. The synergistic combination index (CIx < 1) is shown (replicates/condition n = 3, independent experiments n = 3). Each data point represents the mean and SD.H Percentages of viability of Tsc2-dificient and wild-type MEFs exposed to DMEM 10% or 0.5% FBS with or without L-histidine for 72 hours. The asterisks indicate significant differences with one-sided t-test (10% FBS, P = 0.017; 0.5% FBS, P = 5x10-4; replicates/condition n = 4-6, independent experiments n = 2). The bars indicate mean ± SD.


Figure 6B Inhibition of Tsc2 - deficient 105K tumor growth with different monotherapies , as indicated in the inset . Asterisks indicate significant reductions relative to vehicle ( two - way ANOVA ; clorgyline P = 0 . 015 , loratadine P = 0 . 018 , and rasagiline P = 0 . 049 ) . Each data point represents the mean and SEM . C Further reduction of Tsc2 - deficient 105K tumor growth by rapamycin combined with clorgyline or loratadine , relative to rapamycin alone . Asterisks indicate significant reductions relative to rapamycin alone ( two - way ANOVA ; clorgyline P = 0 . 035 , loratadine P = 0 . 045 ) . Each data point represents the mean and SEM . E Inhibition of Tsc2 - deficient 105K tumor growth with Maoa expression depletion using shRNAs . Asterisks indicate significant reductions relative to control pLKO ( two - way ANOVA ; P = 1x10 - 3 ) . Each data point represents the mean and SEM . F Inhibition of Tsc2 - deficient 105K tumor growth with Hrh1 expression depletion . Asterisk indicates significant reduction relative to control pLKO ( two - way ANOVA ; P = 0 . 035 ) . Each data point represents the mean and SEM . G Inhibition of Tsc2 - deficient 105K tumor growth with administration of α - methyl - DL - histidine . Asterisk indicates significant reduction relative to vehicle ( two - way ANOVA ; P = 0 . 028 ) . Each data point represents the mean and SEM .

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 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 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 O O 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 6B Inhibition of Tsc2-deficient 105K tumor growth with different monotherapies, as indicated in the inset. Asterisks indicate significant reductions relative to vehicle (two-way ANOVA; clorgyline P = 0.015, loratadine P = 0.018, and rasagiline P = 0.049). Each data point represents the mean and SEM.C Further reduction of Tsc2-deficient 105K tumor growth by rapamycin combined with clorgyline or loratadine, relative to rapamycin alone. Asterisks indicate significant reductions relative to rapamycin alone (two-way ANOVA; clorgyline P = 0.035, loratadine P = 0.045). Each data point represents the mean and SEM.E Inhibition of Tsc2-deficient 105K tumor growth with Maoa expression depletion using shRNAs. Asterisks indicate significant reductions relative to control pLKO (two-way ANOVA; P = 1x10-3). Each data point represents the mean and SEM.F Inhibition of Tsc2-deficient 105K tumor growth with Hrh1 expression depletion. Asterisk indicates significant reduction relative to control pLKO (two-way ANOVA; P = 0.035) . Each data point represents the mean and SEM. G Inhibition of Tsc2-deficient 105K tumor growth with administration of α-methyl-DL-histidine. Asterisk indicates significant reduction relative to vehicle (two-way ANOVA; P = 0.028). Each data point represents the mean and SEM.


Figure 7A Top panels , western blot results of HRH1 and phospho - Ser616 DRP1 expression in vehicle control and single - drug treated tumors . Bottom panels show quantifications ; significant differences correspond to one - sided t - test ( * P = 0 . 011 and * * P = 2x10 - 3 ; tumors / group n = 4 ) . Average values are indicated with lilac - colored lines . B Western blot results showing increased HRH1 expression in combinations relative to rapamycin alone , reduced expression of S6 total in the rapamycin - loratadine combination , and raised phospho - Ser616 DRP1 expression in this combination . Bottom panels show quantifications and significant differences with one - sided t - test ( HRH1 , clorgyline P = 0 . 011 , loratadine P = 0 . 049 , rasagiline P = 0 . 021 ; total S6 , loratadine P = 4x10 - 4 ; phospho - Ser616 DRP1 , loratadine P = 0 . 037 ; tumors / group n = 4 ) . Average values are indicated with lilac - colored lines . E Representative images of hematoxylin - eosin - stained Tsc2 - deficient 105K tumors treated with vehicle or monotherapies . Tumors treated with rapamycin tend to have a fascicular growth pattern with bundles of spindle cells and foci of fibrosis , whereas most of the tumors of the other treatment groups showed extensive epithelioid morphology . Scale bars are shown . F Representative images of hematoxylin - eosin - stained Tsc2 - deficient 105K tumors treated with rapamycin alone or rapamycin combinations . With the addition of a second drug to rapamycin , tumors more frequently tended to show glandular differentiation and less atypia . Scale bars 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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

Figure 7A Top panels, western blot results of HRH1 and phospho-Ser616 DRP1 expression in vehicle control and single-drug treated tumors. Bottom panels show quantifications; significant differences correspond to one-sided t-test (*P = 0.011 and **P = 2x10-3; tumors/group n = 4). Average values are indicated with lilac-colored lines. B Western blot results showing increased HRH1 expression in combinations relative to rapamycin alone, reduced expression of S6 total in the rapamycin-loratadine combination, and raised phospho-Ser616 DRP1 expression in this combination. Bottom panels show quantifications and significant differences with one-sided t-test (HRH1, clorgyline P = 0.011, loratadine P = 0.049, rasagiline P = 0.021; total S6, loratadine P = 4x10-4; phospho-Ser616 DRP1, loratadine P = 0.037; tumors/group n = 4). Average values are indicated with lilac-colored lines. E Representative images of hematoxylin-eosin-stained Tsc2-deficient 105K tumors treated with vehicle or monotherapies. Tumors treated with rapamycin tend to have a fascicular growth pattern with bundles of spindle cells and foci of fibrosis, whereas most of the tumors of the other treatment groups showed extensive epithelioid morphology. Scale bars are shown.F Representative images of hematoxylin-eosin-stained Tsc2-deficient 105K tumors treated with rapamycin alone or rapamycin combinations. With the addition of a second drug to rapamycin, tumors more frequently tended to show glandular differentiation and less atypia. Scale bars are shown.


Figure 1Quantification of fibroblast density ( number of PDGFRαH2BEGFP + cells , right ; n = 3 biological replicates per time point ) and dermis volume ( left ) with age ( n = 7 E12 . 5 ; n = 8 P0 , P6 ; n = 9 E16 . 5 , E17 . 5 , P4 ; n = 10 P10 , P50 ; n = 11 E10 . 5 ; n = 12 E18 . 5 , P2 , P21 ; n = 20 P12 biological replicates ) . Changes in fibroblast proliferation and collagen deposition during dermal development in mice . Immunofluorescence staining for Ki67 ( red ) of PDGFRαH2BEGFP ( green ) back skin at indicated developmental time points ( upper panel ) . Polarised light images of Picrosirius red stained back skin sections shown in binary images at indicated time points ( lower panel ) . Percentage of PDGFRαH2BEGFP + cells in G1 cell cycle phase with age ( n = 1 E12 . 5 ; n = 2 P2 , P10 ; n = 3 E16 . 5 , E17 , P0 ; n = 4 E10 . 5 ; n = 5 E17 . 5 , E18 . 5 , P50 ; n = 7 P4 biological replicates ) fitted using an exponential model . Quantification of fibroblast proliferation ( left ; n = 4 E16 . 5 , P10 ; n = 3 E10 . 5 , E18 . 5 , P2 , P21 , P44 , P50 ; n = 2 E17 . 5 , P0 biological replicates ) and collagen density ( right ; n = 4 E16 . 5 , P2 , P6 , P10 , P14 , P16 , P21 , P50 ; n = 3 E10 . 5 , E18 . 5 , P2 , P21 , P44 , P50 biological replicates ) with age . Immunofluorescence image of R26Fucci2a x Dermo1Cre back skin at P2 where mVenus - hGem ( green ) expressing cells are in S / G2 / M phase and mCherry - hCdt1 ( red ) positive cells are arrested in G1 phase . Percentage of labelled cells in S / G2 / M cell cycle phase in the upper and lower dermis at P0 ( n = 3 biological replicates ) and P2 ( n = 4 biological replicates ) . Changes in dermal cell proliferation and collagen deposition in human skin . ( G ) Immunofluorescence staining for Vimentin ( red ) and Ki67 ( green ) of back skin at indicated time points ( upper panel ) . Polarised light image of Picrosirius red stained back skin section shown as binary image at indicated time points ( lower panel ) . ( H ) Quantification of proliferating cells in the upper and lower dermis ( n = 3 biological replicates per time point ) . ( I ) Quantification of dermal cell proliferation ( Ki67 + ) ( left ) and collagen density ( right ) with age ( n = 3 biological replicates per time point ) . Note that cells in upper human dermis were more proliferative than cells in the lower dermis at all developmental time points analysed and that fibroblasts entered a quiescent , non - proliferative state before collagen was efficiently deposited . Data shown are means ± s . d . Nuclei were labelled with DAPI ( blue in B , E , G ) . Scale bars , 100µm . wk , week .

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 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 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 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 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

Figure 1Quantification of fibroblast density (number of PDGFRαH2BEGFP+ cells, right; n=3 biological replicates per time point) and dermis volume (left) with age (n=7 E12.5; n=8 P0, P6; n=9 E16.5, E17.5, P4; n=10 P10, P50; n=11 E10.5; n=12 E18.5, P2, P21; n=20 P12 biological replicates).Changes in fibroblast proliferation and collagen deposition during dermal development in mice. Immunofluorescence staining for Ki67 (red) of PDGFRαH2BEGFP (green) back skin at indicated developmental time points (upper panel). Polarised light images of Picrosirius red stained back skin sections shown in binary images at indicated time points (lower panel).Percentage of PDGFRαH2BEGFP+ cells in G1 cell cycle phase with age (n=1 E12.5; n=2 P2, P10; n=3 E16.5, E17, P0; n=4 E10.5; n=5 E17.5, E18.5, P50; n=7 P4 biological replicates) fitted using an exponential model.Quantification of fibroblast proliferation (left; n= 4 E16.5, P10; n= 3 E10.5, E18.5, P2, P21, P44, P50; n= 2 E17.5, P0 biological replicates) and collagen density (right; n= 4 E16.5, P2, P6, P10, P14, P16, P21, P50; n= 3 E10.5, E18.5, P2, P21, P44, P50 biological replicates) with age.Immunofluorescence image of R26Fucci2a x Dermo1Cre back skin at P2 where mVenus-hGem (green) expressing cells are in S/G2/M phase and mCherry-hCdt1 (red) positive cells are arrested in G1 phase. Percentage of labelled cells in S/G2/M cell cycle phase in the upper and lower dermis at P0 (n=3 biological replicates) and P2 (n=4 biological replicates).Changes in dermal cell proliferation and collagen deposition in human skin. (G) Immunofluorescence staining for Vimentin (red) and Ki67 (green) of back skin at indicated time points (upper panel). Polarised light image of Picrosirius red stained back skin section shown as binary image at indicated time points (lower panel). (H) Quantification of proliferating cells in the upper and lower dermis (n=3 biological replicates per time point). (I) Quantification of dermal cell proliferation (Ki67+) (left) and collagen density (right) with age (n= 3 biological replicates per time point). Note that cells in upper human dermis were more proliferative than cells in the lower dermis at all developmental time points analysed and that fibroblasts entered a quiescent, non-proliferative state before collagen was efficiently deposited. Data shown are means ±s.d. Nuclei were labelled with DAPI (blue in B,E,G). Scale bars, 100µm. wk, week.


Figure 2Two independent primary mouse fibroblast isolations FB1 ( black ) and FB2 ( grey ) were cultured in ( triangle ) and outside ( circle ) collagen gels for up to 5 days . Cell number was measured by CellTiterGlo assay at indicated time points ( n = 3 technical replicates of one representative experiment repeated 3 times ) . Two independent primary mouse fibroblast isolations FB1 ( black ) and FB2 ( grey ) were cultured in collagen gels for 3 days before being released by collagenase treatment and plated on TCP for 4 days ( triangle ) . Controls were kept on TCP for 3 days before plating ( circle ) . Fold increase in confluency 24 hours after plating of collagen gel released cells and cells kept on TCP is shown ( n = 3 technical replicates ) . Repopulation of DED organotypic cultures recapitulates adult dermal homeostasis . Immunofluorescence staining for Krt14 ( green ) , Ki67 ( red ) and Vimentin ( white ) of DED organotypic cultures with and without fibroblasts at indicated time points after seeding . Quantification of Ki67 positive fibroblasts at indicated time point after seeding ( n = 8 section per condition of one representative experiment repeated two times ) . ECM degradation in DED organotypic cultures by collagenase treatment . Immunofluorescence staining for Krt14 ( green ) , Ki67 ( red ) and Vimentin ( white ) of DED organotypic cultures at indicated time points after collagenase treatment . Quantification of Ki67 positive fibroblasts at indicated time point after collagenase treatment ( n = 8 section per condition of one representative experiment repeated two times ) . Note that fibroblasts around the injection sides start to proliferate after 48 hours . Nuclei were labelled with DAPI ( blue ) ( D , F ) . Scale bars , 100µm ( D , F ) . Data shown are means ± s . d . Fb , fibroblasts ; PD , post digestion ; TCP , tissue culture plastic ; h , hours .

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 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 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 2Two independent primary mouse fibroblast isolations FB1 (black) and FB2 (grey) were cultured in (triangle) and outside (circle) collagen gels for up to 5 days. Cell number was measured by CellTiterGlo assay at indicated time points (n= 3 technical replicates of one representative experiment repeated 3 times).Two independent primary mouse fibroblast isolations FB1 (black) and FB2 (grey) were cultured in collagen gels for 3 days before being released by collagenase treatment and plated on TCP for 4 days (triangle). Controls were kept on TCP for 3 days before plating (circle). Fold increase in confluency 24 hours after plating of collagen gel released cells and cells kept on TCP is shown (n= 3 technical replicates).Repopulation of DED organotypic cultures recapitulates adult dermal homeostasis. Immunofluorescence staining for Krt14 (green), Ki67 (red) and Vimentin (white) of DED organotypic cultures with and without fibroblasts at indicated time points after seeding. Quantification of Ki67 positive fibroblasts at indicated time point after seeding (n= 8 section per condition of one representative experiment repeated two times).ECM degradation in DED organotypic cultures by collagenase treatment. Immunofluorescence staining for Krt14 (green), Ki67 (red) and Vimentin (white) of DED organotypic cultures at indicated time points after collagenase treatment. Quantification of Ki67 positive fibroblasts at indicated time point after collagenase treatment (n= 8 section per condition of one representative experiment repeated two times). Note that fibroblasts around the injection sides start to proliferate after 48 hours. Nuclei were labelled with DAPI (blue) (D,F). Scale bars, 100µm (D,F). Data shown are means ± s.d. Fb, fibroblasts; PD, post digestion; TCP, tissue culture plastic; h, hours.


Figure 3Resulting simulation of the system of equations


Figure 4Live imaging of adult PDGFRαH2BEGFP mouse back skin . Representative time - lapse images of adult PDGFRαH2BEGFP ( green ) dermis with collagen shown as second harmonic generation ( SHG ) in purple at indicated imaging time points . Scale bar , 100 µm . In silico lineage tracing of fibroblasts . ( E ) Labelling of fibroblasts close to the epidermis at indicated MCS . ( F ) Labelling of fibroblasts in the middle of the dermis at indicated MCS . ( G ) Labelling of fibroblasts close to the inside of the body at indicated MCS .

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 O O

Figure 4Live imaging of adult PDGFRαH2BEGFP mouse back skin. Representative time-lapse images of adult PDGFRαH2BEGFP (green) dermis with collagen shown as second harmonic generation (SHG) in purple at indicated imaging time points. Scale bar, 100 µm.In silico lineage tracing of fibroblasts. (E) Labelling of fibroblasts close to the epidermis at indicated MCS. (F) Labelling of fibroblasts in the middle of the dermis at indicated MCS. (G) Labelling of fibroblasts close to the inside of the body at indicated MCS.


Figure 5Fibroblast activation during wound repair . Immunofluorescence staining for α − sma ( red ) of PDGFRαH2BEGFP ( green ) wound sites at indicated post wounding ( PW ) times Nuclei were labelled with DAPI ( blue ) Fibroblast activation during wound repair . quantification of α − sma fluorescence intensity in pixel per area inside the upper and lower wound bed ( n = 4 PW0 , PW2 , PW7 , PW21 ; n = 3 PW10 , PW14 ; n = 2 PW4 biological replicates ) Changes in fibroblast density , proliferation and collagen deposition during wound repair . Immunostaining for Ki67 ( red ) of PDGFRαH2BEGFP ( green ) back skin sections at indicated wound healing time points . Nuclei were labelled with DAPIChanges in fibroblast density , proliferation and collagen deposition during wound repair . Quantification of fibroblast proliferation in the upper and lower dermis inside the wound bed over time ( n = 4 PW0 , PW2 , PW21 ; n = 3 PW4 , PW10 ; n = 2 PW7 , PW14 biological replicates ) . Quantification of fibroblast density during wound repair inside and outside the wound bed ( n = 4 PW0 , PW2 , PW21 ; n = 3 PW4 , PW10 ; n = 2 PW7 , PW14 biological replicates ) . Polarised light image of Picrosirius red stained back skin section shown as binary image at indicated time points after wounding . Quantification of proliferating ( Ki67 positive ) keratinocytes and fibroblasts during wound repair over time ( n = 4 PW0 , PW2 , PW21 ; n = 3 PW4 , PW10 ; n = 2 PW7 , PW14 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 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 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 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 B-PANEL_START 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

Figure 5Fibroblast activation during wound repair. Immunofluorescence staining for α−sma (red) of PDGFRαH2BEGFP (green) wound sites at indicated post wounding (PW) times Nuclei were labelled with DAPI (blue)Fibroblast activation during wound repair. quantification of α−sma fluorescence intensity in pixel per area inside the upper and lower wound bed (n= 4 PW0, PW2, PW7, PW21; n= 3 PW10, PW14; n= 2 PW4 biological replicates)Changes in fibroblast density, proliferation and collagen deposition during wound repair. Immunostaining for Ki67 (red) of PDGFRαH2BEGFP (green) back skin sections at indicated wound healing time points. Nuclei were labelled with DAPIChanges in fibroblast density, proliferation and collagen deposition during wound repair. Quantification of fibroblast proliferation in the upper and lower dermis inside the wound bed over time (n= 4 PW0, PW2, PW21; n= 3 PW4, PW10; n= 2 PW7, PW14 biological replicates).Quantification of fibroblast density during wound repair inside and outside the wound bed (n= 4 PW0, PW2, PW21; n= 3 PW4, PW10; n= 2 PW7, PW14 biological replicates).Polarised light image of Picrosirius red stained back skin section shown as binary image at indicated time points after wounding.Quantification of proliferating (Ki67 positive) keratinocytes and fibroblasts during wound repair over time (n= 4 PW0, PW2, PW21; n= 3 PW4, PW10; n= 2 PW7, PW14 biological replicates).


Figure 6Quantification of fibroblast proliferation ( left ; n = 4 PW0 , PW2 , PW21 ; n = 3 PW4 , PW10 ; n = 2 PW7 , PW14 biological replicates ) and collagen density ( right ; n = 4 PW0 , PW2 ; n = 3 PW4 , PW7 , PW10 , PW14 ; n = 2 PW21 biological replicates ) inside ( A ) and outside ( B ) the wound bed over time . Temporal evolution of proliferating fibroblasts ( PF ) and dermal ECM density over time .

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

Figure 6Quantification of fibroblast proliferation (left; n= 4 PW0, PW2, PW21; n= 3 PW4, PW10; n= 2 PW7, PW14 biological replicates) and collagen density (right; n= 4 PW0, PW2; n= 3 PW4, PW7, PW10, PW14; n= 2 PW21 biological replicates) inside (A) and outside (B) the wound bed over time.Temporal evolution of proliferating fibroblasts (PF) and dermal ECM density over time.


Figure 7Live imaging of adult PDGFRαH2BEGFP back skin during wound healing . Representative wound bed Z - stack stitched maximum projections of the entire wound bed ( upper panel ) showing fibroblasts ( green ) and collagen with second harmonic generation ( SHG ) in purple at indicated time points after wounding and before live imaging . Dotted line indicates wound edge and boxed area indicates the position of the magnified area below . Live imaging of adult PDGFRαH2BEGFP back skin during wound healing . Representative time - lapse images of adult wound bed at PW4 , where Z - directional movement is visualized by pseudo colouring the fibroblast nuclei ( red , movement towards the basement membrane ; blue , movement away from the basement membrane ) . In vivo lineage tracing of upper ( Blimp1Cre ) and lower ( Dlk1CreER ) dermal fibroblasts during wound healing . Immunofluorescence image of Blimp1Cre x Confetti ( upper panels ) and Dlk1CreER x Confetti ( lower panel ) back skin inside and outside the wound bed at 10 days post wounding . Colours show YFP ( green ) , RFP ( red ) and CFP ( blue ) with bright field image overlaid . ( G ) Clone size quantification of Blimp1Cre labelled fibroblasts inside and outside the wound bed at 10 days post wounding ( n = 400 clones of 4 biological replicates ) . ( H ) Clone size quantification of Blimp1Cre labelled fibroblasts inside and outside the wound bed at 10 days post wounding ( n = 300 clones of 2 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 O O O O O O O O O O O O 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 7Live imaging of adult PDGFRαH2BEGFP back skin during wound healing. Representative wound bed Z-stack stitched maximum projections of the entire wound bed (upper panel) showing fibroblasts (green) and collagen with second harmonic generation (SHG) in purple at indicated time points after wounding and before live imaging. Dotted line indicates wound edge and boxed area indicates the position of the magnified area below.Live imaging of adult PDGFRαH2BEGFP back skin during wound healing. Representative time-lapse images of adult wound bed at PW4, where Z-directional movement is visualized by pseudo colouring the fibroblast nuclei (red, movement towards the basement membrane; blue, movement away from the basement membrane).In vivo lineage tracing of upper (Blimp1Cre) and lower (Dlk1CreER) dermal fibroblasts during wound healing. Immunofluorescence image of Blimp1Cre x Confetti (upper panels) and Dlk1CreER x Confetti (lower panel) back skin inside and outside the wound bed at 10 days post wounding. Colours show YFP (green), RFP (red) and CFP (blue) with bright field image overlaid. (G) Clone size quantification of Blimp1Cre labelled fibroblasts inside and outside the wound bed at 10 days post wounding (n= 400 clones of 4 biological replicates). (H) Clone size quantification of Blimp1Cre labelled fibroblasts inside and outside the wound bed at 10 days post wounding (n=300 clones of 2 biological replicates).


Figure 2 ( B ) Bar diagram representation of the relative SUMO conjugation activity of Nse2 , full - length Smc5 - Nse2 , ΔHinge / Smc5 - Nse2 , ΔHead / Smc5 - Nse2 and Arm / Smc5 - Nse2 truncation constructs ( schematic representation above ) . Orange bars indicate the presence of ssDNA ( virion ϕx174 ) and red bars absence of ssDNA . Reaction rates were performed at least in three different independent experiments ( see Fig EV2 ) . Data values are mean ± s . e . m . ; and n = 3 technical replicates . Significance was measured by a two - tailed unpaired t - test relative to wild - type . * * P < 0 . 01 ( C ) SYPRO - stained ( left ) and Western blot ( right ) time course SUMO conjugation reaction using and ΔHead / Smc5 - Nse2 truncation construct in the presence of ssDNA . T7 - tagged Smc5 and E1 were immunodetected by an anti - T7 antibody . Reactions were run at 30 ° C and stopped at indicated times by adding SDS - loading buffer ( D ) SYPRO - stained ( left ) and Western blot ( right ) time course SUMO conjugation reaction using and ΔHinge / Smc5 - Nse2 truncation construct in the presence of ssDNA . T7 - tagged Smc5 and E1 were immunodetected by an anti - T7 antibody . Reactions were run at 30 ° C and stopped at indicated times by adding SDS - loading buffer ( E ) Western blot of the time - course SUMO conjugation reaction in the presence of ssDNA ( virion ϕx174 ) using either Nse2 ( left ) or Arm / Smc5 - Nse2 complex ( right ) . The reactions were run in the presence or absence of cNse4 external substrate . Reaction was run at 30 ° C and stopped at indicated minutes by adding SDS - loading buffer ( N - S2 , cNse4 - SUMO2 ; N - 2S2 , cNse4 - 2SUMO2 ; N - 3S2 , cNse4 - 3SUMO2 and pS2 , poly - SUMO2 )

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 (B) Bar diagram representation of the relative SUMO conjugation activity of Nse2, full-length Smc5-Nse2, ΔHinge/Smc5-Nse2, ΔHead/Smc5-Nse2 and Arm/Smc5-Nse2 truncation constructs (schematic representation above). Orange bars indicate the presence of ssDNA (virion ϕx174) and red bars absence of ssDNA. Reaction rates were performed at least in three different independent experiments (see Fig EV2). Data values are mean ± s.e.m.; and n=3 technical replicates. Significance was measured by a two-tailed unpaired t-test relative to wild-type. **P<0.01  (C) SYPRO-stained (left) and Western blot (right) time course SUMO conjugation reaction using and ΔHead/Smc5-Nse2 truncation construct in the presence of ssDNA. T7-tagged Smc5 and E1 were immunodetected by an anti-T7 antibody. Reactions were run at 30 °C and stopped at indicated times by adding SDS-loading buffer  (D) SYPRO-stained (left) and Western blot (right) time course SUMO conjugation reaction using and ΔHinge/Smc5-Nse2 truncation construct in the presence of ssDNA. T7-tagged Smc5 and E1 were immunodetected by an anti-T7 antibody. Reactions were run at 30 °C and stopped at indicated times by adding SDS-loading buffer  (E) Western blot of the time-course SUMO conjugation reaction in the presence of ssDNA (virion ϕx174) using either Nse2 (left) or Arm/Smc5-Nse2 complex (right). The reactions were run in the presence or absence of cNse4 external substrate. Reaction was run at 30°C and stopped at indicated minutes by adding SDS-loading buffer (N-S2, cNse4-SUMO2; N-2S2, cNse4-2SUMO2; N-3S2, cNse4-3SUMO2 and pS2, poly-SUMO2)


Figure 3 ( D ) Bar diagram representation of the SUMO conjugation rates of activity assays of Arm / Smc5 - Nse2 KE mutants in the presence ( orange bars ) or absence ( red bars ) of ssDNA ( virion ϕx174 ) , relative to wild - type ( set to 1 ) . Reactions rates were performed at least in three different independent experiments . Data values are mean ± s . e . m . and n = 3 technical replicates . Significance was measured by a two - tailed unpaired t - test relative to wild - type . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 (D) Bar diagram representation of the SUMO conjugation rates of activity assays of Arm/Smc5-Nse2 KE mutants in the presence (orange bars) or absence (red bars) of ssDNA (virion ϕx174), relative to wild-type (set to 1). Reactions rates were performed at least in three different independent experiments. Data values are mean ± s.e.m. and n=3 technical replicates. Significance was measured by a two-tailed unpaired t-test relative to wild-type. *P<0.05, **P<0.01, ***P<0.001