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 .

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

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 ) .

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-CONTROLLED_VAR O O O O O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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 ) .

O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O 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-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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 .

O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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.


Morphology of wild - type ( Tefm + / + ) and Tefm homozygous knockout ( Tefm - / - ) embryos at embryonic day 8 . 5 ( analyzed embryos , n = 43 ) . Scale bar , 500 μm .

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

Morphology 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 .

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

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 .

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

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 .

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

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.


Transmission 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 ) .

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

Transmission 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 .

O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O 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-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-MEASURED_VAR O

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 ) .

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

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 .

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

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 ) .

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

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

Northern 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.


In 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 .

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

In 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 .

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 .

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

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 .

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

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

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.


Strand - 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 .

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

Strand-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 .

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

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

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).


Volcano 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 .

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

Volcano 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.


B 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 .

B 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 .

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

C 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 .

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

Effects of the 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 .

O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

F 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.


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 B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A Representative 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).


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 ) .

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

A Representative 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).


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 .

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

A Representative 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 .

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

C,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 .

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

E Munc18-1 mutants retain interaction with Syx1 shown by GST pull-down experiment. A representative gel displayed is from one of three independent replicates.


B 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 .

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 .

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.


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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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.


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 .

O O O O O O O O O O O O O O O O O O O O O O O O O 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 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.


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 .

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

A 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.


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 .

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

A 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.


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

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.


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 .

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.


B Munc18 - 1 , Habc and H3 construct a tertiary complex .

B Munc18-1, Habc and H3 construct a tertiary complex.


C The tertiary complex globally resembles the Munc18 - 1 / Syx1 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 .

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

D 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 .

E Munc18-1 displays interaction with Syx1-Habc.


F QKAA , KKEE , and L348R all retained interaction with Syx1 - Habc .

F QKAA, KKEE, and L348R all retained interaction with Syx1-Habc.


C 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 ) .

O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

C 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 .

O O O B-MEASURED_VAR O O O O O O B-CONTROLLED_VAR O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

E 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O 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-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O B-MEASURED_VAR O O O O O O O B-MEASURED_VAR O O O O O O

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.


A 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 .

O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A 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.


A 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 .

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

A Representative 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 ) .

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 ) .

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

C 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 O O O 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-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

D 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


A 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 .

A 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 ) .

O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

D 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 ) .

O 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-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

F 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 .

G Representative images of immunohistochemical detection of acrolein in LAM lung lesions.


A 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 .

O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A 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

O O O O O 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-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

D 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 setting


E Representative images of immunohistochemical detection of HRH1 in LAM lung lesions .

E 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 .

O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

F 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 O O 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-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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.


B 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 .

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B 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 .

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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 .

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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 .

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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.


A 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 .

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A 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 .

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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 .

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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.


Quantification 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 ) .

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Quantification 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 ) .

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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).


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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.


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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 ) .

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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 .

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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.


Two 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 ) .

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Two 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 ) .

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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 ) .

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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 .

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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.


Resulting simulation of the system of equations


Live 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 .

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Live 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 .

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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.


Fibroblast 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 )

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Fibroblast 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 )

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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

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 DAPI


Changes 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 ) .

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Changes 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 ) .

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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 .

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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 ) .

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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).


Quantification 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 .

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Quantification 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 .

Temporal evolution of proliferating fibroblasts (PF) and dermal ECM density over time.


Live 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 .

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Live 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 ) .

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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 ) .

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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).


( 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

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-CONTROLLED_VAR O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) 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

O O O O O O O O O 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-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(C) 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

(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 B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(E) 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)


( 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 B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(D) 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


( C ) Degree of conformational changes in CM induced by 60b linear ssDNA . Blue bar , Arm / Smc5 - Nse2 ( wild - type ) ; grey bar , Arm / Smc5 - Nse2 ( K333E / K344E ) ; green bar , Arm / Smc5 - Nse2 ( K764E ) ; pink bar , Arm / Smc5 - Nse2 ( K333E / K344E / K764E ) ; red bar , Arm / Smc5 - Nse2 ( K743E / K745E ) . Reactions were performed at least in three different independent experiments . Data values are mean ± s . d . and n = 3 technical replicates . Significance was measured by a two - tailed unpaired t - test relative to wild - type . * * * * P < 0 . 0001

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

(C) Degree of conformational changes in CM induced by 60b linear ssDNA. Blue bar, Arm/Smc5-Nse2 (wild-type); grey bar, Arm/Smc5-Nse2 (K333E/K344E); green bar, Arm/Smc5-Nse2 (K764E); pink bar, Arm/Smc5-Nse2 (K333E/K344E/K764E); red bar, Arm/Smc5-Nse2 (K743E/K745E). Reactions were performed at least in three different independent experiments. Data values are mean ± s.d. and n=3 technical replicates. Significance was measured by a two-tailed unpaired t-test relative to wild-type. ****P < 0.0001


( B ) Growth test analysis of wild - type , nse2 - CH and the indicated smc5 - KE mutants ; 10 - fold serial dilutions of the liquid cultures were spotted in YPD and pictures taken after 48 hours

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(B) Growth test analysis of wild-type, nse2-CH and the indicated smc5-KE mutants; 10-fold serial dilutions of the liquid cultures were spotted in YPD and pictures taken after 48 hours


(C) Same as in (B) but using the nse2-CH, smc5-3KE, smc5-K743,745E, smc5-K743,745R, smc5-7KE and smc5-7KR mutants


( A ) Protein extracts from exponentially growing wild - type , smc5 - 3KE , smc5 - K743 , 745E , smc5 - 4KE and smc5 - K743 , 745R cells were prepared under denaturing conditions ; 6xHis - Flag tagged SUMO ( HF - SUMO ) was pulled down from protein extracts to purify sumoylated species and analyzed by western blot . A strain with no HF - tag was used as control ; arrow points to unmodified form of the protein , vertical bar to sumoylated forms

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

(A) Protein extracts from exponentially growing wild-type, smc5-3KE, smc5-K743,745E, smc5-4KE and smc5-K743,745R cells were prepared under denaturing conditions; 6xHis-Flag tagged SUMO (HF-SUMO) was pulled down from protein extracts to purify sumoylated species and analyzed by western blot. A strain with no HF-tag was used as control; arrow points to unmodified form of the protein, vertical bar to sumoylated forms


( B ) Left , same as in ( A ) , but using wild - type and two independent clones of smc5 - 7KE ; right , same as in ( A ) , but using wild - type , smc5 - 7KE and smc5 - 7KR mutant cells

(B) Left, same as in (A), but using wild-type and two independent clones of smc5-7KE; right, same as in (A), but using wild-type, smc5-7KE and smc5-7KR mutant cells


( C ) Quantification of Smc5 sumoylated species in pull downs from the indicated smc5 - KE mutants , relative to wild - type controls from at least three independent experiments . Boxes , 25 - 75 % data range ; whiskers , total data range ; black bar , median ; grey cross , mean . * * * * P < 0 . 0001 ; * * * P < 0 . 001 ; * * P < 0 . 01 ; * P < 0 . 05 . 1 - way ANOVA , n = number of samples analyzed

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

(C) Quantification of Smc5 sumoylated species in pull downs from the indicated smc5-KE mutants, relative to wild-type controls from at least three independent experiments. Boxes, 25-75% data range; whiskers, total data range; black bar, median; grey cross, mean. ****P < 0.0001; ***P<0.001; **P < 0.01; *P<0.05. 1-way ANOVA, n = number of samples analyzed


( D ) Co - immunoprecipitation analysis of the Smc5 - Nse2 interactions . NSE2 - 6HA cells were transformed with centromeric plasmids expressing the indicated SMC5 alleles , and protein extracts from exponentially growing cells subjected to anti - HA immunoprecipitation . A strain with no HA tag was used as control

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

(D) Co-immunoprecipitation analysis of the Smc5-Nse2 interactions. NSE2-6HA cells were transformed with centromeric plasmids expressing the indicated SMC5 alleles, and protein extracts from exponentially growing cells subjected to anti-HA immunoprecipitation. A strain with no HA tag was used as control


( E ) Same as in ( D ) , but using SMC6 - 6HA cells

(E) Same as in (D), but using SMC6-6HA cells


( F ) Quantification of Smc5 - 6HA signal from immunofluorescence on chromosome spreads prepared from exponentially growing cultures of the indicated genotypes . The mean value on wild - type spreads was arbitrarily set to 1 ; each dot represents one nucleus ; red line , median . 1 - way ANOVA , n = number of nuclei analyzed . * * * * P < 0 . 0001

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

(F) Quantification of Smc5-6HA signal from immunofluorescence on chromosome spreads prepared from exponentially growing cultures of the indicated genotypes. The mean value on wild-type spreads was arbitrarily set to 1; each dot represents one nucleus; red line, median. 1-way ANOVA, n=number of nuclei analyzed. ****P < 0.0001


a , Northern blot analysis of lamp - 2 expression . Total RNA was hybridized using a lamp - 2 cDNA2 probe and a murine glyceraldehyde - 3 - phosphate dehydrogenase probe .

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

a, Northern blot analysis of lamp-2 expression. Total RNA was hybridized using a lamp-2 cDNA2 probe and a murine glyceraldehyde-3-phosphate dehydrogenase probe.


b , Western blot analysis of LAMP - 2 expression . In control liver and kidney extracts the glycosylated LAMP - 2 molecules were detected . In LAMP - 2 - / - tissues no LAMP - 2 product was found .

b, Western blot analysis of LAMP-2 expression. In control liver and kidney extracts the glycosylated LAMP-2 molecules were detected. In LAMP-2-/- tissues no LAMP-2 product was found.


c , Increased mortality in LAMP - 2 deficient outbred ( C57B6 / Jx129SV ) mice . Forty - two control ( closed circles ) and LAMP - 2 - deficient mice ( open circles ) were monitored over 300 days .

c, Increased mortality in LAMP-2 deficient outbred (C57B6/Jx129SV) mice. Forty-two control (closed circles) and LAMP-2-deficient mice (open circles) were monitored over 300 days.


d , Loss of weight in early dying ( n = 7 males ) and surviving LAMP - 2 - deficient mice ( n = 11 males ) in comparison with control mice ( n = 12 males ) monitored for 60 days after birth .

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

d, Loss of weight in early dying (n = 7 males) and surviving LAMP-2-deficient mice (n = 11 males) in comparison with control mice ( n = 12 males) monitored for 60 days after birth.


a , Ultrastructure of autophagic vacuoles ( arrowheads ) in pancreatic acinar gland cells from a 6 . 5 - month - old LAMP - 2 - deficient mouse .

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

a, Ultrastructure of autophagic vacuoles (arrowheads) in pancreatic acinar gland cells from a 6.5-month-old LAMP-2-deficient mouse.


b , Autophagic vacuoles containing glycogen ( g ) , cytosol and cellular constituents in a hepatocyte of an 8 - day - old LAMP - 2 - / - mouse . m , mitochrondria ; bc , bile canaliculus . c , Accumulation of numerous early autophagic vacuoles in cultured LAMP - 2 - / - hepatocytes . Arrow , large late autophagic vacuole containing partially degraded material . Insert , magnification of the region indicated by an asterisk with typical early autophagic vacuoles containing undigested cellular constituents . Scale bars : a , 3 . 5 µm ; b , 0 . 2 µm ; c , 1 . 3 µm ; insert , 0 . 9 µm .

O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, Autophagic vacuoles containing glycogen (g), cytosol and cellular constituents in a hepatocyte of an 8-day-old LAMP-2-/- mouse. m, mitochrondria; bc, bile canaliculus. c, Accumulation of numerous early autophagic vacuoles in cultured LAMP-2-/- hepatocytes. Arrow, large late autophagic vacuole containing partially degraded material. Insert, magnification of the region indicated by an asterisk with typical early autophagic vacuoles containing undigested cellular constituents. Scale bars: a, 3.5 µm; b, 0.2 µm; c, 1.3 µm; insert, 0.9 µm.


a , Vacuole in a fibre of the soleus muscle . b , Ultrastructure of a vacuole in a cardiomyocyte . Scale bars : a , 1 µm ; b , 1 . 5 µm .

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

a, Vacuole in a fibre of the soleus muscle. b, Ultrastructure of a vacuole in a cardiomyocyte. Scale bars: a, 1 µm; b, 1.5 µm.


c , Typical original twitch recordings of developed force of heart muscles from control ( thin line ) and LAMP - 2 - deficient ( thick line ) mice . d , Average values of developed force . Preparations of LAMP - 2 deficient mice had a significantly lower baseline developed force at a stimulation frequency of 4 Hz and 10 Hz ; P 0 . 01 .

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

c, Typical original twitch recordings of developed force of heart muscles from control (thin line) and LAMP-2-deficient (thick line) mice. d, Average values of developed force. Preparations of LAMP-2 deficient mice had a significantly lower baseline developed force at a stimulation frequency of 4 Hz and 10 Hz; P  0.01.


a , Volume density of early ( Avi ) and late ( Avd ) autophagic vacuoles from four independent cultures . Examples of Avi and Avd are shown in b and c , respectively . Bars represent 0 . 4 µm .

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

a, Volume density of early (Avi) and late (Avd) autophagic vacuoles from four independent cultures. Examples of Avi and Avd are shown in b and c, respectively. Bars represent 0.4 µm.


d , Labelling density of cathepsin - D as estimated in two independent experiments . e , Labelling density of LAMP - 1 .

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

d, Labelling density of cathepsin-D as estimated in two independent experiments. e, Labelling density of LAMP-1.


f , Degradation of long - lived proteins followed over 24 h in amino acid and fetal - calf - serum - containing medium . g , Effect of amino - acid ( AA ) deprivation and 3 - methyladenine ( MA ) on protein degradation during 4 h of chase . The error bars in a , d , e and g represent s . e . m .

O O O O O O 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-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

f, Degradation of long-lived proteins followed over 24 h in amino acid and fetal-calf-serum-containing medium. g, Effect of amino-acid (AA) deprivation and 3-methyladenine (MA) on protein degradation during 4 h of chase. The error bars in a, d, e and g represent s.e.m.


B ) Percentage specific binding of [ 3H ] dofetilide to the hERG channel after 6 minutes of dissociation induced by 10 µM dofetilide in the absence ( control ) or presence of 10 and 50 µM of LUF compounds . The specific binding of [ 3H ] dofetilide in the absence of test compounds was set as Bcontrol , while the specific binding in their presence was set as B . * = p < 0 . 05 vs control ; N : 3 - 4 .

O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O B-MEASURED_VAR 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-MEASURED_VAR O O O O O O O O O O O O O O O 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) Percentage specific binding of [3H]dofetilide to the hERG channel after 6 minutes of dissociation induced by 10 µM dofetilide in the absence (control) or presence of 10 and 50 µM of LUF compounds. The specific binding of [3H]dofetilide in the absence of test compounds was set as Bcontrol, while the specific binding in their presence was set as B. * = p < 0.05 vs control; N: 3-4.


C ) Representative traces of hERG activation ( left ) , inactivation ( middle ) , and deactivation ( right ) measured in HEK293 hERG cells under baseline conditions ( black ) and in the presence of 3 µM LUF7346 ( red ) . Insets : voltage clamp protocols .

O O O O O O O O O O O O O O O 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-CONTROLLED_VAR O O O O O O O O O O

C) Representative traces of hERG activation (left), inactivation (middle), and deactivation (right) measured in HEK293 hERG cells under baseline conditions (black) and in the presence of 3 µM LUF7346 (red). Insets: voltage clamp protocols.


D ) Steady - state activation ( empty symbols ) and inactivation ( filled symbols ) curves for IKr under baseline conditions ( black ) and in the presence of 3 µM LUF7346 ( red ) . The corresponding Boltzmann ' s fittings are superimposed to data points . * = p < 0 . 05 vs baseline . N : 11 - 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 B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

D) Steady-state activation (empty symbols) and inactivation (filled symbols) curves for IKr under baseline conditions (black) and in the presence of 3 µM LUF7346 (red). The corresponding Boltzmann's fittings are superimposed to data points.* = p < 0.05 vs baseline. N: 11-14.


E ) Plot of the time constants ( τ ) of deactivation derived from biexponential fittings under baseline conditions ( black ) and in the presence of 3 µM LUF7346 ( red ) . * = p < 0 . 05 vs respective baseline . N : 8 .

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E) Plot of the time constants (τ) of deactivation derived from biexponential fittings under baseline conditions (black) and in the presence of 3 µM LUF7346 (red). * = p < 0.05 vs respective baseline. N: 8.


B ) Baseline QT interval ( top ) , baseline RR interval ( middle ) , and baseline QT interval corrected with the Bazett ' s formula ( bottom ) . Bar graphs are divided by isogenic pairs ( LQT1R594Q and JLNSR594Q , left ; LQT2corr , LQT2N996I , hESCWT , and hESC - LQT2N996I , middle ; LQT1corr and LQT1R190Q , right ) and in each graph the unrelated WT is shown as a comparison . N : 17 - 53 . * = p < 0 . 05 . The colour of the symbol indicates comparisons and relative statistical significance .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Baseline QT interval (top), baseline RR interval (middle), and baseline QT interval corrected with the Bazett's formula (bottom). Bar graphs are divided by isogenic pairs (LQT1R594Q and JLNSR594Q, left; LQT2corr, LQT2N996I, hESCWT, and hESC-LQT2N996I, middle; LQT1corr and LQT1R190Q, right) and in each graph the unrelated WT is shown as a comparison. N: 17-53. * = p<0.05. The colour of the symbol indicates comparisons and relative statistical significance.