( A ) Survival rate and life span of WT , Pdia4 - / - ( KO ) , and Pdia4tg / tg ( TG ) mice on Leprdb / db background from birth to 120 weeks Data information : The number of mice ( n ) is indicated in parentheses . Data from over 3 experiments are presented as the mean ± SD . Log rank test were used for statistical analysis of differences between groups , and P ( * ) < 0 . 05 ; P ( * * ) < 0 . 01 and P ( * * * ) < 0 . 001 are considered statistically significant .

O O O O O O O O O O O B-CONTROLLED_VAR O O O 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Survival rate and life span of WT, Pdia4-/-(KO), and Pdia4tg/tg (TG) mice on Leprdb/db background from birth to 120 weeks Data information: The number of mice (n) is indicated in parentheses. Data from over 3 experiments are presented as the mean ± SD. Log rank test were used for statistical analysis of differences between groups, and P (*) < 0.05; P (**) < 0.01 and P (***) < 0.001 are considered statistically significant.


( B ) Pancreata of 18 - week - old WT and Pdia4 - / - ( KO ) mice on BKS or Leprdb / db background were stained with anti - insulin ( αIns ) antibody and dihydroethidium ( DHE ) ( left ) . Islet area ( μm2 ) and relative fluorescence intensity ( RFI ) were quantified ( right ) . Scale bar : 100 μm . The dash circles indicate islet regions . Data information : Data from over 3 experiments are presented as the mean ± SD . one - way ANOVA test were used for statistical analysis of differences between groups , and P ( * ) < 0 . 05 ; P ( * * ) < 0 . 01 and P ( * * * ) < 0 . 001 are considered statistically significant .

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 B-CONTROLLED_VAR 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 O O O O O O O O O O O O O O O O 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) Pancreata of 18-week-old WT and Pdia4-/- (KO) mice on BKS or Leprdb/db background were stained with anti-insulin (αIns) antibody and dihydroethidium (DHE) (left). Islet area (μm2) and relative fluorescence intensity (RFI) were quantified (right). Scale bar: 100 μm. The dash circles indicate islet regions. Data information: Data from over 3 experiments are presented as the mean ± SD. one-way ANOVA test were used for statistical analysis of differences between groups, and P (*) < 0.05; P (**) < 0.01 and P (***) < 0.001 are considered statistically significant.


( D ) The same batch mice as in ( B ) were given water containing BrdU . The BrdU + cells of the islets were visualized and quantified ( BrdU + labeling ) . TUNEL - positive cells in the islets of the mice ( B ) were visualized and quantified ( TUNEL assay ) . BrdU + cells per islet area ( 0 . 01 mm2 ) and TUNEL + cells per islet area ( 0 . 05 mm2 ) are expressed in arbitrary units ( AU ) . The dash circles indicate islet regions and the black arrowheads indicate TUNEL + cells . Data information : Data from over 3 experiments are presented as the mean ± SD . one - way ANOVA test were used for statistical analysis of differences between groups , and P ( * ) < 0 . 05 ; P ( * * ) < 0 . 01 and P ( * * * ) < 0 . 001 are considered statistically significant .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) The same batch mice as in (B) were given water containing BrdU. The BrdU+ cells of the islets were visualized and quantified (BrdU+ labeling). TUNEL-positive cells in the islets of the mice (B) were visualized and quantified (TUNEL assay). BrdU+ cells per islet area (0.01 mm2) and TUNEL+ cells per islet area (0.05 mm2) are expressed in arbitrary units (AU). The dash circles indicate islet regions and the black arrowheads indicate TUNEL+ cells. Data information: Data from over 3 experiments are presented as the mean ± SD. one-way ANOVA test were used for statistical analysis of differences between groups, and P (*) < 0.05; P (**) < 0.01 and P (***) < 0.001 are considered statistically significant.


F ) insulin in supernatants of the mouse islets ( F ) in GSIS assays . High glucose ( HG , 16 . 7 mM ) or low glucose ( LG , 3 . 3 ) were used in GSIS assays . Data information : Data from over 3 experiments are presented as the mean ± SD . one - way ANOVA test were used for statistical analysis of differences between groups , and P ( * ) < 0 . 05 ; P ( * * ) < 0 . 01 and P ( * * * ) < 0 . 001 are considered statistically significant .

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

F) insulin in supernatants of the mouse islets (F) in GSIS assays. High glucose (HG, 16.7 mM) or low glucose (LG, 3.3) were used in GSIS assays. Data information: Data from over 3 experiments are presented as the mean ± SD. one-way ANOVA test were used for statistical analysis of differences between groups, and P (*) < 0.05; P (**) < 0.01 and P (***) < 0.001 are considered statistically significant.


( B ) Pancreata of the same batch of mice as in ( A ) , at the age of 14 weeks , were stained with anti - insulin ( αIns ) antibody and DHE ( left ) . Islet area ( μm2 ) and relative fluorescence intensity ( RFI ) were quantified ( right ) . Scale bar = 100 μm . The dash circles indicate islet regions . Data information : Data from 3 experiments more are presented as the mean ± SD . One - way ANOVA test was used for statistical analysis of differences between groups , and P ( * ) < 0 . 05 ; P ( * * ) < 0 . 01 and P ( * * * ) < 0 . 001 are considered statistically significant .

O O O O 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 O O O O O O O O O O O O O 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) Pancreata of the same batch of mice as in (A), at the age of 14 weeks, were stained with anti-insulin (αIns) antibody and DHE (left). Islet area (μm2) and relative fluorescence intensity (RFI) were quantified (right). Scale bar = 100 μm. The dash circles indicate islet regions. Data information: Data from 3 experiments more are presented as the mean ± SD. One-way ANOVA test was used for statistical analysis of differences between groups, and P (*) < 0.05; P (**) < 0.01 and P (***) < 0.001 are considered statistically significant.


( D ) The islets of the mice from ( C ) were tested for GSIS . High glucose ( HG , 16 . 7 mM ) or low glucose ( LG , 3 . 3 mM ) were used in GSIS assays . Data information : Data from 3 experiments more are presented as the mean ± SD . One - way ANOVA test was used for statistical analysis of differences between groups , and P ( * ) < 0 . 05 ; P ( * * ) < 0 . 01 and P ( * * * ) < 0 . 001 are considered statistically significant .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) The islets of the mice from (C) were tested for GSIS. High glucose (HG, 16.7 mM) or low glucose (LG, 3.3 mM) were used in GSIS assays. Data information: Data from 3 experiments more are presented as the mean ± SD. One-way ANOVA test was used for statistical analysis of differences between groups, and P (*) < 0.05; P (**) < 0.01 and P (***) < 0.001 are considered statistically significant.


( A ) The islets of WT , Pdia4 - / - ( KO ) and Pdia4tg / tg ( TG ) BKS mice were isolated and grown in complete DMEM medium containing 3 . 3 mM ( LG ) and 30 mM glucose ( HG ) for 12 h . The islets stained with propidium iodide ( PI ) , photographed , and quantified . Scale bar = 50 μm . Data information : Data from 3 experiments are presented as the mean ± SD . One - way ANOVA test was used for statistical analysis of differences between groups , and P ( * ) < 0 . 05 ; P ( * * ) < 0 . 01 and P ( * * * ) < 0 . 001 are considered statistically significant .

O O O 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) The islets of WT, Pdia4-/- (KO) and Pdia4tg/tg (TG) BKS mice were isolated and grown in complete DMEM medium containing 3.3 mM (LG) and 30 mM glucose (HG) for 12 h. The islets stained with propidium iodide (PI), photographed, and quantified. Scale bar = 50 μm. Data information: Data from 3 experiments are presented as the mean ± SD. One-way ANOVA test was used for statistical analysis of differences between groups, and P (*) < 0.05; P (**) < 0.01 and P (***) < 0.001 are considered statistically significant.


The islets from the mice ( A ) were incubated with MitoGreen plus MitoSOX in the presence of glucose at 3 . 3 mM ( LG ) and 16 . 7 mM ( HG ) . Mitochondrial ROS in the islets were visualized and quantified . Scale bar = 100 μm . Data information : Data from 3 experiments are presented as the mean ± SD . One - way ANOVA test was used for statistical analysis of differences between groups , and P ( * ) < 0 . 05 ; P ( * * ) < 0 . 01 and P ( * * * ) < 0 . 001 are considered statistically significant .

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

The islets from the mice (A) were incubated with MitoGreen plus MitoSOX in the presence of glucose at 3.3 mM (LG) and 16.7 mM (HG). Mitochondrial ROS in the islets were visualized and quantified. Scale bar = 100 μm. Data information: Data from 3 experiments are presented as the mean ± SD. One-way ANOVA test was used for statistical analysis of differences between groups, and P (*) < 0.05; P (**) < 0.01 and P (***) < 0.001 are considered statistically significant.


The islets from the mice ( A ) were incubated with Hoechst 33342 ( Ho ) plus CM - H2DCFDA in the presence of glucose at 3 . 3 mM ( LG ) and 16 . 7 mM ( HG ) . cytosolic ROS in the islets were visualized and quantified . Scale bar = 100 μm . Data information : Data from 3 experiments are presented as the mean ± SD . One - way ANOVA test was used for statistical analysis of differences between groups , and P ( * ) < 0 . 05 ; P ( * * ) < 0 . 01 and P ( * * * ) < 0 . 001 are considered statistically significant .

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

The islets from the mice (A) were incubated with Hoechst 33342 (Ho) plus CM-H2DCFDA in the presence of glucose at 3.3 mM (LG) and 16.7 mM (HG). cytosolic ROS in the islets were visualized and quantified. Scale bar = 100 μm. Data information: Data from 3 experiments are presented as the mean ± SD. One-way ANOVA test was used for statistical analysis of differences between groups, and P (*) < 0.05; P (**) < 0.01 and P (***) < 0.001 are considered statistically significant.


( E ) Mitochondria isolated from the mouse islets ( A ) were measured for the activity of ETC CI ( left ) , CII / III ( middle ) and CIV ( right ) using MitoCheck assays . Data information : Data from 3 experiments are presented as the mean ± SD . One - way ANOVA test was used for statistical analysis of differences between groups , and P ( * ) < 0 . 05 ; P ( * * ) < 0 . 01 and P ( * * * ) < 0 . 001 are considered statistically significant .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Mitochondria isolated from the mouse islets (A) were measured for the activity of ETC CI (left), CII/III (middle) and CIV (right) using MitoCheck assays. Data information: Data from 3 experiments are presented as the mean ± SD. One-way ANOVA test was used for statistical analysis of differences between groups, and P (*) < 0.05; P (**) < 0.01 and P (***) < 0.001 are considered statistically significant.


( A ) Ultrastructure of mitochondria and insulin granules in typical β - cells of 3 mouse lines on BKS and Leprdb / db backgrounds . Arrows indicate the mitochondria whereas arrowheads indicate insulin granules . Scale bar = 1 μm . Data information : A total of 15 - 23 images per group were analyzed . Data from each group are presented as the mean ± SD .

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

(A) Ultrastructure of mitochondria and insulin granules in typical β-cells of 3 mouse lines on BKS and Leprdb/db backgrounds. Arrows indicate the mitochondria whereas arrowheads indicate insulin granules. Scale bar = 1 μm. Data information: A total of 15-23 images per group were analyzed. Data from each group are presented as the mean ± SD.


( E ) The construct encoding Flag - Pdia4 or its mutants and that expressing Myc / Flag - tagged Ndufs3 ( left ) or p22phox ( right ) were co - transfected into 293T cells . Total lysates ( TL ) and anti - Myc immunoprecipitates ( Myc IP ) underwent immunoblotting analysis . N - terminal Flag tag , catalytic domains ( a , a ' and a ' ' ) , non - catalytic domains ( b and b ' ) , and C - terminal KEEL are indicated . Pdia4 * is a mutant of Pdia4 whose 3 CGHC motifs were changed into 3 SGHS motifs ( a * , a ' * and a ' ' * ) .

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

(E) The construct encoding Flag-Pdia4 or its mutants and that expressing Myc/Flag-tagged Ndufs3 (left) or p22phox (right) were co-transfected into 293T cells. Total lysates (TL) and anti-Myc immunoprecipitates (Myc IP) underwent immunoblotting analysis. N-terminal Flag tag, catalytic domains (a, a' and a''), non-catalytic domains (b and b'), and C-terminal KEEL are indicated. Pdia4* is a mutant of Pdia4 whose 3 CGHC motifs were changed into 3 SGHS motifs (a*, a'* and a''*).


( F ) The construct encoding Myc / Flag - tagged p22phox , Ndufs3 or their mutants and that expressing Flag - tagged Pdia4 were co - transfected into 293T cells . Total lysates ( TL ) and anti - Myc immunoprecipitates ( Myc IP ) underwent immunoblotting analysis using anti - Flag antibody .

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

(F) The construct encoding Myc/Flag-tagged p22phox, Ndufs3 or their mutants and that expressing Flag-tagged Pdia4 were co-transfected into 293T cells. Total lysates (TL) and anti-Myc immunoprecipitates (Myc IP) underwent immunoblotting analysis using anti-Flag antibody.


( A ) Min6 cells infected with a lentivirus expressing a scramble RNAi ( GK ) , a Pdia4 RNAi ( KD ) and a Pdia4 cDNA ( OVE ) were sorted and tested for the Pdia4 protein level ( left ) . The cells were incubated with NAC ( 1 mM ) and then stained with MitoSOX or CellROX in response to 0 . 5 mM ( LG ) and 25 mM ( HG ) glucose for an additional 30 min . Signal from MitoSOX ( middle ) and CellROX ( right ) was re - plotted into histograms . Data information : Data from 3 experiments are presented as the mean ± SD . One - way ANOVA test was used for statistical analysis of differences between groups , and P ( * ) < 0 . 05 ; P ( * * ) < 0 . 01 and P ( * * * ) < 0 . 001 are considered statistically significant .

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 B-CONTROLLED_VAR 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Min6 cells infected with a lentivirus expressing a scramble RNAi (GK), a Pdia4 RNAi (KD) and a Pdia4 cDNA (OVE) were sorted and tested for the Pdia4 protein level (left). The cells were incubated with NAC (1 mM) and then stained with MitoSOX or CellROX in response to 0.5 mM (LG) and 25 mM (HG) glucose for an additional 30 min. Signal from MitoSOX (middle) and CellROX (right) was re-plotted into histograms. Data information: Data from 3 experiments are presented as the mean ± SD. One-way ANOVA test was used for statistical analysis of differences between groups, and P (*) < 0.05; P (**) < 0.01 and P (***) < 0.001 are considered statistically significant.


( B ) Min6 cells infected with a lentivirus expressing a scramble RNAi ( GK ) and an RNAi of Ndufs3 ( Ndufs3 KD ) or p22phox ( p22phox KD ) were selected and tested for levels of Ndufs3 ( 1st column ) , mitochondrial ROS ( 2nd column , MitoSOX ) , p22phox ( 3rd column ) , and cytosolic ROS ( 4th column , CellROX ) . The cells were grown in the presence of 0 . 5 mM ( LG ) and 25 mM ( HG ) glucose . ( C ) The same experiments as ( B ) were conducted except that Min6 cells were infected with a lentivirus expressing a cDNA of truncated Ndufs3 ( tNdufs3 ) and p22phox ( tp22phox ) . ( Data information : Data from 3 experiments are presented as the mean ± SD . One - way ANOVA test was used for statistical analysis of differences between groups , and P ( * ) < 0 . 05 ; P ( * * ) < 0 . 01 and P ( * * * ) < 0 . 001 are considered statistically significant .

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

(B) Min6 cells infected with a lentivirus expressing a scramble RNAi (GK) and an RNAi of Ndufs3 (Ndufs3 KD) or p22phox (p22phox KD) were selected and tested for levels of Ndufs3 (1st column), mitochondrial ROS (2nd column, MitoSOX), p22phox (3rd column), and cytosolic ROS (4th column, CellROX). The cells were grown in the presence of 0.5 mM (LG) and 25 mM (HG) glucose. (C) The same experiments as (B) were conducted except that Min6 cells were infected with a lentivirus expressing a cDNA of truncated Ndufs3 (tNdufs3) and p22phox (tp22phox). ( Data information: Data from 3 experiments are presented as the mean ± SD. One-way ANOVA test was used for statistical analysis of differences between groups, and P (*) < 0.05; P (**) < 0.01 and P (***) < 0.001 are considered statistically significant.


( D ) 293T cells , which were transfected with the construct encoding Flag - Pdia4 and that expressing Myc / Flag - tagged p22phox or Ndufs3 , were treated with GHTT ( 28 μM ) for 30 min . The cells were lysed and incubated with anti - Pdia4 antibodies plus protein G beads . Their total lysates ( TL ) and immunoprecipitates ( IP ) underwent immunoblotting analysis with anti - Flag antibody .

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 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 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

(D) 293T cells, which were transfected with the construct encoding Flag-Pdia4 and that expressing Myc/Flag-tagged p22phox or Ndufs3, were treated with GHTT (28 μM) for 30 min. The cells were lysed and incubated with anti-Pdia4 antibodies plus protein G beads. Their total lysates (TL) and immunoprecipitates (IP) underwent immunoblotting analysis with anti-Flag antibody.


( F ) The sections of pancreata of the mice ( E ) were stained with anti - insulin ( αIns ) antibody and dihydroethidium ( DHE ) ( left ) . Islet area ( μm2 ) and relative fluorescence intensity ( RFI ) were quantified ( right ) . Scale bar : 100 μm . The dash circles indicate islet regions . Data information : Data from 3 experiments are presented as the mean ± SD . one - way ANOVA test were used for statistical analysis of differences between groups , and P ( * ) < 0 . 05 ; P ( * * ) < 0 . 01 and P ( * * * ) < 0 . 001 are considered statistically significant .

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 O O O O O O O O O O O O O O O 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) The sections of pancreata of the mice (E) were stained with anti-insulin (αIns) antibody and dihydroethidium (DHE) (left). Islet area (μm2) and relative fluorescence intensity (RFI) were quantified (right). Scale bar: 100 μm. The dash circles indicate islet regions. Data information: Data from 3 experiments are presented as the mean ± SD. one-way ANOVA test were used for statistical analysis of differences between groups, and P (*) < 0.05; P (**) < 0.01 and P (***) < 0.001 are considered statistically significant.


( B ) Comparison of investigation duration and recognition index between the light ( L , n = 12 animals ) and dark control ( D , n = 11 animals ) groups . A significant reduction in the recognition index is observed in the L group compared to the D group ( * p = 0 . 0156 , Mann - Whitney test ) , while no significant difference is observed in investigation duration during the first trial . Data shown as mean ± SEM .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Comparison of investigation duration and recognition index between the light (L, n = 12 animals) and dark control (D, n = 11 animals) groups. A significant reduction in the recognition index is observed in the L group compared to the D group (* p = 0.0156, Mann-Whitney test), while no significant difference is observed in investigation duration during the first trial. Data shown as mean ± SEM.


( E ) No statistically significant difference is observed in the investigation duration and recognition index between the L ( n = 8 animals ) and D ( n = 7 animals ) groups ( p = 0 . 9551 , Mann - Whitney test ) . Data shown as mean ± SEM .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) No statistically significant difference is observed in the investigation duration and recognition index between the L (n = 8 animals) and D (n = 7 animals) groups (p = 0.9551, Mann-Whitney test). Data shown as mean ± SEM.


( C ) OTA treatment ( n = 6 animals ) significantly reduces the recognition index in mice compared to the PBS injection control ( n = 6 animals ) mice ( * p = 0 . 0260 , Mann - Whitney test ) . Data shown as mean ± SEM .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) OTA treatment (n = 6 animals) significantly reduces the recognition index in mice compared to the PBS injection control (n = 6 animals) mice (* p = 0.0260, Mann-Whitney test). Data shown as mean ± SEM.


( F - G ) Representative confocal images of c - fos staining showing oxytocin neurons in the supraoptic nucleus ( SON ) ( E ) and paraventricular nucleus ( PVN ) ( F ) in the L and D groups . Yellow arrows indicate oxytocin neurons co - labeled with c - fos . High magnification images are 10 μm Z - stack ( 63X , NA = 1 . 4 , pinhole = 1 . 26 AU ) , while low magnification images are 30 μm Z - stack ( 20X , NA = 0 . 7 , pinhole = 1 . 26 AU ) . Scale bar = 100 μm for low magnification images and 50 μm for high magnification images .

O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR 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 B-MEASURED_VAR 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-G) Representative confocal images of c-fos staining showing oxytocin neurons in the supraoptic nucleus (SON) (E) and paraventricular nucleus (PVN) (F) in the L and D groups. Yellow arrows indicate oxytocin neurons co-labeled with c-fos. High magnification images are 10 μm Z-stack (63X, NA=1.4, pinhole = 1.26 AU), while low magnification images are 30 μm Z-stack (20X, NA=0.7, pinhole = 1.26 AU). Scale bar = 100 μm for low magnification images and 50 μm for high magnification images.


( C ) Average delta - F / F signal trace from SON oxytocin neurons with 10 seconds of dim light ( 5 lux ) followed by 30 seconds of bright light ( 800 lux ) in OPN4DTA / DTA ; OxtCre / + mice ( red line , n = 4 animals ) and OPN4 + / + ; OxtCre / + mice ( black line , n = 5 animals ) . The color shade indicates standard deviation .

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

(C) Average delta-F/F signal trace from SON oxytocin neurons with 10 seconds of dim light (5 lux) followed by 30 seconds of bright light (800 lux) in OPN4DTA/DTA; OxtCre/+ mice (red line, n = 4 animals) and OPN4+/+; OxtCre/+ mice (black line, n = 5 animals). The color shade indicates standard deviation.


( G ) The investigation duration and recognition index of the 595nm - control and the 470nm - activation group ( n = 8 animals ) . There is a significant increase in the recognition index in the optogenetic activation group compared to the control ( * * p = 0 . 0078 , Wilcoxon matched - pairs signed rank test ) . Data shown as mean ± SEM .

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

(G) The investigation duration and recognition index of the 595nm-control and the 470nm-activation group (n = 8 animals). There is a significant increase in the recognition index in the optogenetic activation group compared to the control (** p = 0.0078, Wilcoxon matched-pairs signed rank test). Data shown as mean ± SEM.


( B ) Light exposure significantly reduces the recognition index in control Opn4Cre / + mice ( n = 8 animals in both the L and D group ) , similar to WT mice ( * p = 0 . 0499 , Mann - Whitney test ) . Data shown as mean ± SEM . ( C - E ) There is no significant difference in recognition index between light exposure and dark control groups in OPN4DTA / DTA ( C , n = 11 animals in both the L and D group , p = 0 . 7969 ) , OPN4Cre / + ; Brn3bz - DTA / + ( D , n = 9 animals in both the L and D group , p > 0 . 9999 ) , and OPN4Cre / Cre mice ( E , L group n = 9 animals , D group n = 8 animals , p = 0 . 9551 ) using the Mann - Whitney test . Data shown as mean ± SEM .

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 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 B-CONTROLLED_VAR 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 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

(B) Light exposure significantly reduces the recognition index in control Opn4Cre/+ mice (n = 8 animals in both the L and D group), similar to WT mice (*p = 0.0499, Mann-Whitney test). Data shown as mean ± SEM. (C-E) There is no significant difference in recognition index between light exposure and dark control groups in OPN4DTA/DTA (C, n = 11 animals in both the L and D group, p = 0.7969), OPN4Cre/+; Brn3bz-DTA/+ (D, n = 9 animals in both the L and D group, p > 0.9999), and OPN4Cre/Cre mice (E, L group n = 9 animals, D group n = 8 animals, p = 0.9551) using the Mann-Whitney test. Data shown as mean ± SEM.


( H ) During the second trial , the investigation duration of the 470nm - activation group ( red ) was lower than the 595nm - control ( black ) ( n = 7 animals , * p = 0 . 011 , Sidak ' s multiple comparison test ) . Optogenetic activation of ipRGC terminals at SON with 470 nm light significantly enhances the recognition index compared to the 595 nm control ( * p = 0 . 0156 , Wilcoxon matched - pairs signed rank test ) . Data shown as mean ± SEM .

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(H) During the second trial, the investigation duration of the 470nm-activation group (red) was lower than the 595nm-control (black) (n = 7 animals, * p = 0.011, Sidak's multiple comparison test). Optogenetic activation of ipRGC terminals at SON with 470 nm light significantly enhances the recognition index compared to the 595 nm control (* p = 0.0156, Wilcoxon matched-pairs signed rank test). Data shown as mean ± SEM.


( B ) Representative confocal image of the pSON region stained with synaptophysin ( blue ) , CTB - Alexa 568 ( red ) , and GFP from GABAergic neurons ( green ) . Arrowheads indicates colocalization . Scale bar = 10 μm .

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(B) Representative confocal image of the pSON region stained with synaptophysin (blue), CTB-Alexa 568 (red), and GFP from GABAergic neurons (green). Arrowheads indicates colocalization. Scale bar = 10 μm.


( I ) Average delta - F / F signal trace from pSON GABAergic neurons during the transition from baseline to dim light ( 5 lux , black line , n = 7 trials ) and from baseline to bright light ( 800 lux , red line , n = 12 trials ) in vGATCre / + mice ( n = 5 animals ) . The color shade indicates standard deviation .

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(I) Average delta-F/F signal trace from pSON GABAergic neurons during the transition from baseline to dim light (5 lux, black line, n = 7 trials) and from baseline to bright light (800 lux, red line, n = 12 trials) in vGATCre/+ mice (n = 5 animals). The color shade indicates standard deviation.


( D ) Minute - wise investigation duration of the 595nm - control ( black ) and 470nm - activation ( red ) groups ( n = 8 animals ) . Data shown as mean ± SEM for each minute .

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(D) Minute-wise investigation duration of the 595nm-control (black) and 470nm-activation (red) groups (n = 8 animals). Data shown as mean ± SEM for each minute.


( G ) The investigation duration and recognition index of the GFP - control ( n = 4 animals ) and the TeLC - silencing ( n = 6 animals ) group . During the second trial , the investigation duration of the TeLC - silencing group was lower than the GFP - control group ( * p = 0 . 0280 , Sidak ' s multiple comparison test ) . The TeLC - silencing group had a higher recognition index than GFP - control group ( * p = 0 . 0317 , Mann - Whitney test ) . Data shown as mean ± SEM .

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 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 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

(G) The investigation duration and recognition index of the GFP-control (n = 4 animals) and the TeLC-silencing (n = 6 animals) group. During the second trial, the investigation duration of the TeLC-silencing group was lower than the GFP-control group (* p = 0.0280, Sidak's multiple comparison test). The TeLC-silencing group had a higher recognition index than GFP-control group (* p = 0.0317, Mann-Whitney test). Data shown as mean ± SEM.


( C ) Levels of phosphorylation of the Y174 phosphosite of the indicated Vav1 mutant proteins immunoprecipitated from Jurkat cells ( upper panel ) . The total amount of Vav1 immunoprecipitated in each sample is shown in the bottom panel ( n = 3 independent experiments ) .

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(C) Levels of phosphorylation of the Y174 phosphosite of the indicated Vav1 mutant proteins immunoprecipitated from Jurkat cells (upper panel). The total amount of Vav1 immunoprecipitated in each sample is shown in the bottom panel (n = 3 independent experiments).


( C ) Survival curves of mice transplanted with CD4 + T cells expressing the indicated proteins . The number of animals used is indicated in the graph . Note : the mouse transplanted with EGFP - transduced cells that has died in these experiments did not show any sign of tumor development according to anatomopathological

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(C) Survival curves of mice transplanted with CD4+ T cells expressing the indicated proteins. The number of animals used is indicated in the graph. Note: the mouse transplanted with EGFP-transduced cells that has died in these experiments did not show any sign of tumor development according to anatomopathological analyses


( F ) Quantification of CD4 + versus CD8 + T cell ratio in peripheral blood from mice transplanted with CD4 + T cells expressing the indicated proteins ( bottom ) . Each point represents the measurement of an individual mouse . n as in B . Data information : data represent the mean ± SEM . Statistical values obtained using the Student ' s t are given relative to control EGFP + cells . * , P ≤ 0 . 05 ; * * , P ≤ 0 . 01 ; * * * , P ≤ 0 . 001 .

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(F) Quantification of CD4+ versus CD8+ T cell ratio in peripheral blood from mice transplanted with CD4+ T cells expressing the indicated proteins (bottom). Each point represents the measurement of an individual mouse. n as in B. Data information: data represent the mean ± SEM. Statistical values obtained using the Student's t are given relative to control EGFP+ cells. *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001.


( J - L ) Representative flow cytometry analysis ( J ) and quantification of the surface levels of ICOS ( K ) and CD69 ( L ) in TFH cells from mice transplanted with CD4 + T cells expressing the indicated proteins . In J , numbers indicate the relative percentage ( % ) of the cell population that has been interrogated ( boxed ) . In K and L , each point represents the values obtained with a single experimental mouse . n as in H . Data information : data represent the mean ± SEM . Statistical values obtained using the Student ' s t are given relative to control EGFP + cells . * , P ≤ 0 . 05 ; * * , P ≤ 0 . 01 ; * * * , P ≤ 0 . 001 .

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(J-L) Representative flow cytometry analysis (J) and quantification of the surface levels of ICOS (K) and CD69 (L) in TFH cells from mice transplanted with CD4+ T cells expressing the indicated proteins. In J, numbers indicate the relative percentage (%) of the cell population that has been interrogated (boxed). In K and L, each point represents the values obtained with a single experimental mouse. n as in H. Data information: data represent the mean ± SEM. Statistical values obtained using the Student's t are given relative to control EGFP+ cells. *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001.


( M ) Abundance of indicated transcripts ( bottom ) in splenic cells from control ( EGFP + ) , tumor - bearing mice ( expressing EGFP and Vav1 ∆ C ) and mice transplanted with cells transduced with the catalytically dead version of Vav1 ∆ C ( expressing EGFP and Vav1 ∆ C + E201A ) . Values are given relative to the expression of each transcript in samples obtained from EGFP controls ( which was given an arbitrary value of 1 ) . n = 4 animals per class analyzed . a . u . , arbitrary unit . Data information : data represent the mean ± SEM . Statistical values obtained using the Student ' s t are given relative to control EGFP + cells . * , 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 B-CONTROLLED_VAR O O O O O O O O O O O O O O O B-CONTROLLED_VAR 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

(M) Abundance of indicated transcripts (bottom) in splenic cells from control (EGFP+), tumor-bearing mice (expressing EGFP and Vav1∆C) and mice transplanted with cells transduced with the catalytically dead version of Vav1∆C (expressing EGFP and Vav1∆C+E201A). Values are given relative to the expression of each transcript in samples obtained from EGFP controls (which was given an arbitrary value of 1). n = 4 animals per class analyzed. a.u., arbitrary unit. Data information: data represent the mean ± SEM. Statistical values obtained using the Student's t are given relative to control EGFP+ cells. *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001.


( C ) Representative immunoblot showing the abundance of the ectopic Vav1 proteins , EGFP and the endogenous tubulin α ( loading control ) in CD4 + T - cells transduced with retrovirus particles used in these experiments ( top ) .

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(C) Representative immunoblot showing the abundance of the ectopic Vav1 proteins, EGFP and the endogenous tubulin α (loading control) in CD4+ T-cells transduced with retrovirus particles used in these experiments (top).


( E , F ) Representative FACS plots ( E ) and quantification ( F ) of the proliferation of EGFP + CD4 + T cells expressing the indicated proteins using the Cell Trace Violet method . In E , the grey shaded histograms represent the fluorescence obtained from nonstimulated CD4 + T cells before stimulation and retroviral transduction . In F , each point represents the values obtained with a single experimental mouse . n = 6 per each experimental condition , except in the case of Vav1 ∆ N ( n = 3 ) . Data information : values are shown as means ± SEM from three independent experiments .

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

(E, F) Representative FACS plots (E) and quantification (F) of the proliferation of EGFP+ CD4+ T cells expressing the indicated proteins using the Cell Trace Violet method. In E, the grey shaded histograms represent the fluorescence obtained from nonstimulated CD4+ T cells before stimulation and retroviral transduction. In F, each point represents the values obtained with a single experimental mouse. n = 6 per each experimental condition, except in the case of Vav1∆N (n = 3). Data information: values are shown as means ± SEM from three independent experiments.


( G - J ) Flow cytometry determination of Tox ( G ) , ICOS ( H ) , CD25 ( I ) and ICN1 ( J ) expression in EGFP + CD4 + T cells expressing the indicated Vav1 proteins . NSt , Non stimulated cells ( CD4 + T cells before stimulation and retroviral transduction ) . In all panels , each point represents the values obtained for a single experimental condition . f . i . , mean fluorescence intensity relative to the isotype - matched control antibody . n = 6 per each experimental condition , except for the Vav1 ∆ N and NSt conditions ( n = 3 ) . Data information : values are shown as means ± SEM from three independent experiments .

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

(G-J) Flow cytometry determination of Tox (G), ICOS (H), CD25 (I) and ICN1 (J) expression in EGFP+ CD4+ T cells expressing the indicated Vav1 proteins. NSt, Non stimulated cells (CD4+ T cells before stimulation and retroviral transduction). In all panels, each point represents the values obtained for a single experimental condition. f.i., mean fluorescence intensity relative to the isotype-matched control antibody. n = 6 per each experimental condition, except for the Vav1∆N and NSt conditions (n = 3). Data information: values are shown as means ± SEM from three independent experiments.


( K - N ) Example of a flow cytometry analysis ( K and M ) and final quantification of p - Akt ( L ) and p - Erk1 / 2 ( N ) levels in EGFP + CD4 + T cells expressing the indicated Vav1 proteins in primed ( blue bars ) and restimulated ( red bars ) cells . In K and M , the grey shaded histograms represent the fluorescence obtained with the isotype - matched control antibody . In L and N , NSt , nonstimulated , St , stimulated . n = 3 per each experimental condition . Data information : values are shown as means ± SEM from three independent experiments . In panels L and N , P values are given relative to nonstimulated ( blue asterisks ) and stimulated ( red asterisks ) cells expressing Vav1WT . We also included P values for the values exhibited by each Vav1 mutant protein relative to those obtained in nonstimulated condition ( black asterisks ) . * , P ≤ 0 . 05 ; * * , P ≤ 0 . 01 ; * * * , P ≤ 0 . 001 ( Student ' s t test ) .

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(K-N) Example of a flow cytometry analysis (K and M) and final quantification of p-Akt (L) and p-Erk1/2 (N) levels in EGFP+ CD4+ T cells expressing the indicated Vav1 proteins in primed (blue bars) and restimulated (red bars) cells. In K and M, the grey shaded histograms represent the fluorescence obtained with the isotype-matched control antibody. In L and N, NSt, nonstimulated, St, stimulated. n = 3 per each experimental condition. Data information: values are shown as means ± SEM from three independent experiments. In panels L and N, P values are given relative to nonstimulated (blue asterisks) and stimulated (red asterisks) cells expressing Vav1WT. We also included P values for the values exhibited by each Vav1 mutant protein relative to those obtained in nonstimulated condition (black asterisks). *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001 (Student's t test).


B ) quantification of the EGFP + CD4 + T cell proliferation in the different experimental groups n = 3 independent experiments . Data information : values are shown as means ± SEM from three independent experiments . P values are given relative to nontreated ( light blue asterisks ) and treated ( dark blue asterisks ) EGFP + cells . We also include P values for the values exhibited by each experimental group relative to those obtained in nontreated condition ( black asterisks ) . * , P ≤ 0 . 05 ; * * , P ≤ 0 . 01 ; * * * , P ≤ 0 . 001 ( Mann - Whitney U test ) .

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B) quantification of the EGFP+ CD4+ T cell proliferation in the different experimental groups n = 3 independent experiments. Data information: values are shown as means ± SEM from three independent experiments. P values are given relative to nontreated (light blue asterisks) and treated (dark blue asterisks) EGFP+ cells. We also include P values for the values exhibited by each experimental group relative to those obtained in nontreated condition (black asterisks). *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001 (Mann-Whitney U test).


( C - E ) Flow cytometry determination of intracellular Tox ( C ) , intracellular ICN1 ( D ) and surface ICOS ( E ) levels in EGFP + CD4 + T cells expressing the indicated Vav1 proteins . f . i . , mean fluorescence intensity relative to the isotype - matched control antibody . n = 3 independent experiments . Data information : values are shown as means ± SEM from three independent experiments . P values are given relative to nontreated ( light blue asterisks ) and treated ( dark blue asterisks ) EGFP + cells . We also include P values for the values exhibited by each experimental group relative to those obtained in nontreated condition ( black asterisks ) . * , P ≤ 0 . 05 ; * * , P ≤ 0 . 01 ; * * * , P ≤ 0 . 001 ( Mann - Whitney U test ) .

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

(C-E) Flow cytometry determination of intracellular Tox (C), intracellular ICN1 (D) and surface ICOS (E) levels in EGFP+ CD4+ T cells expressing the indicated Vav1 proteins. f.i., mean fluorescence intensity relative to the isotype-matched control antibody. n = 3 independent experiments. Data information: values are shown as means ± SEM from three independent experiments. P values are given relative to nontreated (light blue asterisks) and treated (dark blue asterisks) EGFP+ cells. We also include P values for the values exhibited by each experimental group relative to those obtained in nontreated condition (black asterisks). *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001 (Mann-Whitney U test).


( A ) 12 - lead ECG of patient 1 . ( B ) 12 - lead ECG of patient 2 . ( C ) ECG of patient 2 during epinephrine test ( A - represents baseline ECG ; B - ECG at 17 min and 47 s of the beginning of epinephrine infusion ( 0 . 2 μg / kg of epinephrine ) and C - ECG after the end of epinephrine infusion ) .

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(A) 12-lead ECG of patient 1. (B) 12-lead ECG of patient 2. (C) ECG of patient 2 during epinephrine test (A- represents baseline ECG; B- ECG at 17 min and 47 s of the beginning of epinephrine infusion (0.2 μg/kg of epinephrine) and C- ECG after the end of epinephrine infusion).


( E ) ECG of patient 3 ( subject IV : 13 ) showing mild QTc prolongation .

(E) ECG of patient 3 (subject IV:13) showing mild QTc prolongation.


( F ) ICD interrogation of patient 3 ( subject IV : 13 ) reveals an episode of VT .

(F) ICD interrogation of patient 3 (subject IV:13) reveals an episode of VT.


( A - H ) Expression of Tecrl in mouse development . Expression is not observed in ( A ) the cardiac crescent at E7 . 5 but is prominent in ( B ) the inflow tract ( ift ) region at E8 . 5 , particularly in ( C ) the left sinus venosus ( lsv ) . ( D ) At E9 . 5 , Tecrl expression is observed in all four chambers of the heart but is strongest in the inflow tract . ( E - G ) From E10 onwards , Tecrl is expressed in the somites . ( H ) At E14 . 5 , Tecrl expression is present in the entire myocardium ; [ atrium ( a ) , ventricle ( v ) , neural tube ( nt ) , lung ( lu ) , right atrium ( ra ) , left atrium ( la ) , right ventricle ( rv ) and left ventricle ( lv ) ] .

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(A-H) Expression of Tecrl in mouse development. Expression is not observed in (A) the cardiac crescent at E7.5 but is prominent in (B) the inflow tract (ift) region at E8.5, particularly in (C) the left sinus venosus (lsv). (D) At E9.5, Tecrl expression is observed in all four chambers of the heart but is strongest in the inflow tract. (E-G) From E10 onwards, Tecrl is expressed in the somites. (H) At E14.5, Tecrl expression is present in the entire myocardium; [atrium (a), ventricle (v), neural tube (nt), lung (lu), right atrium (ra), left atrium (la), right ventricle (rv) and left ventricle (lv)].


( I ) mRNA expression analyses of TECR and TECRL by RT - qPCR in human tissues demonstrates preferential expression of Tecrl in the heart ; [ Bone marrow ( Bone M . ) , heart , skeletal muscle ( Sk . Muscle ) , uterus , liver , spleen , thymus , thyroid , prostate , brain , lung , small intestine ( Small I . ) and colon ]

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(I) mRNA expression analyses of TECR and TECRL by RT-qPCR in human tissues demonstrates preferential expression of Tecrl in the heart; [Bone marrow (Bone M.), heart, skeletal muscle (Sk.Muscle), uterus, liver, spleen, thymus, thyroid, prostate, brain, lung, small intestine (Small I.) and colon]


( J ) Localization of MYC - Tecrl in COS - 1 cells . The accumulation of MYC - Tecrl is perinuclear consistent with it being localized to the ER .

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(J) Localization of MYC-Tecrl in COS-1 cells. The accumulation of MYC-Tecrl is perinuclear consistent with it being localized to the ER.


( K - M ) Co - localization of MYC - Tecrl and the ER marker Calnexin in H10 cells . Nuclei stained with DAPI . Scale bar : 10 μm .

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(K-M) Co-localization of MYC-Tecrl and the ER marker Calnexin in H10 cells. Nuclei stained with DAPI. Scale bar: 10 μm.


( A ) Skin fibroblasts ( left ) from IV : 13 , homozygous for the TECRLc . 331 + 1G > A mutation were reprogrammed to hiPSCs ( center ) , which expressed the pluripotency markers NANOG and SSEA4 ( right ) . Scale bars : 100 μm .

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(A) Skin fibroblasts(left) from IV:13, homozygous for the TECRLc.331+1G>A mutation were reprogrammed to hiPSCs(center), which expressed the pluripotency markers NANOG and SSEA4(right). Scale bars: 100 μm.


( B ) TECRLHom - hiPSCs have a normal karyotype ( left ) and PluriTest demonstrates a high pluripotency score and low novelty score for all three hiPSC lines ( right ) .

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(B) TECRLHom-hiPSCs have a normal karyotype(left) and PluriTest demonstrates a high pluripotency score and low novelty score for all three hiPSC lines (right).


( C ) TECRLHom - hiPSCs generate derivatives of mesoderm ( left ) , endoderm ( center ) and ectoderm ( right ) . Scale bars : 25

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(C) TECRLHom-hiPSCs generate derivatives of mesoderm(left), endoderm(center) and ectoderm(right). Scale bars: 25 μm


( F ) ACTN2 immunostaining of CTRL - , TECRLHet - and TECRLHom - hiPSC - CMs . Scale bar : 25 μm .

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(F) ACTN2 immunostaining of CTRL-, TECRLHet- and TECRLHom-hiPSC-CMs. Scale bar: 25 μm.


( G ) Analysis of RT - PCR products by gel electrophoresis . Amplification products of TECRL exons 2 - 4 from CTRL - , TECRLHet - and TECRLHom - hiPSC - CMs ( left ) and coding sequence of TECRL from CTRL - and TECRLHom - hiPSC - CMs ( right ) .

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(G) Analysis of RT-PCR products by gel electrophoresis. Amplification products of TECRL exons 2-4 from CTRL-, TECRLHet- and TECRLHom-hiPSC-CMs(left) and coding sequence of TECRL from CTRL- and TECRLHom-hiPSC-CMs(right).


( A ) Representative traces of [ Ca2 + ] i transients in Indo - 1 AM - loaded hiPSC - CMs paced at 1 Hz . ( B ) Time to reach 50 % of [ Ca2 + ] i transient amplitude , t1 / 2 ; [ Ca2 + ] i concentrations during systole and diastole ( C ) Amplitude and tau decay of the [ Ca2 + ] i transient .

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(A) Representative traces of [Ca2+]i transients in Indo-1 AM-loaded hiPSC-CMs paced at 1 Hz. (B) Time to reach 50% of [Ca2+]i transient amplitude, t1/2; [Ca2+]i concentrations during systole and diastole (C) Amplitude and tau decay of the [Ca2+]i transient.


( A ) Representative traces of [ Ca2 + ] i transients in Indo - 1 AM - loaded hiPSC - CMs paced at 1 Hz , in the presence of caffeine ( caff ) or caffeine and NiCl2 . ( B ) Amplitude of [ Ca2 + ] i in the presence of caffeine and NiCl2and fractional SR release in hiPSC - CMs .

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(A) Representative traces of [Ca2+]i transients in Indo-1 AM-loaded hiPSC-CMs paced at 1 Hz, in the presence of caffeine (caff) or caffeine and NiCl2. (B) Amplitude of [Ca2+]i in the presence of caffeine and NiCl2and fractional SR release in hiPSC-CMs.


( C ) Rate constants of SERCA - , NCX - and slow mechanisms - based [ Ca2 + ] i decay in hiPSC - CMs . ( D ) Relative contribution of SERCA , NCX and slow mechanisms to [ Ca2 + ] i extrusion in hiPSC - CMs .

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(C) Rate constants of SERCA-, NCX- and slow mechanisms-based [Ca2+]i decay in hiPSC-CMs. (D) Relative contribution of SERCA, NCX and slow mechanisms to [Ca2+]i extrusion in hiPSC-CMs.


( A ) AP illustrating the analysed parameters ( B ) Representative APs from control ( CTRL ) , heterozygous ( HET ) and homozygous ( HOM ) CMs . ( C ) RMP , dV / dtmax , APAmax , APAplat , APD20 , APD50 and APD90 of CTRL - , TECRLHet - and TECRLHom - hiPSC - CMs .

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(A) AP illustrating the analysed parameters (B) Representative APs from control (CTRL), heterozygous (HET) and homozygous (HOM) CMs. (C) RMP, dV/dtmax , APAmax, APAplat, APD20, APD50 and APD90 of CTRL-, TECRLHet- and TECRLHom-hiPSC-CMs.


( A ) Representative AP traces and ( B ) averaged activity of triggered and spontaneous activity in hiPSC - CMs in the absence of NA . ( C ) Representative AP traces and ( D ) averaged activity of triggered and spontaneous activity in hiPSC - CMs in the presence of NA .

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(A) Representative AP traces and (B) averaged activity of triggered and spontaneous activity in hiPSC-CMs in the absence of NA. (C) Representative AP traces and (D) averaged activity of triggered and spontaneous activity in hiPSC-CMs in the presence of NA.


( A ) Representative AP trace of a TECRLHom - hiPSC - CM in the presence of NA alone or NA and flecainide . ( B ) Effect of flecainide on AP parameters of hiPSC - CMs . ( C - D ) Addition of 5 μM of flecainide decreased the susceptibility to triggered activity in hiPSC - CMs . Note that the effect of flecainide on triggered activity of TECRLHet - and TECRLHom - hiPSC - CMs is more pronounced than its effect on their spontaneous activity .

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(A) Representative AP trace of a TECRLHom-hiPSC-CM in the presence of NA alone or NA and flecainide. (B) Effect of flecainide on AP parameters of hiPSC-CMs. (C-D) Addition of 5 μM of flecainide decreased the susceptibility to triggered activity in hiPSC-CMs. Note that the effect of flecainide on triggered activity of TECRLHet- and TECRLHom-hiPSC-CMs is more pronounced than its effect on their spontaneous activity.


C Relative mRNA expression of Apopt1 normalized to the expression of GAPDH in skeletal muscle and liver of wild - type , heterozygous and knock - out mice of three months of age . Data are presented as mean ± SEM ( n = 6 mice per genotype ) . The asterisks represent the significance levels calculated by two - way ANOVA with Sidak ' s multiple comparisons test : Muscle - * * * P = 0 . 0001 ( WT vs KO ) , * P = 0 . 0310 ( WT vs het ) , * P = 0 . 0395 ( het vs KO ) , Liver - * * P = 0 . 0022 ( WT vs KO ) , * P = 0 . 0101 ( WT vs het )

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C Relative mRNA expression of Apopt1 normalized to the expression of GAPDH in skeletal muscle and liver of wild-type, heterozygous and knock-out mice of three months of age. Data are presented as mean ± SEM (n = 6 mice per genotype). The asterisks represent the significance levels calculated by two-way ANOVA with Sidak's multiple comparisons test: Muscle - ***P = 0.0001 (WT vs KO), *P = 0.0310 (WT vs het), *P = 0.0395 (het vs KO), Liver - **P = 0.0022 (WT vs KO), *P = 0.0101 (WT vs het)


A Body weight measured in female and male animals at 3 , 6 and 12 months of age . Data are presented as mean ± SEM ( n = 5 per group )

A Body weight measured in female and male animals at 3, 6 and 12 months of age. Data are presented as mean ± SEM (n = 5 per group)


Distance run by the tested female and male mice on the treadmill at 3 and 12 months of age . Data are presented as mean ± SEM ( n = 6 per group ) . The asterisks represent the significance levels calculated by two - way ANOVA with Sidak ' s multiple comparisons test : Females - * * * * P < 0 . 0001 ( 3 months ) , * * * P = 0 . 0004 ( 12 months , Males - * * * * P < 0 . 0001 ( 3 months ) , * * P = 0 . 0019 ( 12 months )

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Distance run by the tested female and male mice on the treadmill at 3 and 12 months of age. Data are presented as mean ± SEM (n = 6 per group). The asterisks represent the significance levels calculated by two-way ANOVA with Sidak's multiple comparisons test: Females - ****P < 0.0001 (3 months), ***P = 0.0004 (12 months, Males - ****P < 0.0001 (3 months), **P = 0.0019 (12 months)


Time in seconds spent by the female and male mice on the Rotarod cylinders before falling at different ages . Data are presented as mean ± SEM ( n = 5 per group ) . The asterisks represent the significance levels calculated by two - way ANOVA with Sidak ' s multiple comparisons test : Females - * * P = 0 . 0059 ( 3 months ) , * * P = 0 . 0083 ( 6 months ) , * P = 0 . 0391 ( 12 months ) , Males - * * P = 0 . 0069 ( 12 months )

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Time in seconds spent by the female and male mice on the Rotarod cylinders before falling at different ages. Data are presented as mean ± SEM (n = 5 per group). The asterisks represent the significance levels calculated by two-way ANOVA with Sidak's multiple comparisons test: Females - **P = 0.0059 (3 months), **P = 0.0083 (6 months), *P = 0.0391 (12 months), Males - **P = 0.0069 (12 months)


Number of entries in each arm of the Y maze performed at different ages . Data are presented as mean ± SEM ( n = 10 per group ) . The asterisks represent the significance levels calculated by two - way ANOVA with Sidak ' s multiple comparisons test : * * * * P < 0 .

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Number of entries in each arm of the Y maze performed at different ages. Data are presented as mean ± SEM (n = 10 per group). The asterisks represent the significance levels calculated by two-way ANOVA with Sidak's multiple comparisons test: ****P < 0.0001


E Total spontaneous horizontal and vertical movements of 12 - month old mice measured in an activity cage . Data are presented as mean ± SEM ( n = 6 per group ) . The asterisks represent the significance levels calculated by two - way ANOVA with Sidak ' s multiple comparisons test : * * * * P < 0 . 0001 ( horizontal )

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E Total spontaneous horizontal and vertical movements of 12-month old mice measured in an activity cage. Data are presented as mean ± SEM (n = 6 per group). The asterisks represent the significance levels calculated by two-way ANOVA with Sidak's multiple comparisons test: ****P < 0.0001 (horizontal)


A Representative Hematoxylin & Eosin staining of skeletal muscle in three - month old individuals

A Representative Hematoxylin & Eosin staining of skeletal muscle in three-month old individuals


B Histochemical reaction specific to COX and SDH in skeletal muscle , cerebellar cortex and kidney of the same individuals

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B Histochemical reaction specific to COX and SDH in skeletal muscle, cerebellar cortex and kidney of the same individuals


C COX ( CIV ) enzymatic activity normalized to the activity of citrate synthase ( CS ) in five animals per genotype as indicated at three and twelve months of age . Data are presented as mean ± SEM ( n = 6 per group ) . The asterisks represent the significance levels calculated by two - way ANOVA with Sidak ' s multiple comparisons test : Muscle - * * * * P < 0 . 0001 ( het vs KO ) , * * * P = 0 . 0001 ( WT vs KO ) , * P = 0 . 0185 ( WT vs het ) , Kidney - * * * * P < 0 . 0001 ( het vs KO ) , * * * P = 0 . 0002 ( WT vs KO ) , Heart - * * * P = 0 . 0003 ( het vs KO ) , * * * P = 0 . 0001 ( WT vs KO ) , Brain and Liver - * * * * P < 0 .

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C COX (CIV) enzymatic activity normalized to the activity of citrate synthase (CS) in five animals per genotype as indicated at three and twelve months of age. Data are presented as mean ± SEM (n = 6 per group). The asterisks represent the significance levels calculated by two-way ANOVA with Sidak's multiple comparisons test: Muscle - ****P < 0.0001 (het vs KO), ***P = 0.0001 (WT vs KO), *P = 0.0185 (WT vs het), Kidney - ****P < 0.0001 (het vs KO), ***P = 0.0002 (WT vs KO), Heart - ***P = 0.0003 (het vs KO), ***P = 0.0001 (WT vs KO), Brain and Liver - ****P < 0.0001


D Western blot analysis of SDS - PAGE of total lysates from liver , skeletal muscle and brain from the indicated genotypes , each lane showing the results for one animal . The graph shows the densitometric quantification of the signals obtained in the liver . Data are presented as mean ± SEM ( n = 2 WT ; n = 2 het ; n = 3 KO ) . The asterisks represent the significance levels calculated by two - way ANOVA with Sidak ' s multiple comparisons : * * * P = 0 . 0002 and * * * * P < 0 . 0001 ( WT vs KO )

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D Western blot analysis of SDS-PAGE of total lysates from liver, skeletal muscle and brain from the indicated genotypes, each lane showing the results for one animal. The graph shows the densitometric quantification of the signals obtained in the liver. Data are presented as mean ± SEM (n=2 WT; n=2 het; n=3 KO). The asterisks represent the significance levels calculated by two-way ANOVA with Sidak's multiple comparisons: ***P = 0.0002 and ****P < 0.0001 (WT vs KO)


E Western blot analysis of 1D - BNGE of mitochondria from skeletal muscle from the indicated genotypes , each lane showing the results from one animal

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E Western blot analysis of 1D-BNGE of mitochondria from skeletal muscle from the indicated genotypes, each lane showing the results from one animal


F Western blot analysis of 2D - BNGE of mitochondria from skeletal muscle from one individual of the indicated genotype

F Western blot analysis of 2D-BNGE of mitochondria from skeletal muscle from one individual of the indicated genotype


A Western blot of SDS - PAGE of different fractions from 143B cells expressing APOPT1HA . Tot : total lysate . Cyto : post - mitochondrial fraction ( cytoplasm ) . Mt : isolated mitochondria . Mt sol : Soluble mitochondrial fraction . Mt memb : mitochondrial membranes . CO32 - pellet : Pellet after carbonate extraction with 0 . 1 M Na2CO3 , pH 10 . 5 for 30 minutes . CO32 - sol : soluble fraction after the carbonate extraction

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A Western blot of SDS-PAGE of different fractions from 143B cells expressing APOPT1HA. Tot: total lysate. Cyto: post-mitochondrial fraction (cytoplasm). Mt: isolated mitochondria. Mt sol: Soluble mitochondrial fraction. Mt memb: mitochondrial membranes. CO32- pellet: Pellet after carbonate extraction with 0.1 M Na2CO3, pH 10.5 for 30 minutes. CO32- sol: soluble fraction after the carbonate extraction


B Western blot of SDS - PAGE of mitochondria used for protease protection assay after digitonin treatment . The experiment was carried out in isolated mitochondria from 143B cells expressing APOPT1HA exposed to increasing amounts of digitonin ( expressed in µg ) and 50 µg / ml trypsin . Complete solubilization with 1 % Triton X - 100 was used as a control of protease sensitivity . TOM20 : Translocase of the outer membrane ( OM ) 20 kDa . ACO2 : Aconitase 2 ( mitochondrial isoform ) . AIF : Apoptosis inducing factor . AK2 : Adenylate kinase 2 . OM : outer mitochondrial membrane . IM : inner mitochondrial membrane . IMS : intermembrane space

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B Western blot of SDS-PAGE of mitochondria used for protease protection assay after digitonin treatment. The experiment was carried out in isolated mitochondria from 143B cells expressing APOPT1HA exposed to increasing amounts of digitonin (expressed in µg) and 50 µg/ml trypsin. Complete solubilization with 1% Triton X-100 was used as a control of protease sensitivity. TOM20: Translocase of the outer membrane (OM) 20 kDa. ACO2: Aconitase 2 (mitochondrial isoform). AIF: Apoptosis inducing factor. AK2: Adenylate kinase 2. OM: outer mitochondrial membrane. IM: inner mitochondrial membrane. IMS: intermembrane space


C Western blot of SDS - PAGE of mitochondria used for protease protection assay after hypotonic shock . The experiment was carried out in isolated mitochondria from 143B cells expressing APOPT1HA incubated either in isotonic ( Iso ) or hypotonic buffers for 5 minutes ( Hypo 5 ' ) or 15 minutes ( Hypo 15 ' ) and 50 µg / ml trypsin . Complete solubilization with 1 % Triton X - 100 was used as a control of protease sensitivity

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C Western blot of SDS-PAGE of mitochondria used for protease protection assay after hypotonic shock. The experiment was carried out in isolated mitochondria from 143B cells expressing APOPT1HA incubated either in isotonic (Iso) or hypotonic buffers for 5 minutes (Hypo 5') or 15 minutes (Hypo 15') and 50 µg/ml trypsin. Complete solubilization with 1% Triton X-100 was used as a control of protease sensitivity


D N - SIM super - resolution micrographs showing 0 . 8 μm Maximum Intensity Projection ( 0 . 15 μm for each Z - stack ) of 143B cells expressing APOPT1GFP shown in green , specific markers for each mitochondrial compartment : TOMM20 ( OM ) , COX8A ( IM ) , MitoTracker ( IM + M ) and matrix - target ( mScarlet ) shown in red . Scale bar : 5 μm . The graph shows Pearson ' s Coefficient in colocalized volume of different sub - compartment combinations with APOPT1GFP . Data show mean ± SEM ( n = 5 ) . One - way ANOVA ( * P = 0 . 0298 and * * P = 0 . 0076 ) was employed for the statistical analysis

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D N-SIM super-resolution micrographs showing 0.8 μm Maximum Intensity Projection (0.15 μm for each Z-stack) of 143B cells expressing APOPT1GFP shown in green, specific markers for each mitochondrial compartment: TOMM20 (OM), COX8A (IM), MitoTracker (IM+M) and matrix-target (mScarlet) shown in red. Scale bar: 5 μm. The graph shows Pearson's Coefficient in colocalized volume of different sub-compartment combinations with APOPT1GFP. Data show mean ± SEM (n = 5). One-way ANOVA (*P = 0.0298 and **P = 0.0076) was employed for the statistical analysis


A APOPT1HA was expressed in S6 and S2 immortalized fibroblasts as shown by the Western blot immunovisualization . The expression levels were tested at different days after transduction . The graph on the right shows the activities of CIV normalized to CS in three biological replicates per cell line . Data are presented as mean ± SEM ( n = 3 ) . The asterisks represent the significance levels calculated by two - way ANOVA with Tukey ' s multiple comparisons test : * * P = 0 . 0030 ( S6 naïve vs S6 APOPT1HA ) , * P = 0 . 0165 ( S6 EV vs S6 APOPT1HA )

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A APOPT1HA was expressed in S6 and S2 immortalized fibroblasts as shown by the Western blot immunovisualization. The expression levels were tested at different days after transduction. The graph on the right shows the activities of CIV normalized to CS in three biological replicates per cell line. Data are presented as mean ± SEM (n = 3). The asterisks represent the significance levels calculated by two-way ANOVA with Tukey's multiple comparisons test: **P = 0.0030 (S6 naïve vs S6 APOPT1HA), *P = 0.0165 (S6 EV vs S6 APOPT1HA )


B APOPT1 GFP was expressed in S6 and S2 immortalized fibroblasts as shown by the Western blot immunovisualization . The graph on the right shows the activities of CIV normalized to CS in four biological replicates per cell line . Data are presented as mean ± SEM ( n = 4 ) . The asterisks represent the significance levels calculated by two - way ANOVA with Tukey ' s multiple comparisons test : * * P = 0 . 0066 ( S6 naïve vs S6 APOPT1GFP ) , * * * P = 0 . 0006 ( S6 EV vs S6 APOPT1GFP ) , * P = 0 . 0169 ( S2 naïve vs S2 APOPT1GFP ) , * * * P = 0 . 0008 ( S2 EV vs S2 APOPT1GFP )

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B APOPT1 GFP was expressed in S6 and S2 immortalized fibroblasts as shown by the Western blot immunovisualization. The graph on the right shows the activities of CIV normalized to CS in four biological replicates per cell line. Data are presented as mean ± SEM (n = 4). The asterisks represent the significance levels calculated by two-way ANOVA with Tukey's multiple comparisons test: **P = 0.0066 (S6 naïve vs S6 APOPT1GFP), ***P = 0.0006 (S6 EV vs S6 APOPT1GFP), *P = 0.0169 (S2 naïve vs S2 APOPT1GFP), ***P = 0.0008 (S2 EV vs S2 APOPT1GFP)


C Western blot analysis of 1D - BNGE of mitoplasts extracted from the indicated cell lines . Fully assembled COX migrating at 200 kDa was immunovisualized with antibodies recognizing MT - CO1 and MT - CO2 . The presence of the assembly intermediates ' MITRAC ' and ' S3 ' was also detected ( arrows )

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C Western blot analysis of 1D-BNGE of mitoplasts extracted from the indicated cell lines. Fully assembled COX migrating at 200 kDa was immunovisualized with antibodies recognizing MT-CO1 and MT-CO2. The presence of the assembly intermediates 'MITRAC' and 'S3' was also detected (arrows)


D Western blot analysis of 2D - BNGE of mitoplasts extracted from the indicated cell lines . Fully assembled COX was immunovisualized with antibodies recognizing MT - CO1 and MT - CO2 . The presence of the assembly intermediates ' MITRAC ' and ' S3 ' was also detected ( arrows ) . Anti - GFP immunodetection revealed the presence of the low molecular weight APOPT1GFP complex in the complemented cells expressing the tagged protein

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D Western blot analysis of 2D-BNGE of mitoplasts extracted from the indicated cell lines. Fully assembled COX was immunovisualized with antibodies recognizing MT-CO1 and MT-CO2. The presence of the assembly intermediates 'MITRAC' and 'S3' was also detected (arrows). Anti-GFP immunodetection revealed the presence of the low molecular weight APOPT1GFP complex in the complemented cells expressing the tagged protein


E L - [ 35S ] - Methionine pulse - chase labeling of mtDNA - encoded proteins . After a two - hour exposure with the radioactive label ( pulse ) , cells were cultured in cold medium for the indicated chase times . The graphs show the densitometric quantification of the bands corresponding to MT - CO1 ( left graph ) and MT - CO2 + MT - CO3 ( right graph ) normalized to the ATP6 band over the indicated time points . Graphs represent the values of three biological replicas for each cell line . Data are presented as mean ± SEM ( n = 4 for controls , n = 2 for S2 / S6 / S2 APOPT1GFP / S6 APOPT1GFP ) . The asterisks represent the significance levels calculated by two - way ANOVA with Tukey ' s multiple comparisons test : MT - CO1 - * * P = 0 . 0039 ( 3 . 5 hours , controls vs S6 ) , * P = 0 . 0202 ( 3 . 5 hours , controls vs S2 ) , * P = 0 . 0163 ( 5 hours , S6vs S6 APOPT1GFP ) , * P = 0 . 0118 ( 5 hours , controls vs S2 ) , * * P = 0 . 0011 ( 20 hours , controls vs S6 ) , * P = 0 . 0343 ( 20 hours , S2 vs S2 APOPT1GFP ) , * * * P = 0 . 0007 ( 20 hours , S6 vs S6 APOPT1GFP ) , * * * P = 0 . 0002 ( 20 hours , controls vs S2 ) , * * * * * P < 0 . 0001 ( 20 hours , controls vs S6 ) , MT - CO2 / MT - CO3 - * P = 0 . 0194 ( 3 . 5 hours , S6 vs S6 APOPTGFP ) , * * * P = 0 . 0003 ( 3 . 5 hours , controls vs S6 ) , * P = 0 . 0375 ( 5 hours , S6 vs S6 APOPT1GFP ) , * P = 0 . 0234 ( 20 hours , S2 vs S2 APOPT1GFP ) , * * * * P < 0 . 0001 ( 20 hours , S6 vs S6 APOPT1GFP ) , * * P = 0 . 0013 ( 20 hours , controls vs S2 ) , * * * * P < 0 . 0001 ( 20 hours , controls vs S6 )

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E L-[35S]-Methionine pulse-chase labeling of mtDNA-encoded proteins. After a two-hour exposure with the radioactive label (pulse), cells were cultured in cold medium for the indicated chase times. The graphs show the densitometric quantification of the bands corresponding to MT-CO1 (left graph) and MT-CO2+MT-CO3 (right graph) normalized to the ATP6 band over the indicated time points. Graphs represent the values of three biological replicas for each cell line. Data are presented as mean ± SEM (n = 4 for controls, n = 2 for S2/S6/S2 APOPT1GFP/S6 APOPT1GFP). The asterisks represent the significance levels calculated by two-way ANOVA with Tukey's multiple comparisons test: MT-CO1 - **P = 0.0039 ( 3.5 hours, controls vs S6), *P = 0.0202 (3.5 hours, controls vs S2), *P = 0.0163 (5 hours, S6vs S6 APOPT1GFP), *P = 0.0118 (5 hours, controls vs S2), **P = 0.0011 (20 hours, controls vs S6), *P = 0.0343 (20 hours, S2 vs S2 APOPT1GFP), ***P = 0.0007 (20 hours, S6 vs S6 APOPT1GFP ), ***P = 0.0002 (20 hours, controls vs S2), *****P < 0.0001 (20 hours, controls vs S6), MT-CO2/MT-CO3 - *P = 0.0194 (3.5 hours, S6 vs S6 APOPTGFP), ***P = 0.0003 (3.5 hours, controls vs S6), *P = 0.0375 (5 hours, S6 vs S6 APOPT1GFP), *P = 0.0234 (20 hours, S2 vs S2 APOPT1GFP), ****P < 0.0001 (20 hours, S6 vs S6 APOPT1GFP), **P = 0.0013 (20 hours, controls vs S2), ****P < 0.0001 (20 hours, controls vs S6)


A Western blot analysis of SDS - PAGE of MGM132 - treated 143B APOPT1HA cells . The graph represents the densitometric quantification of the signals for the precursor and mature protein

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A Western blot analysis of SDS-PAGE of MGM132-treated 143B APOPT1HA cells. The graph represents the densitometric quantification of the signals for the precursor and mature protein


B The upper part of the panel ( Input ) shows Western blot analyses of APOPT1HA in the MGM132 - treated cells . Note the appearance of higher molecular weight bands upon longer exposure in the samples treated with the proteasome inhibitor . The bottom part of the panel ( Purified fractions ) shows the analysis of fractions from the same cells immunoprecipitated with anti - HA . Note that the higher molecular weight species are cross - reacting with both anti - HA and anti - ubiquitin

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B The upper part of the panel (Input) shows Western blot analyses of APOPT1HA in the MGM132-treated cells. Note the appearance of higher molecular weight bands upon longer exposure in the samples treated with the proteasome inhibitor. The bottom part of the panel (Purified fractions) shows the analysis of fractions from the same cells immunoprecipitated with anti-HA. Note that the higher molecular weight species are cross-reacting with both anti-HA and anti-ubiquitin


C Western blot analysis of SDS - PAGE of total lysates from 143B cells overexpressing tagged APOPT1 ( as indicated ) and exposed to 100 µM H2O2 , as illustrated by the scheme ( H2O2 treatment ) , for the indicated times . The graphs represent the densitometric quantification of the tagged APOPT1 signal at each time point . The graph inset shows that the increase of APOPT1 occurs in the first minutes after the exposure to H2O2

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C Western blot analysis of SDS-PAGE of total lysates from 143B cells overexpressing tagged APOPT1 (as indicated) and exposed to 100 µM H2O2, as illustrated by the scheme (H2O2 treatment), for the indicated times. The graphs represent the densitometric quantification of the tagged APOPT1 signal at each time point. The graph inset shows that the increase of APOPT1 occurs in the first minutes after the exposure to H2O2


D Western blot analysis of SDS - PAGE of total lysates from 143B cells overexpressing tagged APOPT1 ( as indicated ) and exposed to 5 µM MitoParaquat ( MitoPQ ) , as illustrated by the scheme ( MitoPQ treatment ) , for the indicated times . The graphs represent the densitometric quantification of the tagged APOPT1 signal at each time point . The graph inset shows that the increase of APOPT1 occurs in the first minutes after the exposure to MitoPQ

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D Western blot analysis of SDS-PAGE of total lysates from 143B cells overexpressing tagged APOPT1 (as indicated) and exposed to 5 µM MitoParaquat (MitoPQ), as illustrated by the scheme (MitoPQ treatment), for the indicated times. The graphs represent the densitometric quantification of the tagged APOPT1 signal at each time point. The graph inset shows that the increase of APOPT1 occurs in the first minutes after the exposure to MitoPQ


A Western blot analysis of SDS - PAGE of different mitochondrial proteins in total lysates from the indicated cell lines treated with 5 µM MitoPQ at the indicated times . UT : untreated cells . B Densitometric quantification of APOPT1GFP signal during the treatment in two biological replicas . C Densitometric quantification of MT - CO1 signal in the non - complemented APOPT1 - less cells ( S6 GFP ) vs . the complemented cells ( S6 APOPT1GFP ) . Three biological replicas ( n = 3 ) were carried out for each cell line . The signals in UT S6 APOPT1 were considered 100 % . The levels of MT - CO1 were significantly lower in the S6 GFP patient cells after 20 hours of MitoPQ treatment compared to the untreated cells ( * P = 0 . 0202 , two - tailed unpaired Student ' s t - test ) . D Densitometric quantification of MT - CO2 signal in the non - complemented APOPT1 - less cells ( S6 GFP ) vs . the complemented cells ( S6 APOPT1GFP ) . Three biological replicas were carried out for each cell line . The signals in UT S6 APOPT1 were considered 100 %

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A Western blot analysis of SDS-PAGE of different mitochondrial proteins in total lysates from the indicated cell lines treated with 5 µM MitoPQ at the indicated times. UT: untreated cells. B Densitometric quantification of APOPT1GFP signal during the treatment in two biological replicas. C Densitometric quantification of MT-CO1 signal in the non-complemented APOPT1-less cells (S6 GFP) vs. the complemented cells (S6 APOPT1GFP). Three biological replicas (n=3) were carried out for each cell line. The signals in UT S6 APOPT1 were considered 100%. The levels of MT-CO1 were significantly lower in the S6 GFP patient cells after 20 hours of MitoPQ treatment compared to the untreated cells (*P = 0.0202, two-tailed unpaired Student's t-test). D Densitometric quantification of MT-CO2 signal in the non-complemented APOPT1-less cells (S6 GFP) vs. the complemented cells (S6 APOPT1GFP). Three biological replicas were carried out for each cell line. The signals in UT S6 APOPT1 were considered 100%


( A , B ) Transmission electron micrographs of negatively stained 2F ( A ) and 3F ( B ) fibrils before sonication and injection into mice . The two fibril polymorphs were grown in vitro from synthetic Aβ40 and show the morphological differences originally reported Fibrils are negatively stained with uranyl acetate . Bars represent 100 nm .

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(A, B) Transmission electron micrographs of negatively stained 2F (A) and 3F (B) fibrils before sonication and injection into mice. The two fibril polymorphs were grown in vitro from synthetic Aβ40 and show the morphological differences originally reported Fibrils are negatively stained with uranyl acetate. Bars represent 100 nm.


( C ) PK resistance profile of 2F and 3F fibrils . Values are expressed as the mean densitometric value at each PK concentration ( technical triplicates ) ± standard error . Statistical analyses were performed by student ' s t - test ( * p < 0 . 05 ) .

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(C) PK resistance profile of 2F and 3F fibrils. Values are expressed as the mean densitometric value at each PK concentration (technical triplicates) ± standard error. Statistical analyses were performed by student's t-test (*p<0.05).


D ) Seeding assays using 100 pM of 2F ( blue ) and 3F ( green ) fibrils as seeds as described in Materials and Methods . Curves were generated by measuring ThT emission values at different time points . Reaction kinetics were statistically different as assessed by using the multiple comparisons method to compare pairs of curves ( * * * p < 0 . 0001 ) . Data in graphs is represented as averages depicting standard errors of five technical replicates .

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D) Seeding assays using 100 pM of 2F (blue) and 3F (green) fibrils as seeds as described in Materials and Methods. Curves were generated by measuring ThT emission values at different time points. Reaction kinetics were statistically different as assessed by using the multiple comparisons method to compare pairs of curves (***p<0.0001). Data in graphs is represented as averages depicting standard errors of five technical replicates.


F - I ) 2F and 3F fibrils were tested for their reactivity against a variety of luminescent conjugated thiophenes able to discriminate among conformational variants of misfolded proteins . Stock solutions of LCOs ( 1 . 5 mM ) were diluted in distilled water to 15 μM and added to the wells to a final concentration of 0 . 3 μM . All wells contained 10 μM of either 2F or 3F fibrils . The samples were incubated at 37 ° C and the emission spectrum of each probe was collected after 30 minutes by exciting the samples at 440 nm ( HS - 68 ) or 535 nm ( HS - 194 , HS - 208 and HS - 212 ) .

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F-I) 2F and 3F fibrils were tested for their reactivity against a variety of luminescent conjugated thiophenes able to discriminate among conformational variants of misfolded proteins. Stock solutions of LCOs (1.5 mM) were diluted in distilled water to 15 μM and added to the wells to a final concentration of 0.3 μM. All wells contained 10 μM of either 2F or 3F fibrils. The samples were incubated at 37°C and the emission spectrum of each probe was collected after 30 minutes by exciting the samples at 440 nm (HS-68) or 535 nm (HS-194, HS-208 and HS-212).


( A - H ) Representative images of hippocampi from mice treated with 2F - , 3F - and Tg2576 - derived seeds after visualization for Aβ deposits using the 4G8 antibody ( A - D ) or ThS ( E - H ) . CA , cornu ammonis 1 ; DG , dentate gyrus . Scale bar in ( B ) , ( D ) , ( F ) and ( H ) represents 500 µm and applies to all panels . Arrows represent regions of interest ( alveus , DG ) .

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(A-H) Representative images of hippocampi from mice treated with 2F-, 3F- and Tg2576- derived seeds after visualization for Aβ deposits using the 4G8 antibody (A-D) or ThS (E-H). CA, cornu ammonis 1; DG, dentate gyrus. Scale bar in (B), (D), (F) and (H) represents 500 µm and applies to all panels. Arrows represent regions of interest (alveus, DG).


( I , J ) Aβ ( I ) and ThS ( J ) burden quantification in hippocampus . Statistical analyses were performed using One - way ANOVA ( * p < 0 . 05 , * * p < 0 . 001 ) . Data in graphs is represented as averages depicting standard errors . Measurements considered five tissue slices per animal , and 4 - 7 animals were included per group .

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(I, J) Aβ (I) and ThS (J) burden quantification in hippocampus. Statistical analyses were performed using One-way ANOVA (*p<0.05, **p<0.001). Data in graphs is represented as averages depicting standard errors. Measurements considered five tissue slices per animal, and 4-7 animals were included per group.


( A - H ) Representative pictures of hippocampi from mice treated with different injectate after visualization for micro - and astro - glial associated signals using anti - GFAP ( A - D ) and anti - Iba - 1 ( E - H ) antibodies . Scale bar in ( D ) and ( H ) represents 500 µm and applies to all panels . CA1 - 3 , cornu ammonis 1 - 3 ; DG , dentate gyrus ; so , striatum oriens ; sp , striatum pyramydale ; sr , striatum radiatum .

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(A-H) Representative pictures of hippocampi from mice treated with different injectate after visualization for micro- and astro-glial associated signals using anti-GFAP (A-D) and anti-Iba-1 (E-H) antibodies. Scale bar in (D) and (H) represents 500 µm and applies to all panels. CA1-3, cornu ammonis 1-3; DG, dentate gyrus; so, striatum oriens; sp, striatum pyramydale; sr, striatum radiatum.


( I - N ) Anti - GFAP burden in hippocampus ( I ) , dentate gyrus ( J ) and alveus ( K ) for all animal groups was quantified for image analysis . Similarly , anti - Iba - 1 burdens for all experimental and control groups were calculated in hippocampus ( L ) , dentate gyrus ( M ) and alveus ( N ) . Statistical analyses were performed by One - way ANOVA ( * p < 0 . 05 , * * p < 0 . 001 ) . . Data in graphs is represented as averages depicting standard errors . Measurements considered five tissue slices per animal , and 4 - 7 animals were included per group .

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(I-N) Anti-GFAP burden in hippocampus (I), dentate gyrus (J) and alveus (K) for all animal groups was quantified for image analysis. Similarly, anti-Iba-1 burdens for all experimental and control groups were calculated in hippocampus (L), dentate gyrus (M) and alveus (N). Statistical analyses were performed by One-way ANOVA (*p<0.05, **p<0.001).. Data in graphs is represented as averages depicting standard errors. Measurements considered five tissue slices per animal, and 4-7 animals were included per group.


( O - R ) Correlations between Aβ burden and glial burden were calculated in the alveus for mice injected with 2F ( O ) and 3F ( P ) fibrils . In the same way , the relationship between glial activation and Aβ burden in the hippocampus was calculated for mice treated with 2F ( Q ) and 3F ( R ) aggregates . Statistical analyses were performed by Pearson correlation coefficients test in which p < 0 . 05 was considered significant .

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(O-R) Correlations between Aβ burden and glial burden were calculated in the alveus for mice injected with 2F (O) and 3F (P) fibrils. In the same way, the relationship between glial activation and Aβ burden in the hippocampus was calculated for mice treated with 2F (Q) and 3F (R) aggregates. Statistical analyses were performed by Pearson correlation coefficients test in which p<0.05 was considered significant.


A ) Panels showing representative epifluorescence microscopy images of the dentate gyrus of mice treated with experimental and control materials and immunostained with both 4G8 ( green ) and anti - GFAP ( red ) antibodies . The third column of panels from left to right depict merged images . Right panels are insets obtained from the merged images ( white punctuated squares ) . Scale bars at the left panels represent 200µm and are applicable to the pictures labeled as " 4G8 " , " GFAP " and " Merge " . Scale bars on the right images ( inset ) represent 50µm . g , granular layer ; h , hilar region ; m , molecular

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A) Panels showing representative epifluorescence microscopy images of the dentate gyrus of mice treated with experimental and control materials and immunostained with both 4G8 (green) and anti-GFAP (red) antibodies. The third column of panels from left to right depict merged images. Right panels are insets obtained from the merged images (white punctuated squares). Scale bars at the left panels represent 200µm and are applicable to the pictures labeled as "4G8", "GFAP" and "Merge". Scale bars on the right images (inset) represent 50µm. g, granular layer; h, hilar region; m, molecular layer


B ) A similar analysis as explained in A ) was performed for the alveus . Images were arranged in the same manner as described above . Scale bars at the left panels represent 200µm and are applicable to the pictures labeled as " 4G8 " , " GFAP " and " Merge " . Scale bars on the right images ( inset ) represent 50µm . Alv , alveus ; so , striatum oriens . One tissue slice per animal was used for these analyses .

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B) A similar analysis as explained in A) was performed for the alveus. Images were arranged in the same manner as described above. Scale bars at the left panels represent 200µm and are applicable to the pictures labeled as "4G8", "GFAP" and "Merge". Scale bars on the right images (inset) represent 50µm. Alv, alveus ; so, striatum oriens. One tissue slice per animal was used for these analyses.