Figure 5A . Representative images of brain acute bleedings detected through hemoglobin IHC in P30 Ctrl and Poly I : C MIA male offspring . Scale bar = 500 µm , enlargement scale bar = 50 µm . B . Representative images of brain acute bleedings detected through hemoglobin IHC in P90 Ctrl and Poly I : C MIA male offspring . Scale bar = 500 µm , enlargement scale bar = 50 µm . C . Quantification of hemoglobin ( Hb ) positive signal in the brain of P30 and P90 male MIA offspring , expressed as percentage of the area . P30 Ctrl = 0 . 00 ± 0 . 00 , P30 Poly I : C = 0 . 01 ± 0 . 00 , P90 Ctrl = 0 . 00 ± 0 . 00 , P90 Poly I : C = 0 . 01 ± 0 . 00 . Mann - Whitney U test , * * * p < . 001 . Data information : Ctrl males = white bars with blu border . Poly I : C males = blu bars with blu border . Numbers in bars indicate the number of animals ( N ) and slices ( n ) . Bars represent mean ± SEM . D . Representative 3D rendering of iron deposits detected by DAB - enhanced Perls staining in P30 Ctrl and Poly I : C MIA male offspring brains . Brain volume is rendered gold - transparent with positive signal for the staining color - coded by surface dimension . Scale bar = 100 µm . E . Quantification of DAB - enhanced histochemical iron stains in the brain of P30 Ctrl ( 0 . 00 ± 0 . 00 ) and Poly I : C ( 0 . 01 ± 0 . 00 ) male MIA offspring , expressed as Perls - positive percentage of the volume . Mann - Whitney U test , * p < . 05 . Data information : Ctrl males = white bars with blu border . Poly I : C males = blu bars with blu border . Numbers in bars indicate the number of animals ( N ) and slices ( n ) . Bars represent mean ± SEM . F . Poisson distribution R value of Perls / DAB positive staining of P30 male MIA offspring , respectively in the Coronal ( Ctrl = 1 . 19 ± 0 . 04 , Poly I : C = 0 . 94 ± 0 . 01 ; Mann - Whitney U test , * * p < . 01 ) , Axial ( Ctrl = 1 . 16 ± 0 . 08 , Poly I : C = 1 . 03 ± 0 . 03 ) , and Sagittal ( Ctrl = 1 . 09 ± 0 . 09 , Poly I : C = 1 . 01 ± 0 . 05 ) planes . Data information : Ctrl males = white bars with blu border . Poly I : C males = blu bars with blu border . Numbers in bars indicate the number of animals ( N ) and slices ( n ) . Bars represent mean ± SEM . G . Representative images of iron deposits detected through Perls staining in P90 Ctrl and Poly I : C MIA male offspring brains . Scale bar = 500 µm , enlargement scale bar = 100 µm . H . Quantification of Perls - positive staining in the brain of P90 Ctrl ( 0 . 25 ± 0 . 05 ) and Poly I : C ( 0 . 59 ± 0 . 10 ) male MIA offspring , expressed as percentage of the area . Mann - Whitney U test , * * p < . 01 . Data information : Ctrl males = white bars with blu border . Poly I : C males = blu bars with blu border . Numbers in bars indicate the number of animals ( N ) and slices ( n ) . Bars represent mean ± SEM . I . Representative image of marble burying test at 0 and 30 minutes . 15 glass marbles were evenly spaced on a 5 x 3 grid . Marbles buried by two - thirds at least are indicated by a red circle or by a cross within a red circle when fully covered by the sawdust . J . Quantification of buried marble ( n ° ) by P90 Ctrl Male ( 3 . 33 ± 1 . 67 ) , Poly I : C Male ( 5 . 72 ± 2 . 10 ) , Ctrl Female ( 3 . 83 ± 1 . 72 ) and Poly I : C Female ( 3 . 00 ± 2 . 00 ) mice . Student ' s t test , * * p < . 01 . Data information : Ctrl males = white bars with blu border . Poly I : C males = blu bars with blu border . Ctrl females = white bars with purple border . Poly I : C females = purple bars with purple border . Numbers in bars indicate the number of animals ( N ) and slices ( n ) . Bars represent mean ± SEM . K . Quantification of self - grooming time ( sec ) by P90 Ctrl Males ( 19 . 34 ± 13 . 00 ) , Poly I : C Males ( 43 . 38 ± 35 . 21 ) , Ctrl Females ( 19 . 86 ± 18 . 06 ) and Poly I : C Females ( 16 . 93 ± 11 . 19 ) mice . Student ' s t test , * p < . 05 . Data information : Ctrl males = white bars with blu border . Poly I : C males = blu bars with blu border . Ctrl females = white bars with purple border . Poly I : C females = purple bars with purple border . Numbers in bars indicate the number of animals ( N ) and slices ( n ) . Bars represent mean ± SEM .

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

Figure 5A. Representative images of brain acute bleedings detected through hemoglobin IHC in P30 Ctrl and Poly I:C MIA male offspring. Scale bar = 500 µm, enlargement scale bar = 50 µm. B. Representative images of brain acute bleedings detected through hemoglobin IHC in P90 Ctrl and Poly I:C MIA male offspring. Scale bar = 500 µm, enlargement scale bar = 50 µm. C. Quantification of hemoglobin (Hb) positive signal in the brain of P30 and P90 male MIA offspring, expressed as percentage of the area. P30 Ctrl = 0.00 ± 0.00, P30 Poly I:C = 0.01 ± 0.00, P90 Ctrl = 0.00 ± 0.00, P90 Poly I:C = 0.01 ± 0.00. Mann-Whitney U test, ***p < .001. Data information: Ctrl males = white bars with blu border. Poly I:C males = blu bars with blu border. Numbers in bars indicate the number of animals (N) and slices (n). Bars represent mean ± SEM.D. Representative 3D rendering of iron deposits detected by DAB-enhanced Perls staining in P30 Ctrl and Poly I:C MIA male offspring brains. Brain volume is rendered gold-transparent with positive signal for the staining color-coded by surface dimension. Scale bar = 100 µm.E. Quantification of DAB-enhanced histochemical iron stains in the brain of P30 Ctrl (0.00 ± 0.00) and Poly I:C (0.01 ± 0.00) male MIA offspring, expressed as Perls-positive percentage of the volume. Mann-Whitney U test, *p < .05. Data information: Ctrl males = white bars with blu border. Poly I:C males = blu bars with blu border. Numbers in bars indicate the number of animals (N) and slices (n). Bars represent mean ± SEM.F. Poisson distribution R value of Perls/DAB positive staining of P30 male MIA offspring, respectively in the Coronal (Ctrl = 1.19 ± 0.04, Poly I:C = 0.94 ± 0.01; Mann-Whitney U test, **p < .01), Axial (Ctrl = 1.16 ± 0.08, Poly I:C = 1.03 ± 0.03), and Sagittal (Ctrl = 1.09 ± 0.09, Poly I:C = 1.01 ± 0.05) planes. Data information: Ctrl males = white bars with blu border. Poly I:C males = blu bars with blu border. Numbers in bars indicate the number of animals (N) and slices (n). Bars represent mean ± SEM.G. Representative images of iron deposits detected through Perls staining in P90 Ctrl and Poly I:C MIA male offspring brains. Scale bar = 500 µm, enlargement scale bar = 100 µm. H. Quantification of Perls-positive staining in the brain of P90 Ctrl (0.25 ± 0.05) and Poly I:C (0.59 ± 0.10) male MIA offspring, expressed as percentage of the area. Mann-Whitney U test, **p < .01. Data information: Ctrl males = white bars with blu border. Poly I:C males = blu bars with blu border. Numbers in bars indicate the number of animals (N) and slices (n). Bars represent mean ± SEM.I. Representative image of marble burying test at 0 and 30 minutes. 15 glass marbles were evenly spaced on a 5 x 3 grid. Marbles buried by two-thirds at least are indicated by a red circle or by a cross within a red circle when fully covered by the sawdust. J. Quantification of buried marble (n°) by P90 Ctrl Male (3.33 ± 1.67), Poly I:C Male (5.72 ± 2.10), Ctrl Female (3.83 ± 1.72) and Poly I:C Female (3.00 ± 2.00) mice. Student's t test, **p < .01. Data information: Ctrl males = white bars with blu border. Poly I:C males = blu bars with blu border. Ctrl females = white bars with purple border. Poly I:C females = purple bars with purple border. Numbers in bars indicate the number of animals (N) and slices (n). Bars represent mean ± SEM.K. Quantification of self-grooming time (sec) by P90 Ctrl Males (19.34 ± 13.00), Poly I:C Males (43.38 ± 35.21), Ctrl Females (19.86 ± 18.06) and Poly I:C Females (16.93 ± 11.19) mice. Student's t test, *p < .05. Data information: Ctrl males = white bars with blu border. Poly I:C males = blu bars with blu border. Ctrl females = white bars with purple border. Poly I:C females = purple bars with purple border. Numbers in bars indicate the number of animals (N) and slices (n). Bars represent mean ± SEM.


Figure 1 ( a ) Representative image of a Control ( left ) and Myo1cKO ( right ) embryo . Top : max projections of embryos stained with Phalloidin ( green ) and DAPI ( gray ) . Bottom : magnifications at dashed rectangles of Phalloidin ( grey ) with white arrows pointing at fragments . Data information : Mann - Whitney test p values are indicated . Scale bars , 10 µm . ( b - c ) Number of fragments without DNA ( b ) per embryo and mean radius ( c ) in Control ( grey ) and Myo1cKO ( salmon ) embryos ( left , Control n = 20 , Myo1cKO n = 20 ) . Boxplot shows median , upper and lower quartiles , min and max values . Whisker plot shows median , upper and lower quartiles . Data information : Mann - Whitney test p values are indicated . Scale bars , 10 µm . ( d ) Representative images of a time - lapse of Myo1cKO embryo undergoing fragmentation outside of division ( top ) and during division ( bottom ) . Max projections of embryos expressing mTmG ( grey ) and mCherry - EB3 ( grey ) imaged with light sheet microscopy are shown . White arrows point at the forming fragments . ( e ) Ratio of fragmentation events during and outside of division ( n = 13 cells from 7 embryos ) . Data information : Mann - Whitney test p values are indicated . Scale bars , 10 µm .

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

Figure 1(a) Representative image of a Control (left) and Myo1cKO (right) embryo. Top: max projections of embryos stained with Phalloidin (green) and DAPI (gray). Bottom: magnifications at dashed rectangles of Phalloidin (grey) with white arrows pointing at fragments. Data information: Mann-Whitney test p values are indicated. Scale bars, 10 µm.(b-c) Number of fragments without DNA (b) per embryo and mean radius (c) in Control (grey) and Myo1cKO (salmon) embryos (left, Control n = 20, Myo1cKO n = 20). Boxplot shows median, upper and lower quartiles, min and max values. Whisker plot shows median, upper and lower quartiles. Data information: Mann-Whitney test p values are indicated. Scale bars, 10 µm.(d) Representative images of a time-lapse of Myo1cKO embryo undergoing fragmentation outside of division (top) and during division (bottom). Max projections of embryos expressing mTmG (grey) and mCherry-EB3 (grey) imaged with light sheet microscopy are shown. White arrows point at the forming fragments. (e) Ratio of fragmentation events during and outside of division (n = 13 cells from 7 embryos). Data information: Mann-Whitney test p values are indicated. Scale bars, 10 µm.


Figure 2 ( a ) Representative images of Control ( left ) and Myo1cKO ( right ) embryos expressing mTmG and mCherry - EB3 ( grey ) shown as max projections . White arrows point at the mitotic spindle . ( b - c ) Spindle persistence in Control ( n = 6 ) and Myo1cKO ( n = 9 ) embryos ( b ) and in non - fragmenting and fragmenting cells of Myo1cKO embryos ( c , n = 42 and 12 cells from 7 Myo1cKO embryos ) . Spindle persistence measures the time from appearance to disassembly of the spindle . Data information : Mann - Whitney test p values are indicated . Boxplots show median , upper and lower quartiles , min and max values . Scale bars , 10 µm . ( f ) Representative images of embryos treated with DMSO ( left ) and 37 . 5 µM Ciliobrevin D ( right ) , stained with Phalloidin . Top : Maximum projection . Bottom : magnifications of a single plane at dashed rectangles of Phalloidin with white arrows pointing at fragments . Data information : Mann - Whitney test p values are indicated . Boxplots show median , upper and lower quartiles , min and max values . Scale bars , 10 µm .

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

Figure 2(a) Representative images of Control (left) and Myo1cKO (right) embryos expressing mTmG and mCherry-EB3 (grey) shown as max projections. White arrows point at the mitotic spindle. (b-c) Spindle persistence in Control (n = 6) and Myo1cKO (n = 9) embryos (b) and in non-fragmenting and fragmenting cells of Myo1cKO embryos (c, n = 42 and 12 cells from 7 Myo1cKO embryos). Spindle persistence measures the time from appearance to disassembly of the spindle. Data information: Mann-Whitney test p values are indicated. Boxplots show median, upper and lower quartiles, min and max values. Scale bars, 10 µm.(f) Representative images of embryos treated with DMSO (left) and 37.5 µM Ciliobrevin D (right), stained with Phalloidin. Top: Maximum projection. Bottom: magnifications of a single plane at dashed rectangles of Phalloidin with white arrows pointing at fragments. Data information: Mann-Whitney test p values are indicated. Boxplots show median, upper and lower quartiles, min and max values. Scale bars, 10 µm.


Figure 3 ( d ) Top : Representative images of a time - lapse of a Myo1cKO embryo expressing Myh9 - GFP ( green ) and mTmG ( magenta ) while labelled with SiRDNA ( grey ) undergoing fragmentation during mitosis shown as max projections . Bottom : magnifications of Myh9 - GFP ( grey ) at dashed rectangles showing cortical accumulation ( middle ) followed by fragmentation ( right ) . Data information Boxplots show median , upper and lower quartiles , min and max values . Scale bars , 10 µm . ( e ) Control ( grey ) and Myo1cKO ( salmon ) embryos Myh9 - GFP Ic / Iref values at DNA - cortex distances < 20 µm and > 20 µm ( Control embryos n = 10 , cells n = 35 ; Myo1cKO embryos n = 9 , cells n = 26 ) Data information : Kruskal - Wallis and pairwise Mann - Whitney tests ( e ) p values are indicated . Boxplots show median , upper and lower quartiles , min and max values . Scale bars , 10 µm . ( f ) Comparison of the increase in Myh9 - GFP intensity in Control and Myo1cKO embryos when DNA is in close proximity to the cortex . The ratio Ic0 - 20 / Ic > 20 is calculated from Myh9 - GFP intensities at the contact region when DNA - cortex distances are < 20 µm ( Ic0 - 20 ) divided by intensities when distances are > 20 µm ( Ic > 20 ) ( Control embryos n = 10 , cells n = 33 ; Myo1cKO n = 9 , cells n = 26 ) . Data information : Mann - Whitney test p values are indicated . Boxplots show median , upper and lower quartiles , min and max values . Scale bars , 10 µm .

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

Figure 3(d) Top: Representative images of a time-lapse of a Myo1cKO embryo expressing Myh9-GFP (green) and mTmG (magenta) while labelled with SiRDNA (grey) undergoing fragmentation during mitosis shown as max projections. Bottom: magnifications of Myh9-GFP (grey) at dashed rectangles showing cortical accumulation (middle) followed by fragmentation (right). Data information Boxplots show median, upper and lower quartiles, min and max values. Scale bars, 10 µm.(e) Control (grey) and Myo1cKO (salmon) embryos Myh9-GFP Ic/Iref values at DNA-cortex distances < 20 µm and > 20 µm (Control embryos n = 10, cells n = 35; Myo1cKO embryos n = 9, cells n = 26) Data information: Kruskal-Wallis and pairwise Mann-Whitney tests (e) p values are indicated. Boxplots show median, upper and lower quartiles, min and max values. Scale bars, 10 µm.(f) Comparison of the increase in Myh9-GFP intensity in Control and Myo1cKO embryos when DNA is in close proximity to the cortex. The ratio Ic0-20/Ic>20 is calculated from Myh9-GFP intensities at the contact region when DNA-cortex distances are < 20 µm (Ic0-20) divided by intensities when distances are > 20 µm (Ic>20) (Control embryos n = 10, cells n = 33; Myo1cKO n = 9, cells n = 26). Data information: Mann-Whitney test p values are indicated. Boxplots show median, upper and lower quartiles, min and max values. Scale bars, 10 µm.


Figure 4 ( a ) Representative images of Control ( left ) and Myo1cKO ( right ) embryos labelled with SiRDNA ( grey ) and expressing a reporter of Cdc42 activity ( yellow ) in one blastomere , which is undergoing mitosis , shown as max projections . The second blastomere is outlined with a dashed line . White arrows point at the accumulation of active Cdc42 at the cortex in close proximity to DNA . ( b ) The ratio of active Cdc42 Ic / Iref was compared between < 20 µm and > 20 µm DNA - cortex distances for Control ( dark yellow ) and Myo1cKO ( yellow ) embryos ( Control embryos n = 10 ; Myo1cKO embryos n = 10 ) . Data information : Kruskal - Wallis and pairwise Mann - Whitney tests ( b Boxplots show median , upper and lower quartiles , min and max values . Scale bars , 10 µm . ( c ) Representative images of Myo1cKO embryos expressing LifeAct - GFP ( green ) alone ( left ) or together with dominant negative Cdc42 ( DNCdc42 , right ) labelled with SiRDNA ( grey ) shown as max projections . White arrows point at the cortex in close proximity to DNA . ( d ) LifeAct - GFP Ic / Iref values at DNA - cortex distances < 20 µm and > 20 µm for Myo1cKO ( salmon ) and Myo1cKO + DNCdc42 ( yellow ) embryos ( Myo1cKO embryos n = 8 , cells n = 19 ; Myo1cKO + DNCdc42 embryos n = 12 , cells n = 30 ) . Data information : Kruskal - Wallis and pairwise Mann - Whitney tests d ) Boxplots show median , upper and lower quartiles , min and max values . Scale bars , 10 µm . ( e ) Representative images of a GFP ( left ) and Ect2 - GFP ( right ) expressing embryo stained with Phalloidin ( green ) and DAPI ( grey ) shown as max projections . White arrows indicate fragments . ( f ) Number of fragments without DNA per embryo in GFP ( grey ) and Ect2 - GFP ( blue ) embryos ( GFP n = 14 ; Ect2 - GFP n = 18 ) . Data information Mann - Whitney test ( f ) p values are indicated . Boxplots show median , upper and lower quartiles , min and max values . Scale bars , 10 µm .

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

Figure 4(a) Representative images of Control (left) and Myo1cKO (right) embryos labelled with SiRDNA (grey) and expressing a reporter of Cdc42 activity (yellow) in one blastomere, which is undergoing mitosis, shown as max projections. The second blastomere is outlined with a dashed line. White arrows point at the accumulation of active Cdc42 at the cortex in close proximity to DNA. (b) The ratio of active Cdc42 Ic/Iref was compared between < 20 µm and > 20 µm DNA-cortex distances for Control (dark yellow) and Myo1cKO (yellow) embryos (Control embryos n = 10; Myo1cKO embryos n = 10). Data information: Kruskal-Wallis and pairwise Mann-Whitney tests (b Boxplots show median, upper and lower quartiles, min and max values. Scale bars, 10 µm.(c) Representative images of Myo1cKO embryos expressing LifeAct-GFP (green) alone (left) or together with dominant negative Cdc42 (DNCdc42, right) labelled with SiRDNA (grey) shown as max projections. White arrows point at the cortex in close proximity to DNA. (d) LifeAct-GFP Ic/Iref values at DNA-cortex distances < 20 µm and > 20 µm for Myo1cKO (salmon) and Myo1cKO + DNCdc42 (yellow) embryos (Myo1cKO embryos n = 8, cells n = 19; Myo1cKO + DNCdc42 embryos n = 12, cells n = 30). Data information: Kruskal-Wallis and pairwise Mann-Whitney tests d) Boxplots show median, upper and lower quartiles, min and max values. Scale bars, 10 µm.(e) Representative images of a GFP (left) and Ect2-GFP (right) expressing embryo stained with Phalloidin (green) and DAPI (grey) shown as max projections. White arrows indicate fragments. (f) Number of fragments without DNA per embryo in GFP (grey) and Ect2-GFP (blue) embryos (GFP n = 14; Ect2-GFP n = 18). Data information Mann-Whitney test (f) p values are indicated. Boxplots show median, upper and lower quartiles, min and max values. Scale bars, 10 µm.


Figure 1 ( A ) Total lysates of different mouse organs underwent immunoblotting analysis using anti - Pdia4 and anti - actin antibodies . Data information : Data from 3 experiments are presented as the mean ± SD . ( C ) Min6 cells were treated with glucose and palmitate at the indicated dosages . Total lysate underwent immunoblotting analysis using the indicated antibodies . ( D ) Human islets were treated with glucose and palmitate at the indicated dosages , followed by immunoblotting analysis . ( Data information : Data from 3 experiments are presented as the mean ± SD . ( G ) Sera from wild - type ( WT ) BKS and Leprdb / db mice , aged 4 , 8 , and 18 weeks , B6 mice , fed with a normal diet ( ND ) and a high fat diet ( HFD ) for 4 weeks , and humans , healthy volunteers and diabetic patients ( T2D ) , were quantified for Pdia4 level using an anti - Pdia4 ELISA kit . 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 . ( H ) Immunoblotting analysis of Pdia4 and markers in the cytosolic ( Cyto ) , nuclear ( Nuc ) , membrane ( Mem ) , mitochondrial ( Mito ) , and ER compartments of Ming 6 cells using the indicated antibodies . Data information : Data from 3 experiments are presented as the mean ± SD .

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

Figure 1(A) Total lysates of different mouse organs underwent immunoblotting analysis using anti-Pdia4 and anti-actin antibodies. Data information: Data from 3 experiments are presented as the mean ± SD.(C) Min6 cells were treated with glucose and palmitate at the indicated dosages. Total lysate underwent immunoblotting analysis using the indicated antibodies. (D) Human islets were treated with glucose and palmitate at the indicated dosages, followed by immunoblotting analysis. ( Data information: Data from 3 experiments are presented as the mean ± SD.(G) Sera from wild-type (WT) BKS and Leprdb/db mice, aged 4, 8, and 18 weeks, B6 mice, fed with a normal diet (ND) and a high fat diet (HFD) for 4 weeks, and humans, healthy volunteers and diabetic patients (T2D), were quantified for Pdia4 level using an anti-Pdia4 ELISA kit. 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.(H) Immunoblotting analysis of Pdia4 and markers in the cytosolic (Cyto), nuclear (Nuc), membrane (Mem), mitochondrial (Mito), and ER compartments of Ming 6 cells using the indicated antibodies. Data information: Data from 3 experiments are presented as the mean ± SD.


Figure 2 ( 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 . ( 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2(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.(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.


Figure 3 ( 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3(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.


Figure 4 ( 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 . 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4(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.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.


Figure 5 ( 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5(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.


Figure 6 ( 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6(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.


Figure 7 ( 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 7(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.


Figure 8 ( 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 8(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.


Figure 1 ( 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1(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.


Figure 2 ( 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2(C) 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.


Figure 3 ( 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3(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.


Figure 4 ( 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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Figure 4(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.


Figure 5 ( 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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Figure 5(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.


Figure 6 ( 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6(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.


Figure 3 ( 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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Figure 3(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).


Figure 4 ( 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 . ( 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4(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.(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.


Figure 6 ( 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 . ( 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 ) .

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

Figure 6(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.(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).


Figure 7B ) 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 ) .

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Figure 7B) 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).


Figure 1 ( 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 . ( F ) ICD interrogation of patient 3 ( subject IV : 13 ) reveals an episode of VT .

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Figure 1(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.(F) ICD interrogation of patient 3 (subject IV:13) reveals an episode of VT.


Figure 2 ( 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 ] ( 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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Figure 2(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](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.


Figure 3 ( 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 ) . ( 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 . ( 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 ) .

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

Figure 3(A) 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).(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.(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).


Figure 4 ( 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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Figure 4(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.


Figure 5 ( 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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Figure 5(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.


Figure 6 ( 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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Figure 6(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.


Figure 7 ( 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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Figure 7(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.


Figure 8 ( 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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Figure 8(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.


Figure 1 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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Figure 1 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)


Figure 2 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 ) 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 . 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 )

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

Figure 2 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)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 < 0.0001 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)


Figure 3 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 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 ) 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

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

Figure 3 A 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  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)  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


Figure 4 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 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

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

Figure 4 A 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  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