( C ) Immunofluorescence micrographs of VAMP8 ( green ) and Syntaxin - 7 ( STX7 ; red ) show strong colocation around 3 µm silica bead phagosomes ( white ) in BMA macrophages . Nuclei are stained with Dapi . Scale bar is 5 µm . To account for people with red - green colour - blindness , ( D ) Colocation of VAMP8 ( green ) and STX7 ( red ) on individual phagosomes between WT and RNF115 KO cells represented by intersection , Manders ' M1 and overlap coefficients . Data is represented as a box and whisker plot of total values across biological duplicate experiments , where the whiskers represent the minimum to maximum values and the central band indicates the median .

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(C) Immunofluorescence micrographs of VAMP8 (green) and Syntaxin-7 (STX7; red) show strong colocation around 3 µm silica bead phagosomes (white) in BMA macrophages. Nuclei are stained with Dapi. Scale bar is 5 µm. To account for people with red-green colour-blindness, (D) Colocation of VAMP8 (green) and STX7 (red) on individual phagosomes between WT and RNF115 KO cells represented by intersection, Manders' M1 and overlap coefficients. Data is represented as a box and whisker plot of total values across biological duplicate experiments, where the whiskers represent the minimum to maximum values and the central band indicates the median.


( D ) Box and whisker plot represents the quantification of the percentage ( % ) of the hepatic lesion area in the livers from six wild - type ( WT ) and six RNF115 KO ( KO ) mice after 48 h infection with S . aureus . Haematoxylin and eosin ( H & E ) - stained liver sections of one representative animal per genotype are shown on the right , dotted line depicts the damaged area .

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(D) Box and whisker plot represents the quantification of the percentage (%) of the hepatic lesion area in the livers from six wild-type (WT) and six RNF115 KO (KO) mice after 48 h infection with S. aureus. Haematoxylin and eosin (H&E)-stained liver sections of one representative animal per genotype are shown on the right, dotted line depicts the damaged area.


( E - F ) Box and whisker plots represent the average cell counts per high power field ( HPF ) of CD3 positive T - cells ( E ) , and CD68 positive macrophages ( F ) in the livers from six wild - type ( WT ) and six RNF115 KO ( KO ) mice after 48 h infection with S . aureus . Representative immunohistochemistry sections are shown on the right .

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(E-F) Box and whisker plots represent the average cell counts per high power field (HPF) of CD3 positive T-cells (E), and CD68 positive macrophages (F) in the livers from six wild-type (WT) and six RNF115 KO (KO) mice after 48 h infection with S. aureus. Representative immunohistochemistry sections are shown on the right.


C Expression of F9 mRNA and protein in hepatic tissue of indicated mouse strains . Data represent means ± SE . M : Marker ; arrowhead : FIX protein ; Note : the bands of FIX in F9383STOP lanes are faint with small molecular weight smears .

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C Expression of F9 mRNA and protein in hepatic tissue of indicated mouse strains. Data represent means ± SE. M: Marker; arrowhead: FIX protein; Note: the bands of FIX in F9383STOP lanes are faint with small molecular weight smears.


D Measurement of coagulation activity by aPTT in mice at 8 weeks of age . n = 5 for each group , Data represent means ± SE .

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D Measurement of coagulation activity by aPTT in mice at 8 weeks of age. n=5 for each group, Data represent means ± SE.


E Measurement of coagulation activity by PT in mice at 8 weeks of age . n = 5 for each group , Data represent means ± SE .

E Measurement of coagulation activity by PT in mice at 8 weeks of age. n=5 for each group, Data represent means ± SE.


F Measurement of blood loss over a 5 - minute period after tail transection in mice at 8 weeks of age . Wild - type , n = 5 ; F9Y381S , n = 6 ; F9Y381D , n = 13 ; F9383STOP , n = 5 .

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F Measurement of blood loss over a 5-minute period after tail transection in mice at 8 weeks of age. Wild-type, n=5; F9Y381S, n=6; F9Y381D, n=13; F9383STOP, n=5.


G Survival rate of mice after the tail - clip challenge . The mice were monitored for two days after tail clipping . Wild - type , n = 5 ; F9Y381S , n = 6 ; F9Y381D , n = 13 ; F9383STOP , n = 5 .

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G Survival rate of mice after the tail-clip challenge. The mice were monitored for two days after tail clipping. Wild-type, n=5; F9Y381S, n=6; F9Y381D, n=13; F9383STOP, n=5.


B Test of clotting activity by aPTT and PT at 8 weeks following hydrodynamic tail vein injection of 120 µg pX458 and 120 µg donor plasmids ( or 120 µg ssODN ) per mouse .

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B Test of clotting activity by aPTT and PT at 8 weeks following hydrodynamic tail vein injection of 120 µg pX458 and 120 µg donor plasmids (or 120 µg ssODN) per mouse.


C Survival rate of mice after the tail - clip challenge . F9Y381D mice were treated with or without Cas9 / sgRNA / donor DNA for 3 months . After tail - clip challenge , the survival rate of each group over 2 days was determined . Wild - type , n = 9 ; untreated F9Y381D , n = 13 ; DNA treated F9Y381D , n = 14 .

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C Survival rate of mice after the tail-clip challenge. F9Y381D mice were treated with or without Cas9/sgRNA/donor DNA for 3 months. After tail-clip challenge, the survival rate of each group over 2 days was determined. Wild-type, n=9; untreated F9Y381D, n=13; DNA treated F9Y381D, n=14.


D Frequency of genetic modification in F9Y381D hepatocytes was determined either by deep sequencing ( Cas9 / donor vector treated group ) or by TA - clone sequencing ( Cas9 / ssODN group ) .

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D Frequency of genetic modification in F9Y381D hepatocytes was determined either by deep sequencing (Cas9/donor vector treated group) or by TA-clone sequencing (Cas9/ssODN group).


F The genome editing efficiency of individual mice is presented .

F The genome editing efficiency of individual mice is presented.


G Plasma aspartate aminotransferase ( AST ) and alanine aminotransferase ( ALT ) level was determined in F9Y381D mice 8 weeks after HTV injection ( n = 5 ) .

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G Plasma aspartate aminotransferase (AST) and alanine aminotransferase (ALT) level was determined in F9Y381D mice 8 weeks after HTV injection (n=5).


H mRNA levels of inflammatory cytokines from liver tissue of naked DNA treated mice were determined by real - time PCR . Data represent means ± SE .

H mRNA levels of inflammatory cytokines from liver tissue of naked DNA treated mice were determined by real-time PCR. Data represent means ± SE.


B In vivo infection efficiency 4 days after Adv injection . Inserts : negative control from untreated mouse liver tissue . Scale bar : 100 μm .

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B In vivo infection efficiency 4 days after Adv injection. Inserts: negative control from untreated mouse liver tissue. Scale bar: 100 μm.


C T7E1 assay was performed on liver DNA obtained from samples in ( B ) , and the frequency of Cas9 - induced indels is indicated as ' % ' below the lane .

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C T7E1 assay was performed on liver DNA obtained from samples in (B), and the frequency of Cas9-induced indels is indicated as '%' below the lane.


D Measurement of clotting activity by aPTTs and PTs 8 weeks after tail vein injection with indicated vectors . Data represent means ± SE .

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D Measurement of clotting activity by aPTTs and PTs 8 weeks after tail vein injection with indicated vectors. Data represent means ± SE.


E In vivo genome editing efficiency of whole liver tissues from mice treated with Adv ( Cas9 + G / T ) . ( Left ) HDR donor used for correction of Y381D mutation , and Illumina sequencing reads ( rn ) are presented . Red text indicates the correction of the F9 mutation , whereas blue text indicates the intended synonymous mutations . ( Right ) The percentage of indel or HDR corrections obtained through sequencing of 235 individual TA - clones ( low dose ) or Illumina sequencing ( high dose ) of treated mouseliver tissues .

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E In vivo genome editing efficiency of whole liver tissues from mice treated with Adv(Cas9+G/T). (Left) HDR donor used for correction of Y381D mutation, and Illumina sequencing reads (rn) are presented. Red text indicates the correction of the F9 mutation, whereas blue text indicates the intended synonymous mutations. (Right) The percentage of indel or HDR corrections obtained through sequencing of 235 individual TA-clones (low dose) or Illumina sequencing (high dose) of treated mouseliver tissues.


Immunofluorescence of early pluripotent marker Sall4 in DMSO - and R406 - treated cells on d12 . n = 5 . Scale bar , 100 μm .

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Immunofluorescence of early pluripotent marker Sall4 in DMSO- and R406- treated cells on d12. n = 5. Scale bar, 100 μm.


RT - qPCR analysis of Sall4 , Cdh1 , Epcam and Esrrb gene expression in MEFs , intermediate cells on d12 treated with DMSO and R406 , and R1 ( mESCs ) . n = 3 .

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RT-qPCR analysis of Sall4, Cdh1, Epcam and Esrrb gene expression in MEFs, intermediate cells on d12 treated with DMSO and R406, and R1 (mESCs). n = 3.


Immunofluorescence of Sall4 in reprogramming intermediates infected with shNC and shSyk . n = 3 . Scale bar , 100 μm .

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Immunofluorescence of Sall4 in reprogramming intermediates infected with shNC and shSyk. n = 3. Scale bar, 100 μm.


ciPSCs express key pluripotent markers Oct4 , Sox2 and Nanog . Scale bar , 100 μm .

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ciPSCs express key pluripotent markers Oct4, Sox2 and Nanog. Scale bar, 100 μm.


Immunofluorescence of Sall4 in WT cells and Ppp3ca knockdown cells on d12 . n = 6 . Scale bar , 100 μm . RT - qPCR analysis of pluripotent genes expression in cells treated with shNC and shPpp3ca . n = 3 .

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Immunofluorescence of Sall4 in WT cells and Ppp3ca knockdown cells on d12. n = 6. Scale bar, 100 μm. RT-qPCR analysis of pluripotent genes expression in cells treated with shNC and shPpp3ca. n = 3.


Immunofluorescence of Sall4 in WT cells and Nfatc1 knockdown cells on d12 . n = 3 . Scale bar , 100 μm . RT - qPCR analysis of pluripotent genes expression in cells treated with shNC and shNfatc1 . n = 3 .

Immunofluorescence of Sall4 in WT cells and Nfatc1 knockdown cells on d12. n = 3. Scale bar, 100 μm. RT-qPCR analysis of pluripotent genes expression in cells treated with shNC and shNfatc1. n = 3.


RT - qPCR analysis of Gldc , Cbs and Tdh expression in R406 - and shSyk - treated cells ( E ) and FK506 - , shPpp3ca - and shNfatc1 - treated cells ( F ) on d8 . n = 3 .

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RT-qPCR analysis of Gldc, Cbs and Tdh expression in R406- and shSyk-treated cells (E) and FK506-, shPpp3ca- and shNfatc1-treated cells (F) on d8. n = 3.


Metabolite abundance of SAM , SAH in samples treated with DMSO and R406 . n = 6 .

Metabolite abundance of SAM, SAH in samples treated with DMSO and R406. n = 6.


Immunofluorescence of Sall4 in reprogramming intermediates treated by R406 + HA and R406 + DPBS on d12 . n = 3 . Scale bar , 100 μm . Immunofluorescence of Sall4 in NAC - and DPBS - treated cells on d12 . n = 5 . Scale bar , 100 μm .

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Immunofluorescence of Sall4 in reprogramming intermediates treated by R406 + HA and R406 + DPBS on d12. n = 3. Scale bar, 100 μm. Immunofluorescence of Sall4 in NAC- and DPBS-treated cells on d12. n = 5. Scale bar, 100 μm.


RT - qPCR analysis of Sall4 , Cdh1 , Epcam and Esrrb gene expression in DPBS - and HA - treated cells on d12 . n = 3 . RT - qPCR analysis of Sall4 , Cdh1 , Epcam and Esrrb gene expression in DPBS - and NAC - treated cells on d12 . n = 3 .

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RT-qPCR analysis of Sall4, Cdh1, Epcam and Esrrb gene expression in DPBS- and HA-treated cells on d12. n = 3. RT-qPCR analysis of Sall4, Cdh1, Epcam and Esrrb gene expression in DPBS- and NAC-treated cells on d12. n = 3.


Immunofluorescence of Sall4 in WT cells and Cbs overexpressing cells on d12 . n = 4 . Scale bar , 100 μm .

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Immunofluorescence of Sall4 in WT cells and Cbs overexpressing cells on d12. n = 4. Scale bar, 100 μm.


Quantification of oxygen consumption rate ( OCR ) of reprogramming intermediates treated with small molecules ( A , C ) and the indicated OCR parameters ( B , D ) . n = 9 .

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Quantification of oxygen consumption rate (OCR) of reprogramming intermediates treated with small molecules (A, C) and the indicated OCR parameters (B, D). n = 9.


Fluorescence images of DCFH - DA staining for ROS levels in cells treated with DMSO , HA , NAC , and R406 on d12 ( E ) and the quantification of the ROS levels ( F ) . n = 4 . Scale bar , 100 μm . MDA quantification in cells treated with DMSO , HA , NAC , and R406 on d12 . n = 9 .

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Fluorescence images of DCFH-DA staining for ROS levels in cells treated with DMSO, HA, NAC, and R406 on d12 (E) and the quantification of the ROS levels (F). n = 4. Scale bar, 100 μm. MDA quantification in cells treated with DMSO, HA, NAC, and R406 on d12. n = 9.


( a , b ) Immunoblot analysis of LAMP - 2A in total lysates ( a ) and quantitative PCR analysis of Lamp2a mRNA ( b ) of naive CD4 + T cells and TH1 and TH2 cells following activation with anti - CD3 and anti - CD28 . β - actin ( a ) serves as a loading control throughout ; mRNA results ( b ) are presented relative to those of resting cells .

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(a,b) Immunoblot analysis of LAMP-2A in total lysates (a) and quantitative PCR analysis of Lamp2a mRNA (b) of naive CD4+ T cells and TH1 and TH2 cells following activation with anti-CD3 and anti-CD28. β-actin (a) serves as a loading control throughout; mRNA results (b) are presented relative to those of resting cells.


( c ) Immunofluorescence microscopy of LAMP - 2A ( green ) and nuclei ( stained with the DNA - binding dye DAPI ; blue ) in resting TH1 cells ( Rest ) and TH1 cells activated for 24 h as in a , b ( Act ) ( left ) , and quantification of lysosomes in cells as at left ( right ) . Original magnification , × 630 ; scale bar , 10 μm . * P = 0 . 013 ( t - test ) .

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(c) Immunofluorescence microscopy of LAMP-2A (green) and nuclei (stained with the DNA-binding dye DAPI; blue) in resting TH1 cells (Rest) and TH1 cells activated for 24 h as in a,b (Act) (left), and quantification of lysosomes in cells as at left (right). Original magnification, ×630; scale bar, 10 μm. *P = 0.013 (t-test).


( d ) Electron microscopy ( left ) of resting TH1 cells and TH1 cells activated for 24 h as in a , b ( top row ) ; below , enlargement of areas outlined above , showing individual lysosomes ( arrows ) . Original magnification ( top row ) , × 12 , 000 ; scale bars ( bottom row ) , 1 μm . Right , morphometric analysis of cells as at left . * P = 0 . 037 and * * P = 0 . 034 ( t - test ) .

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(d) Electron microscopy (left) of resting TH1 cells and TH1 cells activated for 24 h as in a,b (top row); below, enlargement of areas outlined above, showing individual lysosomes (arrows). Original magnification (top row), ×12,000; scale bars (bottom row), 1 μm. Right, morphometric analysis of cells as at left. *P = 0.037 and **P = 0.034 (t-test).


( e ) Distribution of KFERQ - PA - mCherry - 1 in TH1 cells transiently transfected with a CMA reporter vector , photoactivated , and left unstimulated ( Rest ) or stimulated with anti - CD3 and anti - CD28 in the presence ( 3 - MA ) or absence ( vehicle ( veh ) ) of 3 - methyladenine ( left ) ; arrows indicate mCherry + puncta . Original magnification , × 630 . Right , quantification of the results for the cells at left . * P 0 . 05 ( analysis of variance with Tukey ' s post - test ) .

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(e) Distribution of KFERQ-PA-mCherry-1 in TH1 cells transiently transfected with a CMA reporter vector, photoactivated, and left unstimulated (Rest) or stimulated with anti-CD3 and anti-CD28 in the presence (3-MA) or absence (vehicle (veh)) of 3-methyladenine (left); arrows indicate mCherry+ puncta. Original magnification, ×630. Right, quantification of the results for the cells at left. *P 0.05 (analysis of variance with Tukey's post-test).


( f ) Immunoblot analysis of hsc70 in lysosomal fractions of resting ( Lys - R ) and stimulated ( Lys - A ) TH1 cells and in cytosolic fractions of TH1 cells activated as in a , b ( Cyto ) ; LAMP - 2A and LAMP - 1 serve as controls .

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(f) Immunoblot analysis of hsc70 in lysosomal fractions of resting (Lys-R) and stimulated (Lys-A) TH1 cells and in cytosolic fractions of TH1 cells activated as in a,b (Cyto); LAMP-2A and LAMP-1 serve as controls.


( g ) Immunoblot analysis of LAMP - 2A in resting TH1 cells and TH1 cells activated in the presence ( NAc ) or absence ( Veh ) of N - acetylcysteine . ( h ) Quantitative PCR analysis of Lamp2a mRNA in cells as in g ( results presented as in b ) . * P = 0 . 007 ( t test ) .

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(g) Immunoblot analysis of LAMP-2A in resting TH1 cells and TH1 cells activated in the presence (NAc) or absence (Veh) of N-acetylcysteine. (h) Quantitative PCR analysis of Lamp2a mRNA in cells as in g (results presented as in b). *P = 0.007 (t test).


( i ) Luciferase activity in TH1 cells transiently transfected with a vector containing a luciferase reporter under the control of the Lamp2 proximal promoter and stimulated for 0 , 12 or 24 h with plate - bound anti - CD3 and anti - CD28 ; results are presented relative to those of resting cells ( time 0 ) .

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(i) Luciferase activity in TH1 cells transiently transfected with a vector containing a luciferase reporter under the control of the Lamp2 proximal promoter and stimulated for 0, 12 or 24 h with plate-bound anti-CD3 and anti-CD28; results are presented relative to those of resting cells (time 0).


( j ) Luciferase activity in TH1 cells transiently transfected as in i and left unstimulated ( control ( Ctrl ) ) or stimulated for 16 h with 50 μM paraquat ( Pq ) or 1 mM H2O2 ; results are presented in relative light units ( RLU ) .

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(j) Luciferase activity in TH1 cells transiently transfected as in i and left unstimulated (control (Ctrl)) or stimulated for 16 h with 50 μM paraquat (Pq) or 1 mM H2O2; results are presented in relative light units (RLU).


( k ) Immunoblot analysis of LAMP - 2A in total lysates of resting TH1 cells ( − ) and TH1 cells activated for 24 h as in a , b ( + ) , transfected with a control nontargeting siRNA ( with a scrambled sequence ( Scr ) ) or siRNA specific for mRNA encoding Duox1 ( either of two siRNAs : Dx ( 1 ) and Dx ( 2 ) ) or RISP ( Uq ( 1 ) ) .

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(k) Immunoblot analysis of LAMP-2A in total lysates of resting TH1 cells (−) and TH1 cells activated for 24 h as in a,b (+), transfected with a control nontargeting siRNA (with a scrambled sequence (Scr)) or siRNA specific for mRNA encoding Duox1 (either of two siRNAs: Dx(1) and Dx(2)) or RISP (Uq(1)).


( l ) Immunoblot analysis of LAMP - 2A in lysates of resting or activated TH1 cells in the presence ( right half ) or absence ( left half ) of cyclosporine A ( CsA ) .

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(l) Immunoblot analysis of LAMP-2A in lysates of resting or activated TH1 cells in the presence (right half) or absence (left half) of cyclosporine A (CsA).


( m ) Chromatin - immunoprecipitation analysis of the recruitment of NFAT1 to the promoter of Lamp2 or Adad1 ( negative control ) ( left ) or to Il2 ( positive control ) ( right ) in resting and activated TH1 cells ; results are presented relative to input chromatin precipitated with anti - NFAT1 .

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(m) Chromatin-immunoprecipitation analysis of the recruitment of NFAT1 to the promoter of Lamp2 or Adad1 (negative control) (left) or to Il2 (positive control) (right) in resting and activated TH1 cells; results are presented relative to input chromatin precipitated with anti-NFAT1.


( a ) Flow cytometry of T cells transduced with lentivirus expressing GFP and nontargeting control shRNA ( Scr ) or Lamp2a mRNA - specific shRNA ( shL2A ) ( left ) , and immunoblot analysis of LAMP - 2A in sorted GFP + T cells ( right ) . Numbers above bracketed lines ( left ) indicate percent GFP + cells .

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(a) Flow cytometry of T cells transduced with lentivirus expressing GFP and nontargeting control shRNA (Scr) or Lamp2a mRNA-specific shRNA (shL2A) (left), and immunoblot analysis of LAMP-2A in sorted GFP+ T cells (right). Numbers above bracketed lines (left) indicate percent GFP+ cells.


( b ) Enzyme - linked immunosorbent assay of IL - 2 and interferon - γ ( IFN - γ ) in TH1 cells transduced as in a and then left unstimulated ( Rest ) or stimulated for 24 h with anti - CD3 and anti - CD28 ( Act ) . * P = 0 . 038 and * * P = 0 . 031 ( t - test ) .

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(b) Enzyme-linked immunosorbent assay of IL-2 and interferon-γ (IFN-γ) in TH1 cells transduced as in a and then left unstimulated (Rest) or stimulated for 24 h with anti-CD3 and anti-CD28 (Act). *P = 0.038 and **P = 0.031 (t-test).


( c ) Proliferation in TH1 cells transduced as in a and left unstimulated or stimulated for 24 h as in b , assessed by incorporation of the thymidine analog BrdU and presented as absorbance at 495 nm ( A495 ) . * P = 0 . 013 ( t - test ) .

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(c) Proliferation in TH1 cells transduced as in a and left unstimulated or stimulated for 24 h as in b, assessed by incorporation of the thymidine analog BrdU and presented as absorbance at 495 nm (A495). *P = 0.013 (t-test).


( d ) Quantification of annexin V - positive ( AnnV + ) cells among resting or stimulated TH1 cells transduced as in a .

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(d) Quantification of annexin V-positive (AnnV+) cells among resting or stimulated TH1 cells transduced as in a.


( a ) Immunoblot analysis of Itch and RCAN1 in lysosomal fractions isolated from resting cells TH1 cells and TH1 cells activated for 24 h with anti - CD3 and anti - CD28 ; LAMP - 1 serves as a control .

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(a) Immunoblot analysis of Itch and RCAN1 in lysosomal fractions isolated from resting cells TH1 cells and TH1 cells activated for 24 h with anti-CD3 and anti-CD28; LAMP-1 serves as a control.


( b ) Immunoblot analysis of Itch and RCAN1 in lysosomal fractions of TH1 cells activated for 24 h as in a with ( + ) or without ( − ) inhibition of lysosomal proteases during the final 3 h with NH4Cl and leupeptin ( Lys inh ) ; LAMP - 1 serves as a control .

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(b) Immunoblot analysis of Itch and RCAN1 in lysosomal fractions of TH1 cells activated for 24 h as in a with (+) or without (−) inhibition of lysosomal proteases during the final 3 h with NH4Cl and leupeptin (Lys inh); LAMP-1 serves as a control.


( c ) Immunoblot analysis of Itch and RCAN1 in resting TH1 cells and TH1 cells activated for 24 h as in a in the presence ( NAc ) or absence ( Veh ) of N - acetylcysteine .

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(c) Immunoblot analysis of Itch and RCAN1 in resting TH1 cells and TH1 cells activated for 24 h as in a in the presence (NAc) or absence (Veh) of N-acetylcysteine.


( d ) Immunoblot analysis of lysates of TH1 cells transfected with GFP - Itch or GFP - mu - Itch ( top ) or with Myc - RCAN1 or Myc - mu - RCAN1 ( bottom ) and cotransfected with a GFP expression plasmid ( to assess transfection efficiency ) , then activated for 24 h as in a .

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(d) Immunoblot analysis of lysates of TH1 cells transfected with GFP-Itch or GFP-mu-Itch (top) or with Myc-RCAN1 or Myc-mu-RCAN1 (bottom) and cotransfected with a GFP expression plasmid (to assess transfection efficiency), then activated for 24 h as in a.


( e , f ) Proliferation ( e ) and IL - 2 production ( f ) of TH1 cells transfected with plasmid expressing GFP ( Ctrl ) or plasmids as in c and left unstimulated ( Rest ) or activated for 48 h ( e ) or 24 h ( f ) as in a ( assessed as in Fig . 2c ( e ) or Fig . 2b ( f ) ) . * P = 0 . 019 and * * P = 0 . 0034 ( e ) or * P = 0 . 046 and * * P = 0 . 044 ( f ) ( t test ) . Data are representative of four ( a , b ) , two ( c ) or three ( d ) independent experiments or are from three independent experiments ( e , f ; mean and s . e . m . ) .

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(e,f) Proliferation (e) and IL-2 production (f) of TH1 cells transfected with plasmid expressing GFP (Ctrl) or plasmids as in c and left unstimulated (Rest) or activated for 48 h (e) or 24 h (f) as in a (assessed as in Fig. 2c (e) or Fig. 2b (f)). *P = 0.019 and **P = 0.0034 (e) or *P = 0.046 and **P = 0.044 (f) (t test). Data are representative of four (a,b), two (c) or three (d) independent experiments or are from three independent experiments (e,f; mean and s.e.m.).


( a ) Immunoblot analysis of LAMP - 2A in CD4 + T cells ( left ) and liver , kidneys , thymus and lungs ( right ) of L2A - cKO mice ( KO ) and their control littermates ( Ctrl ) .

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(a) Immunoblot analysis of LAMP-2A in CD4+ T cells (left) and liver, kidneys, thymus and lungs (right) of L2A-cKO mice (KO) and their control littermates (Ctrl).


( b ) Quantitative PCR analysis of Lamp2a , Lamp2b and Lamp2c mRNA in T cells from L2A - cKO mice ( right ) ; results are presented relative to those of their control littermates ( left ) , set as 1 .

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(b) Quantitative PCR analysis of Lamp2a, Lamp2b and Lamp2c mRNA in T cells from L2A-cKO mice (right); results are presented relative to those of their control littermates (left), set as 1.


( c ) Immunoblot analysis of the autophagy related protein LC3 in its cytosolic form ( LC3 - I ) and its autophagosome membrane - associated form ( LC3 - II ) in total lysates of CD4 + T cells obtained from L2A - cKO mice or their control littermates and then left unstimulated ( Rest ) or activated for 24 h with anti - CD3 and anti - CD218 ( Act ) , cultured for the final 4 h in the presence ( + ) or absence ( − ) of NH4Cl and leupeptin ( NH4Cl + Leup ) .

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(c) Immunoblot analysis of the autophagy related protein LC3 in its cytosolic form (LC3-I) and its autophagosome membrane-associated form (LC3-II) in total lysates of CD4+ T cells obtained from L2A-cKO mice or their control littermates and then left unstimulated (Rest) or activated for 24 h with anti-CD3 and anti-CD218 (Act), cultured for the final 4 h in the presence (+) or absence (−) of NH4Cl and leupeptin (NH4Cl+Leup).


( d ) Spleens from an L2A - cKO mouse and its control littermate ( left ) , and flow cytometry of peripheral T cells from such mice ( right ) . Numbers in quadrants ( right ) indicate percent CD4 − CD8 + T cells ( top left ) or CD4 + CD8 − T cells ( bottom right ) .

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(d) Spleens from an L2A-cKO mouse and its control littermate (left), and flow cytometry of peripheral T cells from such mice (right). Numbers in quadrants (right) indicate percent CD4−CD8+ T cells (top left) or CD4+CD8− T cells (bottom right).


( e , f ) Proliferation ( e ) and IL - 2 production ( f ) by CD4 + T cells isolated from the spleen of L2A - cKO mice and their control littermates and stimulated ex vivo for 48 h with anti - CD3 and anti - CD28 ( assessed as in Fig . 2c ( e ) or Fig . 2b ( f ) ) . * P = 0 . 0062 ( e ) or 0 . 006 ( f ) ( t - test ) .

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(e,f) Proliferation (e) and IL-2 production (f) by CD4+ T cells isolated from the spleen of L2A-cKO mice and their control littermates and stimulated ex vivo for 48 h with anti-CD3 and anti-CD28 (assessed as in Fig. 2c (e) or Fig. 2b (f)). *P = 0.0062 (e) or 0.006 (f) (t-test).


( g ) Immunoblot analysis of LAMP - 2A , Itch and RCAN1 in total lysates of CD4 + T cells obtained from L2A - cKO mice ( n = 3 ( KO1 , KO2 and KO3 ) ) and their control littermates ( n = 3 ) and activated for 24 h as in e , f . * , nonspecific band .

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(g) Immunoblot analysis of LAMP-2A, Itch and RCAN1 in total lysates of CD4+ T cells obtained from L2A-cKO mice (n = 3 (KO1, KO2 and KO3)) and their control littermates (n = 3) and activated for 24 h as in e,f. *, nonspecific band.


( h ) Quantitative PCR analysis of Rcan1 and Itch mRNA in CD4 + T cells isolated from L2A - cKO mice and their control littermates and activated for 24 h as in e , f ; results are presented relative to those of resting cells .

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(h) Quantitative PCR analysis of Rcan1 and Itch mRNA in CD4+ T cells isolated from L2A-cKO mice and their control littermates and activated for 24 h as in e,f; results are presented relative to those of resting cells.


( i , j ) Recall responses to OVA ( 323 - 339 ) in draining lymph nodes isolated from L2A - cKO mice and their control littermates after immunization with OVA ( 323 - 339 ) , assessed as proliferation ( i ) and IL - 2 production ( j ) ( as in e , f ) . * P = 0 . 0054 ( i ) or 0 . 0052 ( j ) ( t - test ) .

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(i,j) Recall responses to OVA(323-339) in draining lymph nodes isolated from L2A-cKO mice and their control littermates after immunization with OVA(323-339), assessed as proliferation (i) and IL-2 production (j) (as in e,f). *P = 0.0054 (i) or 0.0052 (j) (t-test).


( k , l ) Recall responses to L . monocytogenes in CD4 + T cells isolated from L2A - cKO mice and their control littermates ( n = 6 per group ) previously uninfected ( UI ) or previously infected with L . monocytogenes ( Inf ) , assessed as IFN - γ production ( k ) and proliferation ( l ) ( as in e , f ) . * P = 0 . 032 ( k ) or 0 . 02 ( l ) ( t - test ) .

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(k,l) Recall responses to L. monocytogenes in CD4+ T cells isolated from L2A-cKO mice and their control littermates (n = 6 per group) previously uninfected (UI) or previously infected with L. monocytogenes (Inf), assessed as IFN-γ production (k) and proliferation (l) (as in e,f). *P = 0.032 (k) or 0.02 (l) (t-test).


( m ) L . monocytogenes in spleen and liver of L2A - cKO mice and their control littermates ( n = 3 per genotype ) immunized with L . monocytogenes and then reinfected with a high dose of L . monocytogenes . CFU , colony - forming units . * P = 0 . 008 and * * P = 0 . 001 ( t - test ) .

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(m) L. monocytogenes in spleen and liver of L2A-cKO mice and their control littermates (n = 3 per genotype) immunized with L. monocytogenes and then reinfected with a high dose of L. monocytogenes. CFU, colony-forming units. *P = 0.008 and **P = 0.001 (t-test).


( a ) Immunoblot analysis of LAMP - 2A in CD4 + T cells obtained from 4 - month - old or 22 - month old mice , then left resting or stimulated for 24 h with anti - CD3 plus anti - CD28 .

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(a) Immunoblot analysis of LAMP-2A in CD4+ T cells obtained from 4-month-old or 22-month old mice, then left resting or stimulated for 24 h with anti-CD3 plus anti-CD28.


( b ) Immunoblot analysis of RCAN1 and Itch in CD4 + T cells obtained from 4 - month - old or 22 - month old mice ( n = 3 per group ) , then stimulated for 24 h as in a ( left ) , and quantification of protein in 22 - month old mice , presented relative to that of 4 - month - old mice , set as 100 % . * P = 0 . 044 ( t - test ) .

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(b) Immunoblot analysis of RCAN1 and Itch in CD4+ T cells obtained from 4-month-old or 22-month old mice (n = 3 per group), then stimulated for 24 h as in a (left), and quantification of protein in 22-month old mice, presented relative to that of 4-month-old mice, set as 100%. *P = 0.044 (t-test).


( c ) Immunoblot analysis of LAMP - 2A in total lysates of naive and memory CD4 + T cells left resting or activated for 24 h as in a .

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(c) Immunoblot analysis of LAMP-2A in total lysates of naive and memory CD4+ T cells left resting or activated for 24 h as in a.


( d ) Immunoblot analysis ( bottom ) of LAMP - 2A in total lysates of naive and memory CD4 + T cells obtained from 4 - or 22 - month - old mice and left resting ( R ) or activated for 24 h as in a ( A ) ; above , quantification from results below ( n = 3 mice per group ) . * P = 0 . 009 ( t - test ) .

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(d) Immunoblot analysis (bottom) of LAMP-2A in total lysates of naive and memory CD4+ T cells obtained from 4- or 22-month-old mice and left resting (R) or activated for 24 h as in a (A); above, quantification from results below (n = 3 mice per group). *P = 0.009 (t-test).


( e ) Electrophoretic mobility - shift assay of the binding of NFAT ( N ) and free probe ( FP ) in nuclear extracts of CD4 + T cells isolated from 3 - , 12 - or 22 - month - old mice and activated for 4 h as in a .

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(e) Electrophoretic mobility-shift assay of the binding of NFAT (N) and free probe (FP) in nuclear extracts of CD4+ T cells isolated from 3-, 12- or 22-month-old mice and activated for 4 h as in a.


( f ) ROS production in CD4 + T cells isolated from 4 - or 22 - month - old mice ( n = 3 per group ) and left resting or activated for 12 h as in a , assessed by flow cytometry and presented as the mean fluorescence intensity ( MFI ) of a fluorogenic probe for oxidative stress ( CellROX Green ) .

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(f) ROS production in CD4+ T cells isolated from 4- or 22-month-old mice (n = 3 per group) and left resting or activated for 12 h as in a, assessed by flow cytometry and presented as the mean fluorescence intensity (MFI) of a fluorogenic probe for oxidative stress (CellROX Green).


( g - i ) Immunoblot analysis of LAMP - 2A ( g ) and analysis of the proliferation ( h ) and IL - 2 production ( i ) of TH1 cells obtained from 4 - or 22 - month - old mice and transduced with retrovirus expressing GFP alone ( RV - GFP ) or human LAMP - 2A and GFP ( RV - LAMP2A ) , sorted by GFP expression , cultured for 2 weeks and then stimulated for 24 - 48 h as in a ( assessed as in Fig . 2c ( h ) or Fig . 2b ( i ) ) . * P 0 . 05 ( analysis of variance with Tukey ' s post - test ) .

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(g-i) Immunoblot analysis of LAMP-2A (g) and analysis of the proliferation (h) and IL-2 production (i) of TH1 cells obtained from 4- or 22-month-old mice and transduced with retrovirus expressing GFP alone (RV-GFP) or human LAMP-2A and GFP (RV-LAMP2A), sorted by GFP expression, cultured for 2 weeks and then stimulated for 24-48 h as in a (assessed as in Fig. 2c (h) or Fig. 2b (i)). *P 0.05 (analysis of variance with Tukey's post-test).


( j ) Immunoblot analysis of LAMP - 2A in total lysates of CD4 + T cells obtained from two human donors ( A and B ) and left resting or activated for 24 or 48 h as in a ( above lanes ) .

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(j) Immunoblot analysis of LAMP-2A in total lysates of CD4+ T cells obtained from two human donors (A and B) and left resting or activated for 24 or 48 h as in a (above lanes).


( k ) Real - time PCR analysis of LAMP2A mRNA in naive and memory CD4 + T cells obtained from young human donors ( 26 ± 2 years of age ; n = 3 ) or old human donors ( 72 ± 6 years of age ; n = 6 ) and activated for 24 h as in a ; results were calculated by the change - in - cycle - threshold method ( 2 − ΔCt ) relative to those of the gene encoding β - actin . * P = 0 . 047 and * * P = 0 . 0033 ( Mann - Whitney ) .

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(k) Real-time PCR analysis of LAMP2A mRNA in naive and memory CD4+ T cells obtained from young human donors (26 ± 2 years of age; n = 3) or old human donors (72 ± 6 years of age; n = 6) and activated for 24 h as in a; results were calculated by the change-in-cycle-threshold method (2−ΔCt) relative to those of the gene encoding β-actin. *P = 0.047 and **P = 0.0033 (Mann-Whitney).


( B ) The degree of polymerisation of Z α1 - antitrypsin after 72 hours at 37 ° C , as determined by an end - point immunoassay using the 2C1 monoclonal antibody , in varying concentrations of compound ( shown in panel A ) . Modification of the phenyl and indole heterocycle of GSK425 ( pIC50 6 . 5 ) resulted in an ~ 100 fold increase in potency and the discovery of the 2 - oxindole GSK716 ( pIC50 8 . 3 ) . Data presented as mean ± SD , n = 2 ( GSK425 ) and n = 25 ( GSK716 ) .

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(B) The degree of polymerisation of Z α1-antitrypsin after 72 hours at 37°C, as determined by an end-point immunoassay using the 2C1 monoclonal antibody, in varying concentrations of compound (shown in panel A). Modification of the phenyl and indole heterocycle of GSK425 (pIC50 6.5) resulted in an ~100 fold increase in potency and the discovery of the 2-oxindole GSK716 (pIC50 8.3). Data presented as mean ± SD, n = 2 (GSK425) and n = 25 (GSK716).


( C ) GSK716 binds to Z α1 - antitrypsin with a high affinity mean pKD of 8 . 5 ± 0 . 12 ( n = 18 ) as determined by a competition binding assay with a fluorescently labelled derivative . There was a 50 - fold lower affinity for plasma - purified wild - type M α1 - antitrypsin , with a mean pKD of 6 . 8 ± 0 . 18 ( n = 10 ) . Data presented as mean ± SD .

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(C) GSK716 binds to Z α1-antitrypsin with a high affinity mean pKD of 8.5 ± 0.12 (n = 18) as determined by a competition binding assay with a fluorescently labelled derivative. There was a 50-fold lower affinity for plasma-purified wild-type M α1-antitrypsin, with a mean pKD of 6.8 ± 0.18 (n = 10). Data presented as mean ± SD.


( D ) The compound bound to monomeric but not polymeric Z α1 - antitrypsin ( Z α1 - AT ) as reported by fluorescence polarisation of an Alexa - 488 - labelled variant of GSK716 .

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(D) The compound bound to monomeric but not polymeric Z α1-antitrypsin (Z α1-AT) as reported by fluorescence polarisation of an Alexa-488-labelled variant of GSK716.


( E ) Representative curves reporting the interaction of different concentrations of GSK716 with Z α1 - antitrypsin based on changes in intrinsic tryptophan fluorescence . Based on the concentration dependence ( inset ) , the second - order rate constant of association was found to be 4 . 1 x 104 M - 1 s - 1 . ( F ) The association of GSK716 with M α1 - antitrypsin , giving a second - order rate constant of 2 . 1 x 102 M - 1 s - 1 .

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(E) Representative curves reporting the interaction of different concentrations of GSK716 with Z α1-antitrypsin based on changes in intrinsic tryptophan fluorescence. Based on the concentration dependence (inset), the second-order rate constant of association was found to be 4.1 x 104 M-1 s-1. (F) The association of GSK716 with M α1-antitrypsin, giving a second-order rate constant of 2.1 x 102 M-1 s-1.


( G ) CHO - K1 Tet - On cells expressing Z α1 - antitrypsin were induced with doxycycline ( 0 . 5μg / ml ) for 48 h . Cells were incubated with 10μM GSK716 ( or 0 . 1 % v / v DMSO for the control ) during the induction . Culture media containing either the experimental compound or DMSO were changed every 24 h . After the induction , cells were labelled for 10 minutes with 35S Met / Cys and chased at the indicated times . Culture media were collected and cells lysed in 1 % v / v NP - 40 buffer . Intracellular fractions and culture media from cells expressing Z α1 - antitrypsin were immunoprecipitated either with a mAb against total α1 - antitrypsin ( 3C11 ) or with a polymer - specific mAb ( 2C1 ) . Samples were resolved by 4 - 12 % w / v acrylamide SDS - PAGE and detected by autoradiography . ( H ) The graphs show the effect of GSK716 on intracellular and extracellular Z α1 - antitrypsin ( mean ± SEM , n = 2 )

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(G) CHO-K1 Tet-On cells expressing Z α1-antitrypsin were induced with doxycycline (0.5μg/ml) for 48 h. Cells were incubated with 10μM GSK716 (or 0.1% v/v DMSO for the control) during the induction. Culture media containing either the experimental compound or DMSO were changed every 24 h. After the induction, cells were labelled for 10 minutes with 35S Met/Cys and chased at the indicated times. Culture media were collected and cells lysed in 1% v/v NP-40 buffer. Intracellular fractions and culture media from cells expressing Z α1-antitrypsin were immunoprecipitated either with a mAb against total α1-antitrypsin (3C11) or with a polymer-specific mAb (2C1). Samples were resolved by 4-12% w/v acrylamide SDS-PAGE and detected by autoradiography. (H) The graphs show the effect of GSK716 on intracellular and extracellular Z α1-antitrypsin (mean±SEM, n=2)


( B ) M ( above ) and Z ( below ) α1 - antitrypsin , at a concentration of 0 . 2mg / ml in PBS + 5 % v / v glycerol , were heated at a range of temperatures between 48 ° C - 65 ° C for 4 hours in the presence and absence of GSK716 , as indicated . The oligomerisation state was determined by non - denaturing PAGE electrophoresis ; the lane denoted ' M ' contains the unheated monomeric control .

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(B) M (above) and Z (below) α1-antitrypsin, at a concentration of 0.2mg/ml in PBS+5% v/v glycerol, were heated at a range of temperatures between 48°C-65°C for 4 hours in the presence and absence of GSK716, as indicated. The oligomerisation state was determined by non-denaturing PAGE electrophoresis; the lane denoted 'M' contains the unheated monomeric control.


( C ) M and Z α1 - antitrypsin were subjected to equilibrium unfolding into different concentrations of guanidine hydrochloride ( gdnhcl ) in the presence and absence of GSK716 , with bis - ANS dye added to report the presence of the unfolded intermediate . The normalised fluorescence intensity data were fitted with an equation describing a three - state unfolding curve . Values shown are the mean of three independent experiments and the error bars represent ± SEM .

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(C) M and Z α1-antitrypsin were subjected to equilibrium unfolding into different concentrations of guanidine hydrochloride (gdnhcl) in the presence and absence of GSK716, with bis-ANS dye added to report the presence of the unfolded intermediate. The normalised fluorescence intensity data were fitted with an equation describing a three-state unfolding curve. Values shown are the mean of three independent experiments and the error bars represent ±SEM.


( D ) Top panel , the association of GSK716 induced a marked quenching and blue - shift of the α1 - antitrypsin intrinsic tryptophan fluorescence spectrum with respect to unbound protein ( inset graph ) . The association of 10µM GSK716 with four α1 - antitrypsin variants that vary in their propensity to polymerise , ranging from inert ( M ) , to mild ( S ) , moderate ( Baghdad denoted ' B ' ) and severe ( Z ) . Representative progress curves of the change in intrinsic tryptophan fluorescence at 330nm for 0 . 2mg / ml protein are shown . Bottom panel , the half - time of association calculated from three such independent experiments ( error bars are ± SEM ) show a correspondence with the polymerisation propensity of the four variants . The change in Z α1 - antitrypsin fluorescence was faster than the dead - time of the apparatus ( ~ 10s ) and scaling was with reference to unbound intensity .

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(D) Top panel, the association of GSK716 induced a marked quenching and blue-shift of the α1-antitrypsin intrinsic tryptophan fluorescence spectrum with respect to unbound protein (inset graph). The association of 10µM GSK716 with four α1-antitrypsin variants that vary in their propensity to polymerise, ranging from inert (M), to mild (S), moderate (Baghdad denoted 'B') and severe (Z). Representative progress curves of the change in intrinsic tryptophan fluorescence at 330nm for 0.2mg/ml protein are shown. Bottom panel, the half-time of association calculated from three such independent experiments (error bars are ±SEM) show a correspondence with the polymerisation propensity of the four variants. The change in Z α1-antitrypsin fluorescence was faster than the dead-time of the apparatus (~10s) and scaling was with reference to unbound intensity.


( B ) M α1 - antitrypsin was incubated with different molar ratios of chymotrypsin and resolved by SDS - PAGE . The position of covalent α1 - antitrypsin - chymotrypsin complex ( cpx ) , native ( nat ) and cleaved ( clv ) is shown .

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(B) M α1-antitrypsin was incubated with different molar ratios of chymotrypsin and resolved by SDS-PAGE. The position of covalent α1-antitrypsin-chymotrypsin complex (cpx), native (nat) and cleaved (clv) is shown.


( C ) The inhibitory activity of α1 - antichymotrypsin against chymotrypsin ( ach + chym ) , neuroserpin against trypsin ( neus + tryp ) , and antithrombin against thrombin ( atiii + thrombin ) was determined in the presence and absence of GSK716 . Error bars reflect ± SD of two independent experiments .

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(C) The inhibitory activity of α1-antichymotrypsin against chymotrypsin (ach+chym), neuroserpin against trypsin (neus+tryp), and antithrombin against thrombin (atiii+thrombin) was determined in the presence and absence of GSK716. Error bars reflect ±SD of two independent experiments.


Z α1 - antitrypsin transgenic animals were dosed with 10 , 30 or 100 mg / kg GSK716 three times a day . Total concentrations of GSK716 were determined by LC - MS / MS ( Table 2 ) . ( A ) 100mg / kg GSK716 resulted in a mean 7 - fold increase in circulating monomeric Z α1 - antitrypsin levels demonstrating robust target engagement in the liver . The observed steady state total and free compound levels of GSK716 in the transgenic Z α1 - antitrypsin mouse were well - predicted by the in silico PK model built on : ( 1 ) in vitro metabolic clearance data , ( 2 ) plasma protein binding data , ( 3 ) in vivo PK data from wild type mice and ( 4 ) a term comprising a 5μM circulating sink for drug with an affinity of 1 . 5 nM , representing the Z α1 - antitrypsin within blood . The target free drug concentration was selected based on the observed potency in the in vitro secretion assays in which the total drug approximates to the free drug in the assay . Interestingly , 30mg / kg and 10mg / kg groups also gave significant , dose - dependent increases in circulating Z α1 - antitrypsin despite free concentrations being below the cellular EC50 for secretion for much or all of the dosing period . Significance at p < 0 . 05 by a student ' s t test is denoted by * , shown as pairwise comparisons ( each animal is compared with itself , treated vs pre - treatment ) and between groups ( compound treated vs vehicle ) , with n = 7 , 8 and 8 per group for day 6 dose level 10 , 30 and 100mg / Kg respectively ; day 15 , n = 10 ; day 21 , n = 5 . Data presented as mean ± SD .

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Z α1-antitrypsin transgenic animals were dosed with 10, 30 or 100 mg/kg GSK716 three times a day. Total concentrations of GSK716 were determined by LC-MS/MS (Table 2). (A) 100mg/kg GSK716 resulted in a mean 7-fold increase in circulating monomeric Z α1-antitrypsin levels demonstrating robust target engagement in the liver. The observed steady state total and free compound levels of GSK716 in the transgenic Z α1-antitrypsin mouse were well-predicted by the in silico PK model built on: (1) in vitro metabolic clearance data, (2) plasma protein binding data, (3) in vivo PK data from wild type mice and (4) a term comprising a 5μM circulating sink for drug with an affinity of 1.5 nM, representing the Z α1-antitrypsin within blood. The target free drug concentration was selected based on the observed potency in the in vitro secretion assays in which the total drug approximates to the free drug in the assay. Interestingly, 30mg/kg and 10mg/kg groups also gave significant, dose-dependent increases in circulating Z α1-antitrypsin despite free concentrations being below the cellular EC50 for secretion for much or all of the dosing period. Significance at p<0.05 by a student's t test is denoted by *, shown as pairwise comparisons (each animal is compared with itself, treated vs pre-treatment) and between groups (compound treated vs vehicle), with n= 7, 8 and 8 per group for day 6 dose level 10, 30 and 100mg/Kg respectively; day 15, n=10; day 21, n=5. Data presented as mean ± SD.


A . Flow cytometry of cells isolated from heart tissue when scaled against TMRM staining and also FSC and SSC parameters of flow cytometry .

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A. Flow cytometry of cells isolated from heart tissue when scaled against TMRM staining and also FSC and SSC parameters of flow cytometry.


B . Cardiac troponin I ( cTnI ) and α - Sarcomeric actinin ( α - SA ) staining of the TMRM + + population ( CMs ) and Vimentin staining of the TMRM + population ( NCs ) . The scale bar represents 100 µm .

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B. Cardiac troponin I (cTnI) and α-Sarcomeric actinin (α-SA) staining of the TMRM++ population (CMs) and Vimentin staining of the TMRM+ population (NCs). The scale bar represents 100 µm.


D . EGFP expression in iPSC - like colonies derived from CMs or NCs isolated from triple transgenic mice .

D. EGFP expression in iPSC-like colonies derived from CMs or NCs isolated from triple transgenic mice.


E . EGFP expression combined with immunofluorescence staining of SSEA - 1 and DAPI of iPSC - like colonies derived from CMs isolated from triple transgenic mice .

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E. EGFP expression combined with immunofluorescence staining of SSEA-1 and DAPI of iPSC-like colonies derived from CMs isolated from triple transgenic mice.


F . Morphological changes of isolated CMs and NCs at different time points before AP positive colony formation at day 6 . The scale bar represents 100 µm .

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F. Morphological changes of isolated CMs and NCs at different time points before AP positive colony formation at day 6. The scale bar represents 100 µm.


A . Gene profiling of CM and NC at D0 - D6 as shown clustered into 8 categories . Red indicates up - regulated genes while green indicates down - regulated genes ( n = 3 , n = 2 for NC - D6 ) .

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A. Gene profiling of CM and NC at D0-D6 as shown clustered into 8 categories. Red indicates up-regulated genes while green indicates down-regulated genes (n=3, n=2 for NC-D6).


B . Expression patterns of all genes per cluster by grey lines . Black line shows the mean value .

B. Expression patterns of all genes per cluster by grey lines. Black line shows the mean value.


D . Correlative analysis representing the relationships between CM and NC reprogramming specifically at days 2 , 4 and 6 . Red indicates closely related groups , while green indicates significantly different groups .

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D. Correlative analysis representing the relationships between CM and NC reprogramming specifically at days 2, 4 and 6. Red indicates closely related groups, while green indicates significantly different groups.


A . RNA expression ratio ( D2 - to - D0 ) of candidates selected from day 2 microarray data .

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A. RNA expression ratio (D2-to-D0) of candidates selected from day 2 microarray data.


C . Ki67 , α - MHC , and DAPI staining and quantification of CMs treated with control ( Ctrl ) , or combinations of a FoxM1 up - regulation adenovirus ( F ) , Id1 up - regulation adenovirus ( I ) , Hmgb2 up - regulation adenovirus ( H ) or a small molecule Jnk3 inhibitor ( Ji ) .

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C. Ki67, α-MHC, and DAPI staining and quantification of CMs treated with control (Ctrl), or combinations of a FoxM1 up-regulation adenovirus (F), Id1 up-regulation adenovirus (I), Hmgb2 up-regulation adenovirus (H) or a small molecule Jnk3 inhibitor (Ji).


D . Ki67 , α - MHC , and DAPI staining and quantification of CMs treated with Ctrl or combination of FI with Jnk3 - shRNAs in place of the small molecule ( Ji ) . The arrow head indicates Ki67 + / α - MHC + proliferated CMs .

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D. Ki67, α-MHC, and DAPI staining and quantification of CMs treated with Ctrl or combination of FI with Jnk3-shRNAs in place of the small molecule (Ji). The arrow head indicates Ki67+/α-MHC+ proliferated CMs.


E . Morphology and quantification of FIJs - treated CMs undergoing cytokinesis by H3P and Aurora kinase B ( AURKB ) staining .

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E. Morphology and quantification of FIJs-treated CMs undergoing cytokinesis by H3P and Aurora kinase B (AURKB) staining.


G . RNA expression ratio of Oct4 , FoxM1 , Id1 , or Jnk3 in doxycycline - injected versus control adult CMs isolated from reprogrammable mice .

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G. RNA expression ratio of Oct4, FoxM1, Id1, or Jnk3 in doxycycline-injected versus control adult CMs isolated from reprogrammable mice.


H . Immunofluorescence of H3P and cTnI and quantification of the heart tissue sections from control or doxycycline injected mice .

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H. Immunofluorescence of H3P and cTnI and quantification of the heart tissue sections from control or doxycycline injected mice.


B . The size of hearts isolated from Ctrl or FIJs - treated mice ; heart / body weight ratio of Ctrl or FIJs - treated mice .

B. The size of hearts isolated from Ctrl or FIJs-treated mice; heart/body weight ratio of Ctrl or FIJs-treated mice.


C . H3P , cTnI , and DAPI staining and quantification of heart tissues in Ctrl or FIJs - treated mice . The arrowhead indicates H3P + / cTnI + proliferated CMs and the scale bar represents 50 µm .

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C. H3P, cTnI, and DAPI staining and quantification of heart tissues in Ctrl or FIJs-treated mice. The arrowhead indicates H3P+/cTnI+ proliferated CMs and the scale bar represents 50 µm.


E . Immunofluorescence of H3P and cTnI and quantification of the heart tissue sections from adeno - Ctrl or adeno - FIJs injected mice .

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E. Immunofluorescence of H3P and cTnI and quantification of the heart tissue sections from adeno-Ctrl or adeno-FIJs injected mice.


F . Quantification of BrdU + / EGFP + population % in isolated CMs from adeno - Ctrl or adeno - FIJs injected M / Z mice by flow cytometry .

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F. Quantification of BrdU+/EGFP+ population % in isolated CMs from adeno-Ctrl or adeno-FIJs injected M/Z mice by flow cytometry.


B . RNA expressional changes of FoxM1 , Id1 , and Jnk3 after adeno - Ctrl or adeno - FIJs treatment after MI .

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B. RNA expressional changes of FoxM1, Id1, and Jnk3 after adeno-Ctrl or adeno-FIJs treatment after MI.


C . Immunofluorescence staining of BrdU or H3P and cTnI or EGFP and quantification in the heart tissues after MI . The arrow head indicates cTnI + / BrdU + , cTnI + / H3P + or EGFP + / BrdU + , EGFP + / H3P + proliferated CMs . The scale bar represents 50 µm .

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C. Immunofluorescence staining of BrdU or H3P and cTnI or EGFP and quantification in the heart tissues after MI. The arrow head indicates cTnI+/BrdU+, cTnI+/H3P+ or EGFP+/BrdU+, EGFP+/H3P+proliferated CMs. The scale bar represents 50 µm.


D . Ejection fraction and fraction shortening percentage in Ctrl or FIJs - treated mice 21 days post - MI .

D. Ejection fraction and fraction shortening percentage in Ctrl or FIJs-treated mice 21 days post-MI.


E . Catheterization in Ctrl or FIJs - treated mice 21 days post - MI under baseline or inferior vena cava occlusion condition .

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E. Catheterization in Ctrl or FIJs-treated mice 21 days post-MI under baseline or inferior vena cava occlusion condition.


F . Trichrome staining and quantification showing percentage of fibrotic tissue ( blue ) in Ctrl or FIJs - treated mice 21 days after MI .

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F. Trichrome staining and quantification showing percentage of fibrotic tissue (blue) in Ctrl or FIJs-treated mice 21 days after MI.


A . RNA expression of mitosis checkpoint genes in CMs with FoxM1 or Id1 overexpression , Jnk3 - shRNA , or combined treatment .

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A. RNA expression of mitosis checkpoint genes in CMs with FoxM1 or Id1 overexpression, Jnk3-shRNA, or combined treatment.


B . Transcriptional expression of cyclin - dependent kinases in FoxM1 or Id1 up - regulated , Jnk3 down - regulated , or combined treated CMs .

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B. Transcriptional expression of cyclin-dependent kinases in FoxM1 or Id1 up-regulated, Jnk3 down-regulated, or combined treated CMs.


C . Real - time RT - PCR analysis of CDK inhibitor expression in CMs with FoxM1 or Id1 overexpression , Jnk3 - shRNA or combined treatment .

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C. Real-time RT-PCR analysis of CDK inhibitor expression in CMs with FoxM1 or Id1 overexpression, Jnk3-shRNA or combined treatment.


( B ) Representative large image of a brain slice from R6 / 2 mice after 4 - week striatal infusion of fluorescent cholesterol ( bodipy - chol , green ) . In the inlet , infused striatum is shown . Hoechst ( Ho , blue ) was used to counterstain nuclei . Scale bar : 1000 µm .

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(B) Representative large image of a brain slice from R6/2 mice after 4-week striatal infusion of fluorescent cholesterol (bodipy-chol, green). In the inlet, infused striatum is shown. Hoechst (Ho, blue) was used to counterstain nuclei. Scale bar: 1000 µm.


( C ) Cholesterol content measured by mass spectrometry in infused striatum of untreated wt ( N = 7 ) and R6 / 2 ( N = 4 ) , R6 / 2 ACSF ( N = 5 ) , R6 / 2 chol - low ( N = 5 ) , R6 / 2 chol - medium ( N = 5 ) , and R6 / 2 chol - high ( N = 5 ) mice at 12 weeks of age after 4 - week striatal cholesterol infusion . The low , medium , and high doses respectively correspond to 15 µg , 185 µg , and 369 µg of cholesterol infused in the striatum after 4 weeks . Data information : The data are from five independent trials and shown as scatterplot graphs with means ± standard error . Each dot corresponds to the value obtained from each animal . Statistics : one - way ANOVA with Newman - Keuls post - hoc test ( * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 ; * * * * p < 0 . 0001 ) .

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

(C) Cholesterol content measured by mass spectrometry in infused striatum of untreated wt (N=7) and R6/2 (N=4), R6/2 ACSF (N=5), R6/2 chol-low (N=5), R6/2 chol-medium (N=5), and R6/2 chol-high (N=5) mice at 12 weeks of age after 4-week striatal cholesterol infusion. The low, medium, and high doses respectively correspond to 15 µg, 185 µg, and 369 µg of cholesterol infused in the striatum after 4 weeks. Data information: The data are from five independent trials and shown as scatterplot graphs with means±standard error. Each dot corresponds to the value obtained from each animal. Statistics: one-way ANOVA with Newman-Keuls post-hoc test (*p<0.05; **p<0.01; ***p<0.001; ****p<0.0001).


( D ) Latency to fall ( seconds ) from an accelerating rotarod from 5 weeks ( pre - surgery ; i . e . , before pump implantation ) to 10 weeks of age in wt ( N = 23 at 5 weeks , N = 28 at 8 and 10 weeks ) , R6 / 2 ACSF ( N = 35 at 5 weeks , N = 31 at 8 weeks and N = 30 at 10 weeks ) , R6 / 2 chol - low ( N = 22 at 5 weeks , N = 14 at 8 weeks and N = 13 ) , R6 / 2 chol - medium ( N = 13 at 5 weeks , N = 9 at 8 weeks and N = 8 at 10 weeks ) , and R6 / 2 chol - high ( N = 19 at 5 weeks , N = 17 at 8 and 10 weeks ) mice . The graph shows means ± standard error for each time point . Data information : The data are from five independent trials and shown as scatterplot graphs with means ± standard error . Statistics : one - way ANOVA with Newman - Keuls post - hoc test ( * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 ; * * * * p < 0 . 0001 ) .

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

(D) Latency to fall (seconds) from an accelerating rotarod from 5 weeks (pre-surgery; i.e., before pump implantation) to 10 weeks of age in wt (N=23 at 5 weeks, N=28 at 8 and 10 weeks), R6/2 ACSF (N=35 at 5 weeks, N=31 at 8 weeks and N=30 at 10 weeks), R6/2 chol-low (N=22 at 5 weeks, N=14 at 8 weeks and N=13), R6/2 chol-medium (N=13 at 5 weeks, N=9 at 8 weeks and N=8 at 10 weeks), and R6/2 chol-high (N=19 at 5 weeks, N=17 at 8 and 10 weeks) mice. The graph shows means±standard error for each time point. Data information: The data are from five independent trials and shown as scatterplot graphs with means±standard error. Statistics: one-way ANOVA with Newman-Keuls post-hoc test (*p<0.05; **p<0.01; ***p<0.001; ****p<0.0001).


Global motor activity ( E ) , in an open - field test at 6 weeks of age ( pre - surgery ) and 11 weeks of age ( 4 weeks after infusion ) of wt ( N = 16 at 9 weeks , N = 22 at 11 weeks ) , R6 / 2 ACSF ( N = 27 at 9 weeks , N = 23 at 11 weeks ) , R6 / 2 chol - low ( N = 20 at 9 weeks , N = 13 at 11 weeks ) , R6 / 2 chol - medium ( N = 9 at 9 weeks , N = 7 at 11 weeks ) , and R6 / 2 chol - high ( N = 18 at 9 weeks , N = 16 at 11 weeks ) mice . Data information : The data are from five independent trials and shown as scatterplot graphs with means ± standard error . Each dot corresponds to the value obtained from each animal . Statistics : one - way ANOVA with Newman - Keuls post - hoc test ( * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 ; * * * * p < 0 . 0001 ) .

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

Global motor activity (E), in an open-field test at 6 weeks of age (pre-surgery) and 11 weeks of age (4 weeks after infusion) of wt (N=16 at 9 weeks, N=22 at 11 weeks), R6/2 ACSF (N=27 at 9 weeks, N=23 at 11 weeks), R6/2 chol-low (N=20 at 9 weeks, N=13 at 11 weeks), R6/2 chol-medium (N=9 at 9 weeks, N=7 at 11 weeks), and R6/2 chol-high (N=18 at 9 weeks, N=16 at 11 weeks) mice. Data information: The data are from five independent trials and shown as scatterplot graphs with means±standard error. Each dot corresponds to the value obtained from each animal. Statistics: one-way ANOVA with Newman-Keuls post-hoc test (*p<0.05; **p<0.01; ***p<0.001; ****p<0.0001).


total distance traveled ( F ) , mean velocity ( G ) , stereotyped movements ( H ) , in an open - field test at 6 weeks of age ( pre - surgery ) and 11 weeks of age ( 4 weeks after infusion ) of wt ( N = 16 at 9 weeks , N = 22 at 11 weeks ) , R6 / 2 ACSF ( N = 27 at 9 weeks , N = 23 at 11 weeks ) , R6 / 2 chol - low ( N = 20 at 9 weeks , N = 13 at 11 weeks ) , R6 / 2 chol - medium ( N = 9 at 9 weeks , N = 7 at 11 weeks ) , and R6 / 2 chol - high ( N = 18 at 9 weeks , N = 16 at 11 weeks ) mice . Data information : The data are from five independent trials and shown as scatterplot graphs with means ± standard error . Each dot corresponds to the value obtained from each animal . Statistics : one - way ANOVA with Newman - Keuls post - hoc test ( * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 ; * * * * p < 0 . 0001 ) .

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

total distance traveled (F), mean velocity (G), stereotyped movements (H), in an open-field test at 6 weeks of age (pre-surgery) and 11 weeks of age (4 weeks after infusion) of wt (N=16 at 9 weeks, N=22 at 11 weeks), R6/2 ACSF (N=27 at 9 weeks, N=23 at 11 weeks), R6/2 chol-low (N=20 at 9 weeks, N=13 at 11 weeks), R6/2 chol-medium (N=9 at 9 weeks, N=7 at 11 weeks), and R6/2 chol-high (N=18 at 9 weeks, N=16 at 11 weeks) mice. Data information: The data are from five independent trials and shown as scatterplot graphs with means±standard error. Each dot corresponds to the value obtained from each animal. Statistics: one-way ANOVA with Newman-Keuls post-hoc test (*p<0.05; **p<0.01; ***p<0.001; ****p<0.0001).


number of rearings ( I ) in an open - field test at 6 weeks of age ( pre - surgery ) and 11 weeks of age ( 4 weeks after infusion ) of wt ( N = 16 at 9 weeks , N = 22 at 11 weeks ) , R6 / 2 ACSF ( N = 27 at 9 weeks , N = 23 at 11 weeks ) , R6 / 2 chol - low ( N = 20 at 9 weeks , N = 13 at 11 weeks ) , R6 / 2 chol - medium ( N = 9 at 9 weeks , N = 7 at 11 weeks ) , and R6 / 2 chol - high ( N = 18 at 9 weeks , N = 16 at 11 weeks ) mice . Data information : The data are from five independent trials and shown as scatterplot graphs with means ± standard error . Each dot corresponds to the value obtained from each animal . Statistics : one - way ANOVA with Newman - Keuls post - hoc test ( * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 ; * * * * p < 0 . 0001 ) .

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

number of rearings (I) in an open-field test at 6 weeks of age (pre-surgery) and 11 weeks of age (4 weeks after infusion) of wt (N=16 at 9 weeks, N=22 at 11 weeks), R6/2 ACSF (N=27 at 9 weeks, N=23 at 11 weeks), R6/2 chol-low (N=20 at 9 weeks, N=13 at 11 weeks), R6/2 chol-medium (N=9 at 9 weeks, N=7 at 11 weeks), and R6/2 chol-high (N=18 at 9 weeks, N=16 at 11 weeks) mice. Data information: The data are from five independent trials and shown as scatterplot graphs with means±standard error. Each dot corresponds to the value obtained from each animal. Statistics: one-way ANOVA with Newman-Keuls post-hoc test (*p<0.05; **p<0.01; ***p<0.001; ****p<0.0001).


( J , K ) Representative track plots ( J ) generated from the open - field test from wt ( N = 16 at 9 weeks , N = 22 at 11 weeks ) , R6 / 2 ACSF ( N = 26 at 9 weeks , N = 23 at 11 weeks ) , R6 / 2 chol - low ( N = 11 at 9 weeks , N = 7 at 11 weeks ) , R6 / 2 chol - medium ( N = 11 at 9 weeks , N = 7 at 11 weeks ) , and R6 / 2 chol - high mice ( N = 18 at 9 weeks , N = 16 at 11 weeks ) and relative quantification ( K ) of the time spent in the center and in the periphery ( % ) of the arena . Data information : The data are from five independent trials and shown as scatterplot graphs with means ± standard error . Statistics : one - way ANOVA with Newman - Keuls post - hoc test ( * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 ; * * * * p < 0 . 0001 ) .

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

(J, K) Representative track plots (J) generated from the open-field test from wt (N=16 at 9 weeks, N=22 at 11 weeks), R6/2 ACSF (N=26 at 9 weeks, N=23 at 11 weeks), R6/2 chol-low (N=11 at 9 weeks, N=7 at 11 weeks), R6/2 chol-medium (N=11 at 9 weeks, N=7 at 11 weeks), and R6/2 chol-high mice (N=18 at 9 weeks, N=16 at 11 weeks) and relative quantification (K) of the time spent in the center and in the periphery (%) of the arena. Data information: The data are from five independent trials and shown as scatterplot graphs with means±standard error. Statistics: one-way ANOVA with Newman-Keuls post-hoc test (*p<0.05; **p<0.01; ***p<0.001; ****p<0.0001).


( L ) Discrimination index ( DI ; % ) in the novel object recognition test at 6 weeks of age ( before pump implantation ) and 11 weeks of age ( 4 weeks after cholesterol infusion ) of wt ( N = 16 at 9 weeks , N = 21 at 11 weeks ) , R6 / 2 ACSF ( N = 28 at 9 weeks , N = 23 at 11 weeks ) , R6 / 2 chol - low ( N = 21 at 9 weeks , N = 13 at 11 weeks ) , R6 / 2 chol - medium ( N = 10 at 9 weeks , N = 7 at 11 weeks ) , and R6 / 2 chol - high mice ( N = 18 at 9 weeks , N = 16 at 11 weeks ) . DI above zero indicates a preference for the novel object ; DI below zero indicates a preference for the familiar object . Data information : The data are from five independent trials and shown as scatterplot graphs with means ± standard error . Each dot corresponds to the value obtained from each animal . Statistics : one - way ANOVA with Newman - Keuls post - hoc test ( * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 ; * * * * p < 0 . 0001 ) .

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

(L) Discrimination index (DI; %) in the novel object recognition test at 6 weeks of age (before pump implantation) and 11 weeks of age (4 weeks after cholesterol infusion) of wt (N=16 at 9 weeks, N=21 at 11 weeks), R6/2 ACSF (N=28 at 9 weeks, N=23 at 11 weeks), R6/2 chol-low (N=21 at 9 weeks, N=13 at 11 weeks), R6/2 chol-medium (N=10 at 9 weeks, N=7 at 11 weeks), and R6/2 chol-high mice (N=18 at 9 weeks, N=16 at 11 weeks). DI above zero indicates a preference for the novel object; DI below zero indicates a preference for the familiar object. Data information: The data are from five independent trials and shown as scatterplot graphs with means±standard error. Each dot corresponds to the value obtained from each animal. Statistics: one-way ANOVA with Newman-Keuls post-hoc test (*p<0.05; **p<0.01; ***p<0.001; ****p<0.0001).


( B ) Spontaneous EPSCs were recorded from striatal MSNs ( wt = 6 ; R6 / 2 ACSF = 5 ; R6 / 2 chol - high = 5 ) at a holding potential of - 70 mV .

O O O O O O O O O O O O O O O O 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) Spontaneous EPSCs were recorded from striatal MSNs (wt=6; R6/2 ACSF=5; R6/2 chol-high=5) at a holding potential of -70 mV.


( C - D ) Average amplitude ( C ) and average frequency ( D ) of EPSCs from wt , R6 / 2 ACSF , and R6 / 2 chol - high MSNs . Data information : The data are shown as scatterplot graphs with means ± standard error . Each dot corresponds to the value of each cell recorded Statistics : one - way ANOVA with Newman - Keuls post - hoc test ( * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 ; * * * * p < 0 . 0001 ) .

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

(C-D) Average amplitude (C) and average frequency (D) of EPSCs from wt, R6/2 ACSF, and R6/2 chol-high MSNs. Data information: The data are shown as scatterplot graphs with means±standard error. Each dot corresponds to the value of each cell recorded Statistics: one-way ANOVA with Newman-Keuls post-hoc test (*p<0.05; **p<0.01; ***p<0.001; ****p<0.0001).


( E - G ) Number of excitatory synapses per volume of striatum by using FIB / SEM followed by 3D reconstruction . ( E ) Representative tissue block of striatum ( 10 µm x 15 µm x 10 µm ) used for the 3D analysis . ( F ) Representative FIB - SEM segmentation and reconstruction of excitatory synapses ( yellow ) in 200 serial sections of striatum for a total volume of 500 µm3 , of wt , R6 / 2 , R6 / 2 ACSF , and R6 / 2 chol - high mice . ( G ) Density of excitatory synapses in at least 1500 μm3 of striatal tissue from wt , R6 / 2 , R6 / 2 ACSF , and R6 / 2 chol - high mice ( N = 3 animals / group ) . Data information : The data are shown as scatterplot graphs with means ± standard error . Each dot corresponds to the value of , the number of synapses counted / µm3 in different blocks of striatal tissue ( G ) Statistics : one - way ANOVA with Newman - Keuls post - hoc test ( * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 ; * * * * p < 0 . 0001 ) .

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

(E-G) Number of excitatory synapses per volume of striatum by using FIB/SEM followed by 3D reconstruction. (E) Representative tissue block of striatum (10 µm x 15 µm x 10 µm) used for the 3D analysis. (F) Representative FIB-SEM segmentation and reconstruction of excitatory synapses (yellow) in 200 serial sections of striatum for a total volume of 500 µm3, of wt, R6/2, R6/2 ACSF, and R6/2 chol-high mice. (G) Density of excitatory synapses in at least 1500 μm3 of striatal tissue from wt, R6/2, R6/2 ACSF, and R6/2 chol-high mice (N=3 animals/group). Data information: The data are shown as scatterplot graphs with means±standard error. Each dot corresponds to the value of , the number of synapses counted/µm3 in different blocks of striatal tissue (G) Statistics: one-way ANOVA with Newman-Keuls post-hoc test (*p<0.05; **p<0.01; ***p<0.001; ****p<0.0001).


( H - I ) TEM images ( H ) and 2D profile ( I ) of pre - synaptic terminal sections contain SVs and mitochondria ( m ) . Docked vesicles are indicated by a white arrowhead ( H ) and black circle ( I ) and are defined as a vesicle with its center located within 20 nm from the presynaptic membrane . Scale bar : 100 nm .

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

(H-I) TEM images (H) and 2D profile (I) of pre-synaptic terminal sections contain SVs and mitochondria (m). Docked vesicles are indicated by a white arrowhead (H) and black circle (I) and are defined as a vesicle with its center located within 20 nm from the presynaptic membrane. Scale bar: 100 nm.


( J , K ) Quantification showing the total SVs / µm2 ( J ) and the docked SVs / µm ( K ) in R6 / 2 , R6 / 2 ACSF , and wt striatal synapses ( N = 3 animals / group ) . N = 60 excitatory synapses / group were counted . Data information : The data are shown as scatterplot graphs with means ± standard error . Each dot corresponds to the number of total SVs / µm2 ( J ) and docked SVs / µm of active zone ( K ) counted in each synapse . Statistics : one - way ANOVA with Newman - Keuls post - hoc test ( * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 ; * * * * p < 0 . 0001 ) .

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

(J, K) Quantification showing the total SVs/µm2 (J) and the docked SVs/µm (K) in R6/2, R6/2 ACSF, and wt striatal synapses (N=3 animals/group). N=60 excitatory synapses/group were counted. Data information: The data are shown as scatterplot graphs with means±standard error. Each dot corresponds to the number of total SVs/µm2 (J) and docked SVs/µm of active zone (K) counted in each synapse. Statistics: one-way ANOVA with Newman-Keuls post-hoc test (*p<0.05; **p<0.01; ***p<0.001; ****p<0.0001).


( A ) Spontaneous IPSCs were recorded from striatal MSNs ( wt = 6 ; R6 / 2 ACSF = 5 ; R6 / 2 chol - high = 5 ) at a holding potential of 0 mV .

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

(A) Spontaneous IPSCs were recorded from striatal MSNs (wt=6; R6/2 ACSF=5; R6/2 chol-high=5) at a holding potential of 0 mV.


( B - C ) Average amplitude ( B ) and average frequency ( C ) of IPSCs from wt , R6 / 2 ACSF , and R6 / 2 chol - high MSNs . Data information : The data are shown as scatterplot graphs with means ± standard error . Each dot corresponds to the value of each cell recorded ( B , C ) Statistics : one - way ANOVA with Newman - Keuls post - hoc test ( * * p < 0 . 01 ; * * * p < 0 . 001 ) .

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

(B-C) Average amplitude (B) and average frequency (C) of IPSCs from wt, R6/2 ACSF, and R6/2 chol-high MSNs. Data information: The data are shown as scatterplot graphs with means±standard error. Each dot corresponds to the value of each cell recorded (B, C) Statistics: one-way ANOVA with Newman-Keuls post-hoc test (**p<0.01; ***p<0.001).


( D - E ) Evaluation of the number of inhibitory synapses per volume of striatum by using FIB / SEM followed by 3D reconstruction . ( D ) Representative FIB - SEM segmentation and reconstruction of inhibitory synapses ( green ) in 200 serial sections of striatum for a total volume of 500 µm3 in wt , R6 / 2 , R6 / 2 ACSF , and R6 / 2 chol - high mice . ( E ) Density of inhibitory synapses in at least 2000 µm3 of striatal tissue of wt , R6 / 2 , R6 / 2 ACSF , and R6 / 2 chol - high mice ( N = 3 mice / group ) . Data information : The data E ) are shown as scatterplot graphs with means ± standard error . Each dot corresponds to the number of synapses counted / µm3 in different blocks of tissue for each group of animals ( N = 3 animals / group ) ( E ) . Statistics : one - way ANOVA with Newman - Keuls post - hoc test ( * * p < 0 . 01 ; * * * p < 0 . 001 ) .

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

(D-E) Evaluation of the number of inhibitory synapses per volume of striatum by using FIB/SEM followed by 3D reconstruction. (D) Representative FIB-SEM segmentation and reconstruction of inhibitory synapses (green) in 200 serial sections of striatum for a total volume of 500 µm3 in wt, R6/2, R6/2 ACSF, and R6/2 chol-high mice. (E) Density of inhibitory synapses in at least 2000 µm3 of striatal tissue of wt, R6/2, R6/2 ACSF, and R6/2 chol-high mice (N=3 mice/ group). Data information: The data E) are shown as scatterplot graphs with means±standard error. Each dot corresponds to the number of synapses counted /µm3 in different blocks of tissue for each group of animals (N=3 animals/group) (E). Statistics: one-way ANOVA with Newman-Keuls post-hoc test (**p<0.01; ***p<0.001).


( B ) 24S - OHC level measured by mass spectrometry in the infused and contralateral striatum of wt , R6 / 2 ‐ ACSF , and R6 / 2 chol - high mice at 12 weeks of age after a 4 - week striatal infusion of cholesterol ( N = 4 / group ) . All values are expressed as % above the mean of wt , and these data are shown as scatterplots with means ± standard error . Each dot corresponds to the value obtained from each animal .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) 24S-OHC level measured by mass spectrometry in the infused and contralateral striatum of wt, R6/2‐ACSF, and R6/2 chol-high mice at 12 weeks of age after a 4-week striatal infusion of cholesterol (N=4/group). All values are expressed as % above the mean of wt, and these data are shown as scatterplots with means±standard error. Each dot corresponds to the value obtained from each animal.


( C - E ) Lanosterol ( C ) , lathosterol ( D ) , and desmosterol ( E ) level measured by mass spectrometry in the infused and contralateral striatum of wt , R6 / 2 ACSF , and R6 / 2 chol - high mice at 12 weeks of age after 4 - week striatal infusion of cholesterol ( N = 4 / group ) . All values are expressed as % above the mean of wt , and these data are shown as scatterplots with means ± standard error . Each dot corresponds to the value obtained from each animal .

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

(C-E) Lanosterol (C), lathosterol (D), and desmosterol (E) level measured by mass spectrometry in the infused and contralateral striatum of wt, R6/2 ACSF, and R6/2 chol-high mice at 12 weeks of age after 4-week striatal infusion of cholesterol (N=4/group). All values are expressed as % above the mean of wt, and these data are shown as scatterplots with means±standard error. Each dot corresponds to the value obtained from each animal.


( F - I ) Nuclear ( white arrows ) and perinuclear ( yellow arrows ) localization of endogenous SREBP2 in the striatum of wt , R6 / 2 and R6 / 2 - chol mice . Representative confocal image ( F and H ) and relative quantification ( G and I ) of SREBP2 ( red ) in wt and R6 / 2 mice and in the infused and contralateral striatum of R6 / 2 chol - high mice ( N = 4 ) . Hoechst ( Ho , blue ) was used to counterstain nuclei . Scale bar in F and H : 10 µm . Graphs in ( G and I ) represents the intensity of SREBP2 normalized on nuclei ( % ) . Data are shown as scatterplots with means ± standard error . Each dot corresponds to the value obtained from each image . Statistics : Student ' s t - test ( * * p < 0 . 01 ; * * * * p < 0 . 0001 ) .

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

(F-I) Nuclear (white arrows) and perinuclear (yellow arrows) localization of endogenous SREBP2 in the striatum of wt, R6/2 and R6/2-chol mice. Representative confocal image (F and H) and relative quantification (G and I) of SREBP2 (red) in wt and R6/2 mice and in the infused and contralateral striatum of R6/2 chol-high mice (N=4). Hoechst (Ho, blue) was used to counterstain nuclei. Scale bar in F and H: 10 µm. Graphs in (G and I) represents the intensity of SREBP2 normalized on nuclei (%). Data are shown as scatterplots with means±standard error. Each dot corresponds to the value obtained from each image. Statistics: Student's t-test (**p<0.01; ****p<0.0001).


J , K Representative high - magnification confocal images of immunostaining against SREBP2 ( red ) and NeuN ( J ) or GFAP ( K ) ( green ) on coronal sections of brains from R6 / 2 chol - high mice . A cell in the infused striatum , positive for NeuN and GFAP respectively , is shown . Hoechst ( Ho , blue ) was used to counterstain nuclei . Scale bars : 1 µm .

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

J, K Representative high-magnification confocal images of immunostaining against SREBP2 (red) and NeuN (J) or GFAP (K) (green) on coronal sections of brains from R6/2 chol-high mice. A cell in the infused striatum, positive for NeuN and GFAP respectively, is shown. Hoechst (Ho, blue) was used to counterstain nuclei. Scale bars: 1 µm.