Figure 5Tracks show Ets1 and Runx1 ChIP - seq data from WT and Batf KO Th17 - polarized cells at the indicated gene loci ( D ; WT , grey ; Batf KO , red ) . Arrows indicate differential Ets1 and Runx1 binding with ( red ) or without ( blue ) the presence of Batf binding comparing WT and Batf KO cells . Ets1 ( E ) or Runx1 ( F ) peaks from WT Th1 , Treg , or both ( Th1 - Treg ) were integrated with enriched Ets1 or Runx1 peaks in Batf KO compared to WT Th17 cells ( Fig . 4C ) to separate peaks into unique and overlapping clusters .

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Figure 5Tracks show Ets1 and Runx1 ChIP-seq data from WT and Batf KO Th17-polarized cells at the indicated gene loci (D; WT, grey; Batf KO, red). Arrows indicate differential Ets1 and Runx1 binding with (red) or without (blue) the presence of Batf binding comparing WT and Batf KO cells. Ets1 (E) or Runx1 (F) peaks from WT Th1, Treg, or both (Th1-Treg) were integrated with enriched Ets1 or Runx1 peaks in Batf KO compared to WT Th17 cells (Fig. 4C) to separate peaks into unique and overlapping clusters.


Figure 6Naïve WT and Batf KO CD4 + CD62Lhi T cells cultured under Th17 - polarizing conditions were treated with or without 1 uM Stat5 inhibitor . On day 5 , cells were harvested and used for RNA extraction to assess gene expression ( A ; data were normalized to WT DMSO ; Foxp3 , unstimulated ; Il17a and Ifng , 6 h 2 ug / ml anti - CD3 restimulation ) Data information : Data are mean ± s . e . m . of 4 independent experiments . ( * p < 0 . 05 ; two - sided t - test or one - way ANOVA followed by Tukey ' s test ) . a . u . , arbitrary unit . Naïve WT and Batf KO CD4 + CD62Lhi T cells cultured under Th17 - polarizing conditions were treated with or without 1 uM Stat5 inhibitor . restimulated with 2 ug / ml anti - CD3 for 24 h to assess cytokine production by ELISA ( B ) . Data information : Data are mean ± s . e . m . of 4 independent experiments . ( * p < 0 . 05 ; two - sided t - test or one - way ANOVA followed by Tukey ' s test ) . a . u . , arbitrary unit . Naïve WT and Batf KO CD4 + CD62Lhi T cells cultured under Th17 - polarizing conditions were treated with or without 1 uM Stat5 inhibitor . WT and Batf KO Th17 - polarized cells were collected on day 5 of culture and used to performed ChIP using antibodies to Stat5 , Batf , Ets1 , Runx1 , Stat3 and p300 ( C ) . Data information : Data are mean ± s . e . m . of 4 independent experiments . ( * p < 0 . 05 ; two - sided t - test or one - way ANOVA followed by Tukey ' s test ) . a . u . , arbitrary unit . Nuclear extracts from WT Th17 cells were incubated with biotinylated oligonucleotides containing Ets1 - Runx1 - Stat5 - specific binding sites at the indicated genomic regions ( wild type or mutant ( ∆ ) as indicated ) . Immunoblots of precipitated proteins ( D ) E , Nuclear extracts from WT Th17 cells were incubated with biotinylated oligonucleotides containing Ets1 - Runx1 - Stat5 - specific binding sites at the indicated genomic regions ( wild type or mutant ( ∆ ) as indicated ) . densitometry measurements ( E ) . Data information : Data are mean ± s . e . m . of 4 independent experiments . ( * p < 0 . 05 ; two - sided t - test or one - way ANOVA followed by Tukey ' s test ) . a . u . , arbitrary unit .

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

Figure 6Naïve WT and Batf KO CD4+CD62Lhi T cells cultured under Th17-polarizing conditions were treated with or without 1 uM Stat5 inhibitor. On day 5, cells were harvested and used for RNA extraction to assess gene expression (A; data were normalized to WT DMSO; Foxp3, unstimulated; Il17a and Ifng, 6 h 2 ug/ml anti-CD3 restimulation) Data information: Data are mean ± s.e.m. of 4 independent experiments. (*p<0.05; two-sided t-test or one-way ANOVA followed by Tukey's test). a.u., arbitrary unit.Naïve WT and Batf KO CD4+CD62Lhi T cells cultured under Th17-polarizing conditions were treated with or without 1 uM Stat5 inhibitor. restimulated with 2 ug/ml anti-CD3 for 24 h to assess cytokine production by ELISA (B). Data information: Data are mean ± s.e.m. of 4 independent experiments. (*p<0.05; two-sided t-test or one-way ANOVA followed by Tukey's test). a.u., arbitrary unit.Naïve WT and Batf KO CD4+CD62Lhi T cells cultured under Th17-polarizing conditions were treated with or without 1 uM Stat5 inhibitor. WT and Batf KO Th17-polarized cells were collected on day 5 of culture and used to performed ChIP using antibodies to Stat5, Batf, Ets1, Runx1, Stat3 and p300 (C). Data information: Data are mean ± s.e.m. of 4 independent experiments. (*p<0.05; two-sided t-test or one-way ANOVA followed by Tukey's test). a.u., arbitrary unit.Nuclear extracts from WT Th17 cells were incubated with biotinylated oligonucleotides containing Ets1-Runx1-Stat5-specific binding sites at the indicated genomic regions (wild type or mutant (∆) as indicated). Immunoblots of precipitated proteins (D)E, Nuclear extracts from WT Th17 cells were incubated with biotinylated oligonucleotides containing Ets1-Runx1-Stat5-specific binding sites at the indicated genomic regions (wild type or mutant (∆) as indicated). densitometry measurements (E). Data information: Data are mean ± s.e.m. of 4 independent experiments. (*p<0.05; two-sided t-test or one-way ANOVA followed by Tukey's test). a.u., arbitrary unit.


Figure 7Naïve CD4 + CD62Lhi T cells from IL - 17a reporter mice ( Il17aeGFP ) were cultured under Th17 polarizing conditions . On day 5 , eGFP + / IL - 17a + cells were sorted ( Th17 ( R1 ) ) and cultured for two additional rounds in the presence of anti - CD3 alone ( TCR ( R3 ) ) or Th17 polarizing cytokines ( Th17 ( R3 ) ) . Ets1 , Runx1 , and Stat5 binding at the indicated genomic regions by ChIP - qPCR ( C ) . Data information : Data are mean ± s . e . m . of 3 independent experiments . ( * p < 0 . 05 ; * * p < 0 . 01 ; one - way ANOVA followed by Tukey ' s test ) .

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

Figure 7Naïve CD4+CD62Lhi T cells from IL-17a reporter mice (Il17aeGFP) were cultured under Th17 polarizing conditions. On day 5, eGFP+/IL-17a+ cells were sorted (Th17(R1)) and cultured for two additional rounds in the presence of anti-CD3 alone (TCR(R3)) or Th17 polarizing cytokines (Th17(R3)). Ets1,Runx1, and Stat5 binding at the indicated genomic regions by ChIP-qPCR (C). Data information: Data are mean ± s.e.m. of 3 independent experiments. (*p<0.05; **p<0.01; one-way ANOVA followed by Tukey's test).


Figure 1Blue native gel electrophoresis ( BNGdataE ) of mouse ( M ) C57BL / 6J ( 111 ) , CD1 ( 113 ) and zebrafish ( ZF ) skeletal muscle digitonin - solubilized mitochondria . Immunodetection of the indicated proteins after BNGEBlue native gel electrophoresis ( BNGdataE ) of mouse ( M ) C57BL / 6J ( 111 ) , CD1 ( 113 ) and zebrafish ( ZF ) skeletal muscle digitonin - solubilized mitochondria . Immunodetection of the indicated proteins after BNGEBlue native gel electrophoresis ( BNGdataE ) of mouse ( M ) C57BL / 6J ( 111 ) , CD1 ( 113 ) and zebrafish ( ZF ) skeletal muscle digitonin - solubilized mitochondria . ( C ) in - gel activity for CI and ( D ) CIV ( shown is a representative gel from two technical and two biological replicates ) . BNGE of whole - body zebrafish digitonin - solubilized mitochondria of scaf1Δ1 / Δ1 ( - / - ) and its respective scaf1 + / + counterpart . Immunodetection of the indicated proteinsBNGE of whole - body zebrafish digitonin - solubilized mitochondria of scaf1Δ1 / Δ1 ( - / - ) and its respective scaf1 + / + counterpart . ( G ) in - gel activity of CI and ( H ) CIV ( representative gel from two technical and three biological replicates ) . I , 2D BNGE / SDS electrophoresis : 1st dimension with digitonin ( Dig ) and 2nd dimension with SDS , followed by immunoblotting with the indicated antibodies to identify the proteins detected by the commercial anti - SCAF1 antibody . Asterisks indicate missing bands in scaf1Δ1 / Δ1 .

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Figure 1Blue native gel electrophoresis (BNGdataE) of mouse (M) C57BL/6J (111), CD1 (113) and zebrafish (ZF) skeletal muscle digitonin-solubilized mitochondria. Immunodetection of the indicated proteins after BNGEBlue native gel electrophoresis (BNGdataE) of mouse (M) C57BL/6J (111), CD1 (113) and zebrafish (ZF) skeletal muscle digitonin-solubilized mitochondria. Immunodetection of the indicated proteins after BNGEBlue native gel electrophoresis (BNGdataE) of mouse (M) C57BL/6J (111), CD1 (113) and zebrafish (ZF) skeletal muscle digitonin-solubilized mitochondria. (C) in-gel activity for CI and (D) CIV (shown is a representative gel from two technical and two biological replicates).BNGE of whole-body zebrafish digitonin-solubilized mitochondria of scaf1Δ1/Δ1 (-/-) and its respective scaf1+/+ counterpart. Immunodetection of the indicated proteinsBNGE of whole-body zebrafish digitonin-solubilized mitochondria of scaf1Δ1/Δ1 (-/-) and its respective scaf1+/+ counterpart. (G) in-gel activity of CI and (H) CIV (representative gel from two technical and three biological replicates).I, 2D BNGE/SDS electrophoresis: 1st dimension with digitonin (Dig) and 2nd dimension with SDS, followed by immunoblotting with the indicated antibodies to identify the proteins detected by the commercial anti-SCAF1 antibody. Asterisks indicate missing bands in scaf1Δ1/Δ1.


Figure 2A , Quantitative data - independent scanning ( DiS ) mass spectrometry protein profiles for CI , CIII and CIV . Vertical numbers indicate the BNGE gel slices . Left and right profiles correspond to scaf1 + / + and scaf1Δ1 / Δ1 animals , respectively . Red heatmap corresponds to the E - score from two proteotypic Scaf1 - derived tryptic peptides spanning sequences ascribed to the CIII interacting site ( in green ) and to the CIV interacting site ( in yellow ) . Thick blue line , marked with an asterisk , indicates the putative proteolytic site in Scaf1 .

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Figure 2A, Quantitative data-independent scanning (DiS) mass spectrometry protein profiles for CI, CIII and CIV. Vertical numbers indicate the BNGE gel slices. Left and right profiles correspond to scaf1+/+ and scaf1Δ1/Δ1 animals, respectively. Red heatmap corresponds to the E-score from two proteotypic Scaf1-derived tryptic peptides spanning sequences ascribed to the CIII interacting site (in green) and to the CIV interacting site (in yellow). Thick blue line, marked with an asterisk, indicates the putative proteolytic site in Scaf1.


Figure 3A , B , Representative images from scaf1 + / + and scaf1 - / - ( A ) female and ( B ) male adult zebrafish . c - f , Size of scaf1Δ1 / Δ1 and scaf1Δ2 / Δ2 ( scaf1 - / - ) fish in comparison with their respective scaf1 + / + lines , ( C ) length and ( E ) weight of females ( Δ1 + / + n = 10 , Δ1 - / - n = 12 , Δ2 + / + n = 24 , Δ2 - / - n = 18 ) ; ( D ) length and ( F ) weight of males ( Δ1 + / + n = 16 , Δ1 - / - n = 13 , Δ2 + / + n = 13 , Δ2 - / - n = 23 ) . G - K , Adipose tissue measurements on hematoxylin - eosin ( H & E ) - stained adult zebrafish sagittal sections . ( G , I ) Adipose tissue area per total section area ( average of 3 sections / biological replicate ) and ( H , J ) adipocyte size ( average of 20 - 30 adipocytes of ventral adipose tissue per biological replicate ) of females ( G , H ) ( Δ1 n = 8 , Δ2 n = 8 , same number of animals for homozygous mutants and controls ) and males ( I , J ) ( Δ1 n = 8 , Δ2 n = 7 , same number of animals for homozygous mutants and controls ) . K , Representative images of ventral fat deposits in females ( dotted lines ) . L - N , Effect of Scaf1 loss of function on female fertility . ( L ) Number of eggs per clutch ( Δ1 + / + n = 12 , Δ1 - / - n = 13 , Δ2 + / + n = 13 , Δ2 - / - n = 10 ) . ( M ) Quantification of mature ovary follicles per ovary section ( average of three sections / biological replicate ) ( Δ1 n = 5 , Δ2 n = 8 ; same number of animals for homozygous scaf1 + / + and scaf1 - / - ) . ( N ) Representative images of H & E - stained ovaries . Dotted lines delineate adipose tissue .

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Figure 3A, B, Representative images from scaf1+/+ and scaf1-/- (A) female and (B) male adult zebrafish.c-f, Size of scaf1Δ1/ Δ1 and scaf1Δ2/ Δ2 (scaf1-/-) fish in comparison with their respective scaf1+/+ lines, (C) length and (E) weight of females (Δ1 +/+ n=10, Δ1 -/- n=12, Δ2 +/+ n=24, Δ2 -/- n=18); (D) length and (F) weight of males (Δ1 +/+ n=16, Δ1 -/- n=13, Δ2 +/+ n=13, Δ2 -/- n=23).G-K, Adipose tissue measurements on hematoxylin-eosin (H&E)-stained adult zebrafish sagittal sections. (G, I) Adipose tissue area per total section area (average of 3 sections/biological replicate) and (H, J) adipocyte size (average of 20-30 adipocytes of ventral adipose tissue per biological replicate) of females (G, H) (Δ1 n=8, Δ2 n=8, same number of animals for homozygous mutants and controls) and males (I, J)(Δ1 n=8, Δ2 n=7, same number of animals for homozygous mutants and controls). K, Representative images of ventral fat deposits in females (dotted lines).L-N, Effect of Scaf1 loss of function on female fertility. (L) Number of eggs per clutch (Δ1 +/+ n=12, Δ1 -/- n=13, Δ2 +/+ n=13, Δ2 -/- n=10). (M) Quantification of mature ovary follicles per ovary section (average of three sections/biological replicate) (Δ1 n=5, Δ2 n=8; same number of animals for homozygous scaf1+/+ and scaf1-/-). (N) Representative images of H&E-stained ovaries. Dotted lines delineate adipose tissue.


Figure 4Transmission electron microscopy image of cardiac muscle from scaf1Δ1 / Δ1 ( n = 3 ) and scaf1 + / + fish ( n = 3 ) . ( A ) Representative images showing mitochondria . Transmission electron microscopy image of cardiac muscle from scaf1Δ1 / Δ1 ( n = 3 ) and scaf1 + / + fish ( n = 3 ) . ( B ) Mitochondria size ( 100 - 150 mitochondria per biological sample ) . ( C ) Cristae lumen width ( average of three cristae per mitochondria , 20 mitochondria per biological sample ) . Different biological replicates are represented with different color tones . D , E , Mitochondrial DNA copy number per nuclear copy number in muscle in females ( D ) and males ( E ) ( Δ1 n = 6 , Δ2 n = 6 and same number for their respective controls ) . F , Survival curve of 4 days post - fertilization embryos treated with different concentrations of the indicated OXPHOS inhibitors ( three experimental replicates per biological replicate and three biological replicates ) . G , H , Oxygen consumption of 48 hours post - fertilization embryos using the XFe24 Seahorse analyzer , ( G ) Representative oxygen consumption rate ( OCR ) profile along time and ( H ) maximum OCR ( Δ1 n = 11 , Δ2 n = 11 , n = 10 , n = 11 respectively for their controls ) . I , Maximum uncoupled ( FCCP ) OCR in isolated mitochondria from adult fish ( male and females Δ1 n = 4 and Δ2 n = 4 , and same number for their respective controls ) with the indicated site I [ pyruvate ( Pyr ) , glutamate ( Glu ) , malate ( Mal ) ] or site II [ succinate ( Succ ) ] substrates . J , K , Respiratory control ratio ( RCR ) ( State 3 / State4 ) ( J ) and P / O ratio ( K ) in isolated mitochondria from adult fish ( male and females Δ1 n = 4 , and same number for their respective controls ) with the indicated substrates .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 O O O O O O O O O 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 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 4Transmission electron microscopy image of cardiac muscle from scaf1Δ1/Δ1 (n=3) and scaf1+/+ fish (n=3). (A) Representative images showing mitochondria.Transmission electron microscopy image of cardiac muscle from scaf1Δ1/Δ1 (n=3) and scaf1+/+ fish (n=3). (B) Mitochondria size (100-150 mitochondria per biological sample). (C) Cristae lumen width (average of three cristae per mitochondria, 20 mitochondria per biological sample). Different biological replicates are represented with different color tones.D, E, Mitochondrial DNA copy number per nuclear copy number in muscle in females (D) and males (E) (Δ1 n=6, Δ2 n=6 and same number for their respective controls).F, Survival curve of 4 days post-fertilization embryos treated with different concentrations of the indicated OXPHOS inhibitors (three experimental replicates per biological replicate and three biological replicates).G, H, Oxygen consumption of 48 hours post-fertilization embryos using the XFe24 Seahorse analyzer, (G) Representative oxygen consumption rate (OCR) profile along time and (H) maximum OCR (Δ1 n=11, Δ2 n=11, n=10, n=11 respectively for their controls).I, Maximum uncoupled (FCCP) OCR in isolated mitochondria from adult fish (male and females Δ1 n=4 and Δ2 n=4, and same number for their respective controls) with the indicated site I [pyruvate (Pyr), glutamate (Glu), malate (Mal)] or site II [succinate (Succ)] substrates.J, K, Respiratory control ratio (RCR)(State 3/State4) (J) and P/O ratio (K) in isolated mitochondria from adult fish (male and females Δ1 n=4, and same number for their respective controls) with the indicated substrates.


Figure 5A , Representative images of scaf1 - / - and scaf1 + / + fish fed with the indicated diets . B , C , Changes in ( B ) length and ( C ) weight over time ( Δ1 + / + n = 10 , Δ1 - / - n = 10 , Δ2 + / + n = 10 , Δ2 - / - n = 12 - 13 . D - F , Adipose tissue measurements on hematoxylin - eosin ( H & E ) - stained adult zebrafish sagittal sections . ( D ) Adipose tissue area per total section area ( average of three sections / biological replicate ) and ( E ) adipocyte size ( average of 20 - 30 adipocytes of ventral adipose tissue per biological replicate ) ( Δ1 + / + n = 3 , Δ2 n = 8 ) . scaf1Δ1 and scaf1Δ2 are represented with circles and squares , respectively . Adipose tissue measurements on hematoxylin - eosin ( H & E ) - stained adult zebrafish sagittal sections . ( F ) Representative images of ventral fat deposits ( dotted lines ) . G , Number of eggs per clutch ( standard diet Δ1 + / + n = 8 , Δ1 - / - n = 8 , Δ2 + / + n = 7 , Δ2 - / - n = 12 , double diet Δ1 + / + n = 7 , Δ1 - / - n = 8 , Δ2 + / + n = 8 , Δ2 - / - n = 9 ) . H , Representative images of H & E - stained ovaries . Black dotted lines outline the ovaries , blue dotted line indicates a mature follicle . I , Quantification of mature ovary follicles per ovary section ( average of three sections / biological sample ) ( Δ1 n = 2 - 3 , Δ2 n = 6 ) .

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

Figure 5A, Representative images of scaf1-/- and scaf1+/+ fish fed with the indicated diets.B, C, Changes in (B) length and (C) weight over time (Δ1 +/+ n=10, Δ1 -/- n=10, Δ2 +/+ n=10, Δ2 -/- n=12-13.D-F, Adipose tissue measurements on hematoxylin-eosin (H&E)-stained adult zebrafish sagittal sections. (D) Adipose tissue area per total section area (average of three sections/biological replicate) and (E) adipocyte size (average of 20-30 adipocytes of ventral adipose tissue per biological replicate) (Δ1 +/+ n=3, Δ2 n=8). scaf1Δ1 and scaf1Δ2 are represented with circles and squares, respectively.Adipose tissue measurements on hematoxylin-eosin (H&E)-stained adult zebrafish sagittal sections. (F) Representative images of ventral fat deposits (dotted lines).G, Number of eggs per clutch (standard diet Δ1 +/+ n=8, Δ1 -/- n=8, Δ2 +/+ n=7, Δ2 -/- n=12, double diet Δ1 +/+ n=7, Δ1 -/- n=8, Δ2 +/+ n=8, Δ2 -/- n=9).H, Representative images of H&E-stained ovaries. Black dotted lines outline the ovaries, blue dotted line indicates a mature follicle. I, Quantification of mature ovary follicles per ovary section (average of three sections/biological sample) (Δ1 n=2-3, Δ2 n=6).


Figure 6A , B , Representative images of scaf1 - / - and scaf1 + / + females ( A ) and males ( B ) fed with the indicated diets . C , D , Length of females ( C ) and males ( D ) after the indicated diets ( Δ1 + / + n = 5 , Δ1 - / - n = 5 , Δ2 + / + n = 5 , Δ2 - / - n = 5 ) . E , F , Weight of females ( E ) and males ( F ) after the indicated diets ( Δ1 + / + n = 5 , Δ1 - / - n = 5 , Δ2 + / + n = 5 , Δ2 - / - n = 5 ) . G , number of eggs per clutch ( standard diet : Δ1 + / + n = 4 , Δ1 - / - n = 3 , Δ2 + / + n = 3 , Δ2 - / - n = 2 , double diet : Δ1 + / + n = 5 , Δ1 - / - , n = 5 Δ2 + / + n = 1 , Δ2 - / - n = 2 ) . H , Quantification of mature ovary follicles per ovary section in haematoxylin - eosin ( H & E ) histological sections ( average of three sections / biological sample ) ( Δ1 n = 3 , Δ2 n = 3 ) . adipose tissue quantification in H & E sections ( Δ1 n = 3 , Δ2 n = 3 ) in females and males : adipose tissue area ( I , J ) adipose tissue quantification in H & E sections ( Δ1 n = 3 , Δ2 n = 3 ) in females and males adipocyte size ( K , L ) .

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Figure 6A, B, Representative images of scaf1-/- and scaf1+/+ females (A) and males (B) fed with the indicated diets.C, D, Length of females (C) and males (D) after the indicated diets (Δ1 +/+ n=5, Δ1 -/- n=5, Δ2 +/+ n=5, Δ2 -/- n=5).E, F, Weight of females (E) and males (F) after the indicated diets (Δ1 +/+ n=5, Δ1 -/- n=5, Δ2 +/+ n=5, Δ2 -/- n=5).G, number of eggs per clutch (standard diet: Δ1 +/+ n=4, Δ1 -/- n=3, Δ2 +/+ n=3, Δ2 -/- n=2, double diet: Δ1 +/+ n=5, Δ1 -/-, n=5 Δ2 +/+ n=1, Δ2 -/- n=2). H, Quantification of mature ovary follicles per ovary section in haematoxylin-eosin (H&E) histological sections (average of three sections/biological sample) (Δ1 n=3, Δ2 n=3). adipose tissue quantification in H&E sections (Δ1 n=3, Δ2 n=3) in females and males : adipose tissue area (I, J)adipose tissue quantification in H&E sections (Δ1 n=3, Δ2 n=3) in females and males adipocyte size (K, L).


Figure 7A , Immunoblot of the indicated proteins of BNGE from female scaf1 + / + and scaf1 - / - whole zebrafish mitochondria for the indicated diet ( representative of n = 2 ) . B , Maximum uncoupled ( FCCP ) oxygen consumption rate in whole zebrafish mitochondria ( females Δ1 n = 4 and Δ2 n = 4 , and same number for their respective controls ) with glutamate ( Glu ) , malate ( Mal ) and succinate ( Succ ) . RNAseq data from scaf1 + / + and scaf1 - / - skeletal muscle for the indicated diet ( standard diet scaf1 + / + and scaf1 - / - n = 4 , double diet scaf1 + / + n = 3 , scaf1 - / - n = 4 ) . ( C - F ) Volcano plots of differentially expressed genes ( DEGs ) . ( C ) Comparison between scaf1 - / - and scaf1 + / + zebrafish in standard diet . ( D ) Comparison between scaf1 - / - and scaf1 + / + zebrafish in double diet . ( E ) Comparison of scaf1 + / + zebrafish in double diet and standard diet . ( F ) Comparison of scaf1 - / - zebrafish in double diet and standard diet . In blue , significant DEGs ( p - adj < 0 . 05 , log2FC > | 1 | ) , in grey not significant DEGs , red circles represent non - significant differentially regulated OXPHOS genes , green circles represent significant differentially regulated OXPHOS genes , purple scaf1 ( cox7a2l ) .

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Figure 7A, Immunoblot of the indicated proteins of BNGE from female scaf1+/+ and scaf1-/- whole zebrafish mitochondria for the indicated diet (representative of n=2).B, Maximum uncoupled (FCCP) oxygen consumption rate in whole zebrafish mitochondria (females Δ1 n=4 and Δ2 n=4, and same number for their respective controls) with glutamate (Glu), malate (Mal) and succinate (Succ).RNAseq data from scaf1+/+ and scaf1-/- skeletal muscle for the indicated diet (standard diet scaf1+/+ and scaf1-/- n=4, double diet scaf1+/+ n=3, scaf1-/- n=4). (C-F) Volcano plots of differentially expressed genes (DEGs). (C) Comparison between scaf1-/- and scaf1+/+ zebrafish in standard diet. (D) Comparison between scaf1-/- and scaf1+/+ zebrafish in double diet. (E) Comparison of scaf1+/+ zebrafish in double diet and standard diet. (F) Comparison of scaf1-/- zebrafish in double diet and standard diet. In blue, significant DEGs (p-adj < 0.05, log2FC > |1|), in grey not significant DEGs, red circles represent non-significant differentially regulated OXPHOS genes, green circles represent significant differentially regulated OXPHOS genes, purple scaf1 (cox7a2l).


Figure 1B . Detection of sFn14 in conditioned media . HEK293E cells were transfected with either empty vector or a plasmid encoding human Fn14 that bears an N - terminal HA - tag and a C - terminal double FLAG - tag . Conditioned media and lysates of the transfected cells were collected and analyzed by immunoblotting with the indicated antibodies . Calnexin served as a loading control . Shown are representative blots from N = 3 experiments . C . Generation of sFn14 is sensitive to the γ - secretase inhibitor DAPT . HEK293E cells were transfected with either Fn14 or C99 ( C - terminal fragment of APP ) , both containing a N - terminal HA - tag and C - terminal double FLAG - tag . One day after transfection , cells were treated with γ - secretase inhibitor DAPT ( 1 μM ) , broad - spectrum metalloprotease inhibitor TAPI - 1 ( 50 μM ) , or the corresponding amount of vehicle DMSO as indicated . The conditioned media and the lysates were blotted with anti - HA antibody . Shown are representative blots from N = 4 experiments . E . In vitro γ - secretase cleavage assay . HEK293E cells were transfected with epitope - tagged Fn14 or C99 . Cellular membranes were collected and incubated under indicated conditions for the γ - secretase activity assay . Reactions were terminated and ultracentrifuged . Supernatant ( containing γ - secretase cleavage products ) was used for detecting the ICD fragment while the pellet was used to blot for full length proteins Fn14 or C99 . Blotting is done by anti - Flag antibody . Shown are representative blots from N = 3 experiments . F . sFn14 production requires the proteolytic presenilin subunit of γ - secretase . HEK293 cells stably transfected with APP carrying the Swedish double mutation ( K595N / M596L ) and with a CRISPR / CAS9 - mediated knockout of presenilin 1 ( PS1 KO ) or of both PS1 and PS2 ( PS1 / 2 dKO ) , were transiently transfected with epitope - tagged Fn14 . Conditioned media and lysates of the transfected cells were blotted with the indicated antibodies . C83 is a small C - terminal fragment of APP ( containing the C - terminal 83 amino acids ) , which is also subjected to γ - secretase processing . Upon longer exposure , the C99 ( generated by BACE1 from APP ) is also visible in the PS1 / 2 dKO condition . Shown are representative blots from N = 4 experiments . G . Quantification of results from panel F . sFn14 levels in the conditioned media quantified and normalized to WT sFn14 signal . Data Information : Panel ( G ) shows mean ± SEM , along with p - values calculated by ordinary one - way ANOVA test with Tukey ' s multiple comparison test .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 1B. Detection of sFn14 in conditioned media. HEK293E cells were transfected with either empty vector or a plasmid encoding human Fn14 that bears an N-terminal HA-tag and a C-terminal double FLAG-tag. Conditioned media and lysates of the transfected cells were collected and analyzed by immunoblotting with the indicated antibodies. Calnexin served as a loading control. Shown are representative blots from N=3 experiments.C. Generation of sFn14 is sensitive to the γ-secretase inhibitor DAPT. HEK293E cells were transfected with either Fn14 or C99 (C-terminal fragment of APP), both containing a N-terminal HA-tag and C-terminal double FLAG-tag. One day after transfection, cells were treated with γ-secretase inhibitor DAPT (1 μM), broad-spectrum metalloprotease inhibitor TAPI-1 (50 μM), or the corresponding amount of vehicle DMSO as indicated. The conditioned media and the lysates were blotted with anti-HA antibody. Shown are representative blots from N=4 experiments.E. In vitro γ-secretase cleavage assay. HEK293E cells were transfected with epitope-tagged Fn14 or C99. Cellular membranes were collected and incubated under indicated conditions for the γ-secretase activity assay. Reactions were terminated and ultracentrifuged. Supernatant (containing γ-secretase cleavage products) was used for detecting the ICD fragment while the pellet was used to blot for full length proteins Fn14 or C99. Blotting is done by anti-Flag antibody. Shown are representative blots from N=3 experiments.F. sFn14 production requires the proteolytic presenilin subunit of γ-secretase. HEK293 cells stably transfected with APP carrying the Swedish double mutation (K595N/M596L) and with a CRISPR/CAS9-mediated knockout of presenilin 1 (PS1 KO) or of both PS1 and PS2 (PS1/2 dKO), were transiently transfected with epitope-tagged Fn14. Conditioned media and lysates of the transfected cells were blotted with the indicated antibodies. C83 is a small C-terminal fragment of APP (containing the C-terminal 83 amino acids), which is also subjected to γ-secretase processing. Upon longer exposure, the C99 (generated by BACE1 from APP) is also visible in the PS1/2 dKO condition. Shown are representative blots from N=4 experiments. G. Quantification of results from panel F. sFn14 levels in the conditioned media quantified and normalized to WT sFn14 signal. Data Information: Panel (G) shows mean ± SEM, along with p-values calculated by ordinary one-way ANOVA test with Tukey's multiple comparison test.


Figure 3A . Human breast cancer cell line MDA - MB - 231 shows cellular accumulation of Fn14 upon γ - secretase inhibition . The cells were treated overnight with γ - secretase inhibitor DAPT ( 1 μM ) , broad spectrum metalloprotease inhibitor TAPI - 1 ( 50 μM ) , or the corresponding amount of vehicle DMSO as indicated . Lysates were blotted for Fn14 with an antibody that targets the C - terminal end of the protein , or against calnexin as loading control . The asterisk labels an N - terminally truncated form of Fn14 . B . Quantification of blots from panel A . The control condition , where the cells were only treated with vehicle ( DMSO ) , was used as baseline , and its average normalized to 1 . Data Information : All quantification data is shown as mean ± SEM . All the panels have N = 3 biological replicates . For panels B the tested conditions were compared against control ( DMSO ) condition by ordinary one - way ANOVA and Dunnett ' s multiple comparison test . The p - values that are above 0 . 05 have not been included into the panels . C . Human ovarian cancer cell line SKOV - 3 shows cellular accumulation of Fn14 upon γ - secretase inhibition . The cells were treated overnight with γ - secretase inhibitor DAPT ( 1 μM ) , broad spectrum metalloprotease inhibitor TAPI - 1 ( 50 μM ) , or corresponding amount of vehicle DMSO as indicated . Lysates were blotted for Fn14 with an antibody that targets the C - terminal end of the protein , or against calnexin as loading control . The asterisk labels an N - terminally truncated form of Fn14 . D . Quantification of blot from panel C . The control condition , where the cells were only treated with vehicle ( DMSO ) , was used as baseline , and its average normalized to 1 . Data Information : All quantification data is shown as mean ± SEM . All the panels have N = 3 biological replicates . For panels D , the tested conditions were compared against control ( DMSO ) condition by ordinary one - way ANOVA and Dunnett ' s multiple comparison test . The p - values that are above 0 . 05 have not been included into the panels .

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Figure 3A. Human breast cancer cell line MDA-MB-231 shows cellular accumulation of Fn14 upon γ-secretase inhibition. The cells were treated overnight with γ-secretase inhibitor DAPT (1 μM), broad spectrum metalloprotease inhibitor TAPI-1 (50 μM), or the corresponding amount of vehicle DMSO as indicated. Lysates were blotted for Fn14 with an antibody that targets the C-terminal end of the protein, or against calnexin as loading control. The asterisk labels an N-terminally truncated form of Fn14. B. Quantification of blots from panel A. The control condition, where the cells were only treated with vehicle (DMSO), was used as baseline, and its average normalized to 1. Data Information: All quantification data is shown as mean ± SEM. All the panels have N=3 biological replicates. For panels B the tested conditions were compared against control (DMSO) condition by ordinary one-way ANOVA and Dunnett's multiple comparison test. The p-values that are above 0.05 have not been included into the panels.C. Human ovarian cancer cell line SKOV-3 shows cellular accumulation of Fn14 upon γ-secretase inhibition. The cells were treated overnight with γ-secretase inhibitor DAPT (1 μM), broad spectrum metalloprotease inhibitor TAPI-1 (50 μM), or corresponding amount of vehicle DMSO as indicated. Lysates were blotted for Fn14 with an antibody that targets the C-terminal end of the protein, or against calnexin as loading control. The asterisk labels an N-terminally truncated form of Fn14. D. Quantification of blot from panel C. The control condition, where the cells were only treated with vehicle (DMSO), was used as baseline, and its average normalized to 1. Data Information: All quantification data is shown as mean ± SEM. All the panels have N=3 biological replicates. For panels D, the tested conditions were compared against control (DMSO) condition by ordinary one-way ANOVA and Dunnett's multiple comparison test. The p-values that are above 0.05 have not been included into the panels.


Figure 4A . MDA - MB - 231 cells were transfected with an siRNA pool against human Fn14 or non - targeting control ( Ctrl ) siRNA . A day after transfection , the cells were treated with γ - secretase inhibitor DAPT ( 1 μM ) or vehicle overnight . The lysate was collected and blotted against Fn14 ( C - terminal antibody ) or calnexin as loading control . Shown are representative blots from N = 4 experiments . B . Quantification of blot in panel A . The control condition where the cells were only treated with vehicle DMSO and non - targeting siRNA ( DMSO + siCtrl ) was used as baseline , and its average normalized to 1 . N = 4 experiments . Data information : All quantification data is shown as mean ± SEM . The tested conditions were compared against control ( DMSO + siCtrl ) condition by ordinary one - way ANOVA and Dunnett ' s multiple comparison test . The p - values that are above 0 . 05 have not been included into the panels .

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Figure 4A. MDA-MB-231 cells were transfected with an siRNA pool against human Fn14 or non-targeting control (Ctrl) siRNA. A day after transfection, the cells were treated with γ-secretase inhibitor DAPT (1 μM) or vehicle overnight. The lysate was collected and blotted against Fn14 (C-terminal antibody) or calnexin as loading control. Shown are representative blots from N=4 experiments. B. Quantification of blot in panel A. The control condition where the cells were only treated with vehicle DMSO and non-targeting siRNA (DMSO + siCtrl) was used as baseline, and its average normalized to 1. N=4 experiments. Data information: All quantification data is shown as mean ± SEM. The tested conditions were compared against control (DMSO + siCtrl) condition by ordinary one-way ANOVA and Dunnett's multiple comparison test. The p-values that are above 0.05 have not been included into the panels.


Figure 6A . Cellular Fn14 in ex vivo glioblastoma samples increased upon γ - secretase inhibition . Primary cells from four different glioblastomas were treated with DAPT ( 1 μM ) or vehicle overnight . Lysates were blotted against Fn14 and calnexin as loading control . B . Quantification of the blot in panel A . For each glioblastoma , the relative ( rel . ) mean intensity of the normalized Fn14 vehicle condition was used for normalization . Data information : All quantification data is shown as mean ± SEM . The tested conditions were compared against their corresponding control ( DMSO ) condition using two - tailed unpaired t - tests . The p - values that are above 0 . 05 have not been included into the panels . For all the panels , three biological replicates were performed .

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Figure 6A. Cellular Fn14 in ex vivo glioblastoma samples increased upon γ-secretase inhibition. Primary cells from four different glioblastomas were treated with DAPT (1 μM) or vehicle overnight. Lysates were blotted against Fn14 and calnexin as loading control. B. Quantification of the blot in panel A. For each glioblastoma, the relative (rel.) mean intensity of the normalized Fn14 vehicle condition was used for normalization. Data information: All quantification data is shown as mean ± SEM. The tested conditions were compared against their corresponding control (DMSO) condition using two-tailed unpaired t-tests. The p-values that are above 0.05 have not been included into the panels. For all the panels, three biological replicates were performed.


Figure 7B . Plasma samples from 10 controls and GBM patients with an initial diagnosis of GBM ( iGBM , 40 patients ) or a recurrent GBM ( rGBM , 20 patients ) were collected and sFn14 levels were measured by ELISA . Data information : For panel B median of the measurements , along with interquartile range borders are displayed . In panel B , ordinary one - way ANOVA with Tukey ' s multiple comparison test was applied , but no value passed our p - value display threshold of 0 . 05 . C . Plasma samples from 10 ovarian cancer patients with low / middle and 19 patients with high tumor burden were collected and sFn14 levels were measured by ELISA . The tumor burden was clinically determined by computer tomographic scans . Data information : For panel C , median of the measurements , along with interquartile range borders are displayed . For panel C , a two - tailed unpaired t - test was applied , but no value passed our p - value display threshold of 0 . 05 . D . Serum samples from 10 patients with refractory multiple myeloma were collected pre - and post - treatment with 25 mg LY3039478 administered in 3 daily doses over 5 days . sFn14 levels in these samples are displayed ( N = 10 ) . Data information : For panel D , ratio - paired t - test was used to compare the sFn14 levels pre - and post - treatment .

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Figure 7B. Plasma samples from 10 controls and GBM patients with an initial diagnosis of GBM (iGBM, 40 patients) or a recurrent GBM (rGBM, 20 patients) were collected and sFn14 levels were measured by ELISA. Data information: For panel B median of the measurements, along with interquartile range borders are displayed. In panel B, ordinary one-way ANOVA with Tukey's multiple comparison test was applied, but no value passed our p-value display threshold of 0.05.C. Plasma samples from 10 ovarian cancer patients with low/middle and 19 patients with high tumor burden were collected and sFn14 levels were measured by ELISA. The tumor burden was clinically determined by computer tomographic scans. Data information: For panel C, median of the measurements, along with interquartile range borders are displayed. For panel C, a two-tailed unpaired t-test was applied, but no value passed our p-value display threshold of 0.05.D. Serum samples from 10 patients with refractory multiple myeloma were collected pre- and post-treatment with 25 mg LY3039478 administered in 3 daily doses over 5 days. sFn14 levels in these samples are displayed (N=10). Data information: For panel D, ratio-paired t-test was used to compare the sFn14 levels pre- and post-treatment.


Figure 1A Representative IF image of NPCs stained for Nestin , FoxG1 and Pank2 . B NPCs differentiated into neurons by overexpressing Ngn2 ( one representative experiment is shown ) . Two weeks after the infection differentiated NPC were positive for neuronal markers βIII - tubulin ( Tuj1 ) , Map2 , NeuN and humannuclei ( hNu ) and synaptic markers , the voltage - gated Na + channels ( PanNav ) and the vesicular glutamate transporter 1 ( VGlut1 ) . C Western blot of soluble cell homogenates from humanneurons probed with PANK2 and β - actin antibodies ( arrows ) . Asterisk indicates nonspecific band . Data are representative of three independent experiments . D Plots showing the total dendritic length and branching points . Data presented as mean and + SEM from at least three independent experiments . A total of 38 neurons were counted for each sample . Statistics were determined by the t - test and resulted not significant . E Representative example of co - culture containing control ( green ) , PKAN ( red ) humanneurons , and E18 cortical miceneurons . Control and PKAN NPCs were infected with GFP - LV and tdT - LV expressing vectors , respectively , and differentiated for 8 weeks . Scale bars 20 μm . F Examples of electrophysiological properties of humanneurons : control individual ( top ) ; PKAN patient ( bottom ) . Traces on the left represent trains of action potentials induced by injection of a suprathreshold current step through the patch electrode in current - clamp mode . Middle traces show Na + and K + currents ( down - and upward - deflecting from baseline , respectively ) in response to a 60 mV step from a holding voltage of - 70 mV in voltage - clamp mode . Insets on the right display enlarged portions of the traces to magnify fast Na + currents . G Histogram with percentages of recorded cells showing repetitively firing ( z - test ) .

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Figure 1A Representative IF image of NPCs stained for Nestin, FoxG1 and Pank2.B NPCs differentiated into neurons by overexpressing Ngn2 (one representative experiment is shown). Two weeks after the infection differentiated NPC were positive for neuronal markers βIII-tubulin (Tuj1), Map2, NeuN and humannuclei (hNu) and synaptic markers, the voltage-gated Na+ channels (PanNav) and the vesicular glutamate transporter 1 (VGlut1).C Western blot of soluble cell homogenates from humanneurons probed with PANK2 and β-actin antibodies (arrows). Asterisk indicates nonspecific band. Data are representative of three independent experiments.D Plots showing the total dendritic length and branching points. Data presented as mean and + SEM from at least three independent experiments. A total of 38 neurons were counted for each sample. Statistics were determined by the t-test and resulted not significant.E Representative example of co-culture containing control (green), PKAN (red) humanneurons, and E18 cortical miceneurons. Control and PKAN NPCs were infected with GFP-LV and tdT-LV expressing vectors, respectively, and differentiated for 8 weeks. Scale bars 20 μm.F Examples of electrophysiological properties of humanneurons: control individual (top); PKAN patient (bottom). Traces on the left represent trains of action potentials induced by injection of a suprathreshold current step through the patch electrode in current-clamp mode. Middle traces show Na+ and K+ currents (down- and upward-deflecting from baseline, respectively) in response to a 60 mV step from a holding voltage of -70 mV in voltage-clamp mode. Insets on the right display enlarged portions of the traces to magnify fast Na+ currents. G Histogram with percentages of recorded cells showing repetitively firing (z-test).


Figure 2A Representative images of humanneurons cells stained with the mitochondrial membrane potential sensible fluorescent probe TMRM , the neuronal specific anti - NCAM antibody and the nuclear staining Hoechst . Left panel , basal conditions . Right panel , after addition of the mitochondrial uncoupler FCCP . Scale bar 20 μm . Plot showing the quantification of TMRM fluorescence signal from NCAM + neurons . Data presented as means + SEM of three independent experiments ( unpaired , two - tailed t - test ) . B Representative images of ultrastructural analysis of fixed neurons examined with electron microscope . PANK2 panel represents PKAN neuronal cells overexpressing PANK2 protein . Scale bar 500nm . Mitochondrial size was measured at level of the larger diameter along the perpendicular axis for all the mitochondria in > 30 field ( 200 mitochondria in total ) for each sample ( one - way ANOVA ) . C OCR measurements of controls and each PKAN patients analyzed individually and D data obtained by individual analysis and plotted as grouped controls , PKAN patients and PKAN patients overexpressing PANK2 . The plots show OCR normalization to cell number . OCR was measured in basal conditions , and after oligomycin and FCCP addition . Bars indicate means + SEM of three independent experiments ( Two - way ANOVA ) .

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

Figure 2A Representative images of humanneurons cells stained with the mitochondrial membrane potential sensible fluorescent probe TMRM, the neuronal specific anti-NCAM antibody and the nuclear staining Hoechst. Left panel, basal conditions. Right panel, after addition of the mitochondrial uncoupler FCCP. Scale bar 20 μm. Plot showing the quantification of TMRM fluorescence signal from NCAM+neurons. Data presented as means + SEM of three independent experiments (unpaired, two-tailed t-test).B Representative images of ultrastructural analysis of fixed neurons examined with electron microscope. PANK2 panel represents PKAN neuronal cells overexpressing PANK2 protein. Scale bar 500nm. Mitochondrial size was measured at level of the larger diameter along the perpendicular axis for all the mitochondria in >30 field (200 mitochondria in total) for each sample (one-way ANOVA).C OCR measurements of controls and each PKAN patients analyzed individually and D data obtained by individual analysis and plotted as grouped controls, PKAN patients and PKAN patients overexpressing PANK2. The plots show OCR normalization to cell number. OCR was measured in basal conditions, and after oligomycin and FCCP addition. Bars indicate means + SEM of three independent experiments (Two-way ANOVA).


Figure 3A Representative images of neurons stained with the neuron specific anti - NCAM antibody , ROS sensible fluorescent probe DCF , and the nuclei dye Hoechst . Scale bar 20 μm . Plots of the DCF fluorescence signal from NCAM positive controls - and PKAN humanneurons , infected or not with Ngn2 - PANK2 - LV . Data presented as means + SEM of at least three independent experiments ( One - way ANOVA ) . B Representative images of neurons stained with ThiolTracker Violet and the anti Tuj1 . Scale bar 20 μm . ThiolTracker Violet fluorescence signal from Tuj1 positive neurons were quantified and shown in the plots . Data presented as means + SEM of at least three independent experiments ( unpaired , two - tailed t - test ) .

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Figure 3A Representative images of neurons stained with the neuron specific anti-NCAM antibody, ROS sensible fluorescent probe DCF, and the nuclei dye Hoechst. Scale bar 20 μm. Plots of the DCF fluorescence signal from NCAM positive controls- and PKAN humanneurons, infected or not with Ngn2-PANK2-LV. Data presented as means + SEM of at least three independent experiments (One-way ANOVA).B Representative images of neurons stained with ThiolTracker Violet and the anti Tuj1. Scale bar 20 μm. ThiolTracker Violet fluorescence signal from Tuj1 positive neurons were quantified and shown in the plots. Data presented as means + SEM of at least three independent experiments (unpaired, two-tailed t-test).


Figure 4A Upper panel : representative images of in - gel enzymatic activity of mitochondrial and cytosolic aconitase ( mAco and cAco , respectively ) . The lower part of the gel was cut , stained with Coomassie blue , and a protein band was used as loading control ( Loading ) . Lower panel : quantification of mAco and cAco enzymatic activity by densitometry . B Upper panel : Western blot analysis of mitochondrial and cytosolic aconitases . Lower panel : quantification of mAco and cAco by densitometry . C Hemequantification by absorbance at 400 nm of the soluble cell lysates . D Upper panel : western blot analysis of transferrin receptor ( TfR1 ) and ferritin ( FtH ) . Lower panel : quantification of TfR1 or FtH normalized on actin by densitometry . All the data are presented as means + SEM of at least three independent experiments ( unpaired , two - tailed t - test ) .

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

Figure 4A Upper panel: representative images of in-gel enzymatic activity of mitochondrial and cytosolic aconitase (mAco and cAco, respectively). The lower part of the gel was cut, stained with Coomassie blue, and a protein band was used as loading control (Loading). Lower panel: quantification of mAco and cAco enzymatic activity by densitometry.B Upper panel: Western blot analysis of mitochondrial and cytosolic aconitases. Lower panel: quantification of mAco and cAco by densitometry.C Hemequantification by absorbance at 400 nm of the soluble cell lysates.D Upper panel: western blot analysis of transferrin receptor (TfR1) and ferritin (FtH). Lower panel: quantification of TfR1 or FtH normalized on actin by densitometry. All the data are presented as means + SEM of at least three independent experiments (unpaired, two-tailed t-test).


Figure 5A Representative images of co - cultures of control and PKAN NPCs infected with GFP - LV and tdT - LV expressing vectors , respectively , at the beginning ( Day 1 ) , after 120 ( Day 120 ) and 150 days of differentiation ( Day 150 ) . Scale bar 20 μm . Plots show the number of green and red humanneurons counted at different time points . All data are presented as means + SEM of at least three independent experiments ( One - way ANOVA ) . B Example of electrophysiological properties of cultured PKAN humanneurons with or without CoA incubation for 30 days . Repetitive firing activity ( left ) and relatively large Na + and K + currents ( right ) , were restored by CoA . The histogram on the right shows fractions of repetitively firing cells recorded in untreated vs . CoA - treated neurons from control and PKAN patients ( z - test ) . C An example of neurons stained with the ROS sensible fluorescent probe DCF and the nuclei dye Hoechst . Anti - NCAM antibody was used to detect neurons . Scale bar 20 μm . Plots of the DCF fluorescence signal from NCAM + controls - and PKAN humanneurons differentiated or not in the presence of CoA ( 25 μM ) in the medium for 3 weeks . All data are presented as means + SEM of at least three independent experiments ( One - way ANOVA ) . D Oxygen consumption rate ( OCR ) with and without CoA on controls and PKAN patients . Basal and uncoupled ( FCCP ) respiration increased after CoA supplementation . Data presented as means + SEM of 24 independent replicates for each condition ( Two - way ANOVA ) . E Heme quantification by absorbance at 400 nm of the soluble NPC cell lysates . All data are presented as means + SEM of at least three independent experiments ( One - way ANOVA ) .

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Figure 5A Representative images of co-cultures of control and PKAN NPCs infected with GFP-LV and tdT-LV expressing vectors, respectively, at the beginning (Day 1), after 120 (Day 120) and 150 days of differentiation (Day 150). Scale bar 20 μm. Plots show the number of green and red humanneurons counted at different time points. All data are presented as means + SEM of at least three independent experiments (One-way ANOVA).B Example of electrophysiological properties of cultured PKAN humanneurons with or without CoA incubation for 30 days. Repetitive firing activity (left) and relatively large Na+ and K+ currents (right), were restored by CoA. The histogram on the right shows fractions of repetitively firing cells recorded in untreated vs. CoA-treated neurons from control and PKAN patients (z-test).C An example of neurons stained with the ROS sensible fluorescent probe DCF and the nuclei dye Hoechst. Anti-NCAM antibody was used to detect neurons. Scale bar 20 μm. Plots of the DCF fluorescence signal from NCAM+ controls- and PKAN humanneurons differentiated or not in the presence of CoA (25 μM) in the medium for 3 weeks. All data are presented as means + SEM of at least three independent experiments (One-way ANOVA).D Oxygen consumption rate (OCR) with and without CoA on controls and PKAN patients. Basal and uncoupled (FCCP) respiration increased after CoA supplementation. Data presented as means + SEM of 24 independent replicates for each condition (Two-way ANOVA).E Heme quantification by absorbance at 400 nm of the soluble NPC cell lysates. All data are presented as means + SEM of at least three independent experiments (One-way ANOVA).


Figure 1 ( A , B ) Immunoblot analysis of the phosphorylation level of CLIP - 170 , AMPK and ACC in heart ( A ) and liver ( B ) along with the developmental stages . α - tubulin was used as a loading control . ( C - E ) Quantitative analysis of western blotting shown in Fig 1A . ( F - H ) Quantitative analysis of western blotting shown in Fig 1B . ( I ) Immunostained images of adult mouse heart tissue . These were stained with a phosphorylated AMPK antibody ( I , upper ) , a phosphorylated CLIP - 170 antibody ( I , lower ) and a plakoglobin antibody ( middle ) . The representative images from more than 5 analyzed tissues were shown .

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Figure 1(A, B) Immunoblot analysis of the phosphorylation level of CLIP-170, AMPK and ACC in heart (A) and liver (B) along with the developmental stages. α-tubulin was used as a loading control. (C-E) Quantitative analysis of western blotting shown in Fig 1A. (F-H) Quantitative analysis of western blotting shown in Fig 1B. (I) Immunostained images of adult mouse heart tissue. These were stained with a phosphorylated AMPK antibody (I, upper), a phosphorylated CLIP-170 antibody (I, lower) and a plakoglobin antibody (middle). The representative images from more than 5 analyzed tissues were shown.


Figure 2 ( A , B ) Immunostained images of neonatal rat cardiomyocytes 2 hours after treatment with 0 . 01 % DMSO ( Control , upper row ) or 2 μM MYK - 461 ( MYK - 461 , middle row ) and 4 hours after washing out of MYK - 461 ( Wash out , bottom row ) . These cells were stained with a phosphorylated AMPK antibody , an AMPKβ2 antibody and a N - cadherin antibody . ( C , D ) Immunostained images of neonatal rat cardiomyocytes 2 hours after treatment with 0 . 01 % DMSO ( Control , upper row ) or 2 μM MYK - 461 ( MYK - 461 , bottom row ) and stained with a connexin43 antibody , a plakoglobin antibody , an LKB - 1 antibody and an N - cadherin antibody . The representative images from 4 independent experiments were shown . ( E ) Quantification of pAMPK , AMPKβ2 , Connexin43 or LKB1 that was localized with a cell - cell junction marker ( N - cadherin or Plakoglobin ) . Box and whisker plots showed the 25th percentile ( bottom line of each box ) , median ( middle line of each box ) , 75th percentile ( top line of each box ) , maximum and minimum ( each whisker ) . Numbers in the brackets indicate the cells that were analyzed for quantification . Differences among multiple groups were compared by one - way ANOVA , followed by a post hoc comparison using the Tukey method . Two - tailed Student ' s t - test was used to analyze differences between two groups . * , P < 0 . 05 * * , P < 0 . 01 . n . s . , not significant .

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Figure 2(A, B) Immunostained images of neonatal rat cardiomyocytes 2 hours after treatment with 0.01% DMSO (Control, upper row) or 2 μM MYK-461(MYK-461, middle row) and 4 hours after washing out of MYK-461 (Wash out, bottom row). These cells were stained with a phosphorylated AMPK antibody, an AMPKβ2 antibody and a N-cadherin antibody.(C, D) Immunostained images of neonatal rat cardiomyocytes 2 hours after treatment with 0.01% DMSO (Control, upper row) or 2 μM MYK-461(MYK-461, bottom row) and stained with a connexin43 antibody, a plakoglobin antibody, an LKB-1 antibody and an N-cadherin antibody. The representative images from 4 independent experiments were shown.(E) Quantification of pAMPK, AMPKβ2, Connexin43 or LKB1 that was localized with a cell-cell junction marker (N-cadherin or Plakoglobin). Box and whisker plots showed the 25th percentile (bottom line of each box), median (middle line of each box), 75th percentile (top line of each box), maximum and minimum (each whisker). Numbers in the brackets indicate the cells that were analyzed for quantification. Differences among multiple groups were compared by one-way ANOVA, followed by a post hoc comparison using the Tukey method. Two-tailed Student's t-test was used to analyze differences between two groups. *, P<0.05 **, P<0.01. n.s., not significant.


Figure 3 ( A ) GFP time - lapse images of neonatal rat cardiomyocytes expressing EGFP - CLIP - 170 WT 0 min and 15 min after treatment with 20 μM Compound C ( left side panel ) and expressing EGFP - CLIP - 170 S311A ( right side panel ) . Higher magniﬁcation of white square ( upper row ) showing CLIP - 170 migrated longitudinally toward the cell - cell junctions . ( B ) Beeswarm plots of comet length of EGFP - CLIP - 170 . The comet length from multiple cells in different fields was analyzed . Number of comets analyzed , Pre : n = 32 , Cpd . C : n = 36 , CLIP S311A : n = 68 . Data means ± S . D . Differences among multiple groups were compared by one - way ANOVA , followed by a post hoc comparison using the Tukey method . * * , P < 0 . 01 versus Pre . ( C ) GFP time - lapse images of neonatal rat cardiomyocytes expressing EGFP - CLIP - 170 WT treated with control siRNA ( siCL , left ) or siRNA targeting both AMPKα1 and α2 ( siAMPKα1α2 , right ) . White dotted lines in the images showed the connected cardiomyocyte not expressing EGFP - CLIP - 170 WT . ( D ) Beeswarm plots of comet length of EGFP - CLIP - 170 . The comet length from multiple cells from different fields was analyzed . Number of comets analyzed , siCL : n = 178 , siAMPKα1α2 : n = 184 . Data means ± S . D . Two - tailed Student ' s t - test was used to analyze differences between two groups . * * , P < 0 . 01 versus siCL .

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

Figure 3(A) GFP time-lapse images of neonatal rat cardiomyocytes expressing EGFP-CLIP-170 WT 0 min and 15 min after treatment with 20 μM Compound C (left side panel) and expressing EGFP-CLIP-170 S311A (right side panel). Higher magniﬁcation of white square (upper row) showing CLIP-170 migrated longitudinally toward the cell-cell junctions.(B) Beeswarm plots of comet length of EGFP-CLIP-170. The comet length from multiple cells in different fields was analyzed. Number of comets analyzed, Pre: n=32, Cpd. C: n=36, CLIP S311A: n=68. Data means ±S.D. Differences among multiple groups were compared by one-way ANOVA, followed by a post hoc comparison using the Tukey method. **, P<0.01 versus Pre.(C) GFP time-lapse images of neonatal rat cardiomyocytes expressing EGFP-CLIP-170 WT treated with control siRNA (siCL, left) or siRNA targeting both AMPKα1 and α2 (siAMPKα1α2, right). White dotted lines in the images showed the connected cardiomyocyte not expressing EGFP-CLIP-170 WT.(D) Beeswarm plots of comet length of EGFP-CLIP-170. The comet length from multiple cells from different fields was analyzed. Number of comets analyzed, siCL: n=178, siAMPKα1α2: n=184. Data means ±S.D. Two-tailed Student's t-test was used to analyze differences between two groups. **, P<0.01 versus siCL.


Figure 4 ( A ) GFP time - lapse images of neonatal rat cardiomyocytes expressing EGFP - CLIP - 170 WT 0 , 2 and 3 hours after treatment with 2 μM MYK - 461 . ( B ) Immunostained images of neonatal rat cardiomyocytes 4 hours after treatment with 0 . 01 % DMSO ( Control , upper row ) or 2 μM MYK - 461 ( MYK - 461 , bottom row ) . These cells were stained with an α - tubulin ( green ) and a plakoglobin ( red ) antibody . ( C ) Box and whisker plots of the cell size of a cardiomyocyte 2 hours after treatment with or without 2 μM MYK - 461 , showing the 25th percentile ( bottom line of each box ) , median ( middle line of each box ) , 75th percentile ( top line of each box ) , maximum and minimum ( each whisker ) . Adenovirus expressing EGFP - CLIP - 170 ( adCLIP ) mutant was used ( WT , S311A , S311D ) . Numbers in the graph indicate n number of the cells from 2 independent experiments . Control ( - / - ) : n = 784 , MYK - 461 : n = 939 , CLIP WT : n = 656 , CLIP WT + MYK - 461 : n = 619 , CLIP S311A : n = 405 , CLIP S311A + MYK - 461 : n = 635 , CLIP S311D : n = 613 , CLIP S311D + MYK - 461 : n = 735 . Data means ± S . D . Differences among multiple groups were compared by one - way ANOVA , followed by a post hoc comparison using the Tukey method . * * , P < 0 . 01 versus Control , † † , P < 0 . 01 versus CLIP WT , n . s . , not 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) GFP time-lapse images of neonatal rat cardiomyocytes expressing EGFP-CLIP-170 WT 0, 2 and 3 hours after treatment with 2 μM MYK-461.(B) Immunostained images of neonatal rat cardiomyocytes 4 hours after treatment with 0.01% DMSO (Control, upper row) or 2 μM MYK-461(MYK-461, bottom row). These cells were stained with an α-tubulin (green) and a plakoglobin (red) antibody.(C) Box and whisker plots of the cell size of a cardiomyocyte 2 hours after treatment with or without 2 μM MYK-461, showing the 25th percentile (bottom line of each box), median (middle line of each box), 75th percentile (top line of each box), maximum and minimum (each whisker). Adenovirus expressing EGFP-CLIP-170 (adCLIP) mutant was used (WT, S311A, S311D). Numbers in the graph indicate n number of the cells from 2 independent experiments. Control (-/-): n=784, MYK-461: n=939, CLIP WT: n=656, CLIP WT + MYK-461: n=619, CLIP S311A: n=405, CLIP S311A + MYK-461: n=635, CLIP S311D: n=613, CLIP S311D + MYK-461: n=735. Data means ±S.D. Differences among multiple groups were compared by one-way ANOVA, followed by a post hoc comparison using the Tukey method. **, P<0.01 versus Control, ††, P<0.01 versus CLIP WT, n.s., not significant.


Figure 5 ( A ) Beeswarm plots of the echocardiographic parameter ( ejection fraction ) of individual Cre control ( CLIP - 170 S311A + / + ; α - MHC - MerCreMer + / − ) , Control mice ( CLIP - 170 S311A flox / + ; α - MHC - MerCreMer − / − ) , and CLIP - 170 S311A overexpressing mice ( CLIP - 170 S311A flox / + ; α - MHC - MerCreMer + / − ) , before ( Pre ) , 2 weeks ( Tx2w ) , 8 weeks ( Tx8w ) , 26 weeks ( Tx26w ) and over 1 year ( Tx1y ) after tamoxifen induction . Number of mice , ( Cre control , Control , S311A ) , Pre ( 6 , 2 , 3 ) , Tx 2w ( 9 , 5 , 5 ) , Tx 8w ( 9 , 10 , 14 ) , Tx1 2w ( 7 , 10 , 14 ) , Tx 26w ( 3 , 7 , 8 ) , Tx 52w ( 3 , 7 , 9 ) . ( B ) Beeswarm plots of another line of CLIP - 170 S311A overexpressing mice ( line 9 ) . Number of mice , ( Control , S311A ) , Pre ( 7 , 3 ) , Tx 2w ( 4 , 7 ) , Tx 8w ( 6 , 4 ) , Tx 12w ( 5 , 4 ) , Tx 52w ( 3 , 3 ) . Data means ± S . D . Statistical significance was determined by two - way ANOVA followed by post hoc Tukey ' s multiple comparison test . # # : p < 0 . 01 vs Duration - matched Cre Control , * : p < 0 . 05 , * * : p < 0 . 01 vs Duration - matched Control ; † : p < 0 . 05 , † † : p < 0 . 01 , S311A TG mice 12 weeks vs 52 weeks . ( C ) Representative long axis four - chamber cardiac magnetic resonance images of CLIP - 170 S311A TG mice and control mice over 1 year after tamoxifen induction . Left column showed systole images and right column represented diastole images . Three mice from each group were analyzed for cardiac MRI . The representative images were shown . ( D ) Beeswarm plots showed the ejection fraction from cardiac MRI . LV , left ventricular , RV , right ventricular . Number of mice , ( Control , S311A ) , 1y ( 3 , 3 ) . Data means ± S . D . Two - tailed Student ' s t - test was used to analyze differences between two groups . * * , P < 0 . 01 versus Control . ( E ) Beeswarm plots of the echocardiographic parameter ( ejection fraction ) from CLIP - 170 S311D overexpressing mice and the control . Number of mice , ( Control , S311D ) , Tx 2w ( 3 , 4 ) , Tx 8w ( 5 , 11 ) , Tx 26w ( 4 , 11 ) . Data means ± S . D . There was no significant difference .

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 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 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

Figure 5(A) Beeswarm plots of the echocardiographic parameter (ejection fraction) of individual Cre control (CLIP-170 S311A +/+; α-MHC-MerCreMer+/−), Control mice (CLIP-170 S311A flox/+; α-MHC-MerCreMer−/−), and CLIP-170 S311A overexpressing mice (CLIP-170 S311A flox/+; α-MHC-MerCreMer+/−), before (Pre), 2 weeks (Tx2w), 8 weeks (Tx8w), 26 weeks (Tx26w) and over 1 year (Tx1y) after tamoxifen induction. Number of mice, (Cre control, Control, S311A), Pre (6, 2, 3), Tx 2w (9, 5, 5), Tx 8w (9, 10, 14), Tx1 2w (7, 10, 14), Tx 26w (3, 7, 8), Tx 52w (3, 7, 9).(B) Beeswarm plots of another line of CLIP-170 S311A overexpressing mice (line 9). Number of mice, (Control, S311A), Pre (7, 3), Tx 2w (4, 7), Tx 8w (6, 4), Tx 12w (5, 4), Tx 52w (3, 3). Data means ±S.D. Statistical significance was determined by two-way ANOVA followed by post hoc Tukey's multiple comparison test. ##: p < 0.01 vs Duration-matched Cre Control, *: p < 0.05, **: p < 0.01 vs Duration-matched Control; †: p < 0.05, ††: p < 0.01, S311A TG mice 12 weeks vs 52 weeks.(C) Representative long axis four-chamber cardiac magnetic resonance images of CLIP-170 S311A TG mice and control mice over 1 year after tamoxifen induction. Left column showed systole images and right column represented diastole images. Three mice from each group were analyzed for cardiac MRI. The representative images were shown.(D) Beeswarm plots showed the ejection fraction from cardiac MRI. LV, left ventricular, RV, right ventricular. Number of mice, (Control, S311A), 1y (3, 3). Data means ±S.D. Two-tailed Student's t-test was used to analyze differences between two groups. **, P<0.01 versus Control.(E) Beeswarm plots of the echocardiographic parameter (ejection fraction) from CLIP-170 S311D overexpressing mice and the control. Number of mice, (Control, S311D), Tx 2w (3, 4), Tx 8w (5, 11), Tx 26w (4, 11). Data means ±S.D. There was no significant difference.


Figure 6 ( A ) Masson ' s trichrome staining of the heart of CLIP - 170 S311A overexpressing mice and the control mice from 3 months and 1 year after tamoxifen treatment . ( B ) Quantitative analysis of cardiac fibrosis . Quantification was made as percentage of fibrosis area ( in blue ) in the left ventricle . Number of mice , ( Control , S311A ) , 3m ( 7 , 6 ) , 1y ( 5 , 6 ) . * , P < 0 . 05 versus Control . ( C ) Representative immunostained images with an α - tubulin antibody of CLIP - 170 S311A overexpressing mice heart and control mice heart over 1 year after tamoxifen treatment . ( D ) Quantitative analysis of intensity of α - tubulin in ( C ) . Number of mice , ( Control : n = 7 , S311A : n = 8 ) . Average of 5 sections per animal were analyzed . * * , P < 0 . 01 versus Control . ( E ) Representative images of western blot for an α - tubulin antibody . GAPDH was used as a loading control . ( F ) Quantitative analysis of intensity of α - tubulin in ( E ) . Number of mice , ( Control : n = 4 , S311A : n = 5 ) . * , P < 0 . 05 versus Control . ( G ) Representative immunostained images of CLIP - 170 S311A overexpressing mice heart and the control mice heart over 1 year after tamoxifen treatment . These were stained with WGA ( red ) and a plakoglobin antibody ( green ) . ( H ) Quantitative analysis of ( G ) Cell width , length and aspect ratio ( length / width ) were measured . Number of cells analyzed , Control , n = 251 , S311A , n = 310 , * * , P < 0 . 01 versus Control .

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

Figure 6(A) Masson's trichrome staining of the heart of CLIP-170 S311A overexpressing mice and the control mice from 3 months and 1 year after tamoxifen treatment.(B) Quantitative analysis of cardiac fibrosis. Quantification was made as percentage of fibrosis area (in blue) in the left ventricle. Number of mice, (Control, S311A), 3m (7, 6), 1y (5, 6). *, P<0.05 versus Control.(C) Representative immunostained images with an α-tubulin antibody of CLIP-170 S311A overexpressing mice heart and control mice heart over 1 year after tamoxifen treatment. (D) Quantitative analysis of intensity of α-tubulin in (C). Number of mice, (Control: n=7, S311A: n=8). Average of 5 sections per animal were analyzed. **, P<0.01 versus Control. (E) Representative images of western blot for an α-tubulin antibody. GAPDH was used as a loading control. (F) Quantitative analysis of intensity of α-tubulin in (E). Number of mice, (Control: n=4, S311A: n=5). *, P<0.05 versus Control. (G) Representative immunostained images of CLIP-170 S311A overexpressing mice heart and the control mice heart over 1 year after tamoxifen treatment. These were stained with WGA (red) and a plakoglobin antibody (green).(H) Quantitative analysis of (G) Cell width, length and aspect ratio (length/width) were measured. Number of cells analyzed, Control, n=251, S311A, n=310, **, P<0.01 versus Control.


Figure 1Verification of PTPδ - tdTomato reporter mice by Western blot analysis of whole - brain lysates from heterozygote ( HT ) and homozygous ( Ho ) mice ( P21 ) and PTPδ using mCherry antibodies . Note that levels of endogenous PTPδ protein ( ~ 85 kDa ) are decreased , and that PTPδ - tdTomato protein ( ~ 140 kDa ) is detectable only in reporter mice ( HT and Ho ) . Note also that the 85 - kDa band represents the transmembrane domain + cytoplasmic fragment of the full - length PTPδ protein that undergoes proteolytic cleavage at extracellular membrane - proximal sites , which can only be recognized by the PTPδ C - terminal antibody that targets the cytoplasmic region . The PTPδ - tdTomato protein ( ~ 140 kDa ) contains the membrane domain + cytoplasmic fragment of PTPδ ( ~ 85 kDa ) fused to tdTomato ( 55 kDa ) . The N - terminal antibody recognizes equal amounts of the C - terminal cleavage product derived from PTPδ and PTPδ - tdTomato proteins . Representative coronal , horizontal , and sagittal sections from PTPδ - tdTomato mice . Enlarged windows are demarcated by black dotted lines ( Fig EV1A - C ) . Red tdTomato images were converted to grayscale images for clarity . ACA , anterior cingulate area ; ACB , nucleus accumbens ; BLA , basolateral amygdala ; BMA , basomedial amygdala ; CA1 , cornu ammonis area 1 ; CA2 , cornu ammonis area 2 ; CA3 , cornu ammonis area 3 ; CB , cerebellum ; CC , corpus callosum ; CEA , central amygdala ; CP , caudate putamen ; CST , corticospinal tract ; DG , dentate gyrus ; EP , endopiriform nucleus , HY , hypothalamus ; L1 , cortical layer 1 ; L2 / 3 , cortical layer 2 / 3 ; LA , lateral amygdala ; lENT , lateral entorhinal area ; LH , lateral habenula ; MB , midbrain ; MEA , medial amygdala ; mENT , medial entorhinal area ; MH , medial habenula ; PIR1 , piriform molecular layer ; PIR2 , piriform pyramidal layer ; PIR3 , piriform polymorph layer ; PL , prelimbic ; PONS , pons ; PRE , presubiculum ; RSP , retrosplenial area ; SS , somatosensory area ; SUB , subiculum ; TEa , temporal association area ; TH , thalamus ; VIS , visual area ; Scale bar , 500 μm . PTPδ - tdTomato protein is detected in tau - positive axonal compartments , but not in MAP2 - positive dendritic compartments in cultured neurons ( entorhinal cortex + hippocampus ; 1 : 2 mixture ; days in vitro or DIV 24 ) derived from Ptprd - / - mouse embryos . Note that the tdTomato signals envelop , but not overlap with , MAP2 - delineated dendrites , although this does not necessarily suggest that tdTomato signals not in dendrites . Scale bars , 10 μm in main images and 2 μm in enlarged images . Presynaptic PTPδ - tdTomato clusters colocalize more strongly with PSD - 95 clusters ( excitatory postsynaptic marker ) relative to gephyrin ( inhibitory postsynaptic marker ) clusters in cultured neurons ( entorhinal cortex + hippocampus ; DIV24 ) derived from Ptprd - / - mouse embryos . See Fig EV1F for the quantification of the results . Scale bars , 10 μm in main images and 2 μm in enlarged images . Ultrastructural localization of PTPδ - tdTomato protein ( red arrows ; DAB staining ) at axon terminals juxtaposed to electron - dense postsynaptic densities ( purple arrows ; PSDs ) and also at vGlut2 - positive excitatory synaptic axon terminals ( green arrows ; immunogold staining ) but not at GAD67 / 65 - positive inhibitory synaptic axon terminals ( blue arrows ; immunogold staining ) in the stratum lacunosum - moleculare ( SLM ) region of the CA1 hippocampal region ( P21 ) . Note that ~ 85 % of PTPδ - tdTomato signals are present in excitatory presynaptic terminals apposed to PSDs , ~ 10 % at presynaptic terminals not apposed to PSDs , and ~ 5 % in neuropils other than presynaptic terminals . Axon terminals are indicated by pink shades . Scale bar , 500 nm . ( n = 4 areas from 2 mice [ WT and KO ] ) .

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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 1Verification of PTPδ-tdTomato reporter mice by Western blot analysis of whole-brain lysates from heterozygote (HT) and homozygous (Ho) mice (P21) and PTPδ using mCherry antibodies. Note that levels of endogenous PTPδ protein (~85 kDa) are decreased, and that PTPδ-tdTomato protein (~140 kDa) is detectable only in reporter mice (HT and Ho). Note also that the 85-kDa band represents the transmembrane domain + cytoplasmic fragment of the full-length PTPδ protein that undergoes proteolytic cleavage at extracellular membrane-proximal sites, which can only be recognized by the PTPδ C-terminal antibody that targets the cytoplasmic region. The PTPδ-tdTomato protein (~140 kDa) contains the membrane domain + cytoplasmic fragment of PTPδ (~85 kDa) fused to tdTomato (55 kDa). The N-terminal antibody recognizes equal amounts of the C-terminal cleavage product derived from PTPδ and PTPδ-tdTomato proteins.Representative coronal, horizontal, and sagittal sections from PTPδ-tdTomato mice. Enlarged windows are demarcated by black dotted lines (Fig EV1A-C). Red tdTomato images were converted to grayscale images for clarity. ACA, anterior cingulate area; ACB, nucleus accumbens; BLA, basolateral amygdala; BMA, basomedial amygdala; CA1, cornu ammonis area 1; CA2, cornu ammonis area 2; CA3, cornu ammonis area 3; CB, cerebellum; CC, corpus callosum; CEA, central amygdala; CP, caudate putamen; CST, corticospinal tract; DG, dentate gyrus; EP, endopiriform nucleus, HY, hypothalamus; L1, cortical layer 1; L2/3, cortical layer 2/3; LA, lateral amygdala; lENT, lateral entorhinal area; LH, lateral habenula; MB, midbrain; MEA, medial amygdala; mENT, medial entorhinal area; MH, medial habenula; PIR1, piriform molecular layer; PIR2, piriform pyramidal layer; PIR3, piriform polymorph layer; PL, prelimbic; PONS, pons; PRE, presubiculum; RSP, retrosplenial area; SS, somatosensory area; SUB, subiculum; TEa, temporal association area; TH, thalamus; VIS, visual area; Scale bar, 500 μm.PTPδ-tdTomato protein is detected in tau-positive axonal compartments, but not in MAP2-positive dendritic compartments in cultured neurons (entorhinal cortex + hippocampus; 1:2 mixture; days in vitro or DIV 24) derived from Ptprd-/- mouse embryos. Note that the tdTomato signals envelop, but not overlap with, MAP2-delineated dendrites, although this does not necessarily suggest that tdTomato signals not in dendrites. Scale bars, 10 μm in main images and 2 μm in enlarged images.Presynaptic PTPδ-tdTomato clusters colocalize more strongly with PSD-95 clusters (excitatory postsynaptic marker) relative to gephyrin (inhibitory postsynaptic marker) clusters in cultured neurons (entorhinal cortex + hippocampus; DIV24) derived from Ptprd-/- mouse embryos. See Fig EV1F for the quantification of the results. Scale bars, 10 μm in main images and 2 μm in enlarged images.Ultrastructural localization of PTPδ-tdTomato protein (red arrows; DAB staining) at axon terminals juxtaposed to electron-dense postsynaptic densities (purple arrows; PSDs) and also at vGlut2-positive excitatory synaptic axon terminals (green arrows; immunogold staining) but not at GAD67/65-positive inhibitory synaptic axon terminals (blue arrows; immunogold staining) in the stratum lacunosum-moleculare (SLM) region of the CA1 hippocampal region (P21). Note that ~85% of PTPδ-tdTomato signals are present in excitatory presynaptic terminals apposed to PSDs, ~10% at presynaptic terminals not apposed to PSDs, and ~5% in neuropils other than presynaptic terminals. Axon terminals are indicated by pink shades. Scale bar, 500 nm. (n = 4 areas from 2 mice [WT and KO]).