( J ) Fragments of SQST - 1 containing amino acids 351 - 499 and 604 - 693 , but not 500 - 603 , interact with LGG - 1 .

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

(J) Fragments of SQST-1 containing amino acids 351-499 and 604-693, but not 500-603, interact with LGG-1.


( K ) Fragments of SQST - 1 - containing amino acids 418 - 499 and 604 - 630 bind to LGG - 1 . Mutating the putative LIR in the 604 - 630 fragment ( 605YPDL608 to APDA ) does not affect the binding .

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

(K) Fragments of SQST-1-containing amino acids 418-499 and 604-630 bind to LGG-1. Mutating the putative LIR in the 604-630 fragment (605YPDL608 to APDA) does not affect the binding.


( L and M ) SQST - 1 ( del LGG - 1 - 1 + 2 ) : : GFP , in which the two LGG - 1 - binding fragments ( amino acids 418 - 499 and 604 - 630 ) are deleted , is weakly expressed and diffusely localized in the cytoplasm in sqst - 1 ( ok2892 ) mutants . ( N and O ) Loss of function of lgg - 1 dramatically elevates the expression level of SQST - 1 ( del LGG - 1 - 1 + 2 ) : : GFP , which forms a large number of aggregates . ( L and N ) DIC images of the embryos shown in M and O ,

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 and M) SQST-1(del LGG-1-1+2)::GFP, in which the two LGG-1-binding fragments (amino acids 418-499 and 604-630) are deleted, is weakly expressed and diffusely localized in the cytoplasm in sqst-1(ok2892) mutants. (N and O) Loss of function of lgg-1 dramatically elevates the expression level of SQST-1(del LGG-1-1+2)::GFP, which forms a large number of aggregates. (L and N) DIC images of the embryos shown in M and O, respectively


( P ) SQST - 1 ( del LGG - 1 - 1 + 2 ) : : GFP aggregates colocalize with EPG - 7 in lgg - 1 ; sqst - 1 mutants . Insets show a magnified view .

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

(P) SQST-1(del LGG-1-1+2)::GFP aggregates colocalize with EPG-7 in lgg-1;sqst-1 mutants. Insets show a magnified view.


Formation of ATG - 9 puncta in various autophagy mutants . ( A and B ) In wild - type embryos , ATG - 9 : : GFP is diffusely localized in the cytoplasm . ( A ) DIC image of the embryo shown in B . Insets show a magnified view . ( C ) ATG - 9 : : GFP forms a large number of small , intense puncta in epg - 1 mutants . ( D and E ) Accumulation of ATG - 9 : : GFP puncta in unc - 51 ( D ) and epg - 1 ( E ) mutants is independent of lgg - 1 . ( F ) The ATG - 9 : : GFP puncta largely disappear in epg - 1 ; epg - 7 mutants . ( G ) ATG - 9 : : GFP accumulates into large punctate structures in epg - 6 mutants . ( H ) Loss of function of lgg - 1 suppresses the accumulation of ATG - 9 : : GFP puncta in epg - 6 mutants . ( I ) Loss of function of epg - 7 has no effect on the formation of ATG - 9 : : GFP puncta in epg - 6 mutants . The ATG - 9 : : GFP puncta are smaller in epg - 6 ; epg - 7 mutants than those in epg - 6 single mutants . ( J - L ) ATG - 9 : : GFP accumulates into large punctate structures in epg - 4 mutants ( J ) and the accumulation is suppressed by simultaneously depleting the activity of lgg - 1 ( K ) or epg - 7 ( L ) . ( M and N ) In epg - 8 mutants , ATG - 9 accumulates into aggregates ( M ) that are suppressed by loss of lgg - 1 activity ( N ) . ( O ) epg - 7 mutants exhibit the same distribution pattern of ATG - 9 : : GFP as wild - type embryos . ( P ) Percentage of ATG - 9 : : GFP puncta colocalized with EPG - 7 and SQST - 1 aggregates in indicated autophagy mutants .

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

Formation of ATG-9 puncta in various autophagy mutants. (A and B) In wild-type embryos, ATG-9::GFP is diffusely localized in the cytoplasm. (A) DIC image of the embryo shown in B. Insets show a magnified view. (C) ATG-9::GFP forms a large number of small, intense puncta in epg-1 mutants. (D and E) Accumulation of ATG-9::GFP puncta in unc-51(D) and epg-1 (E) mutants is independent of lgg-1. (F) The ATG-9::GFP puncta largely disappear in epg-1;epg-7 mutants. (G) ATG-9::GFP accumulates into large punctate structures in epg-6 mutants. (H) Loss of function of lgg-1 suppresses the accumulation of ATG-9::GFP puncta in epg-6 mutants. (I) Loss of function of epg-7 has no effect on the formation of ATG-9::GFP puncta in epg-6 mutants. The ATG-9::GFP puncta are smaller in epg-6;epg-7 mutants than those in epg-6 single mutants. (J-L) ATG-9::GFP accumulates into large punctate structures in epg-4 mutants (J) and the accumulation is suppressed by simultaneously depleting the activity of lgg-1 (K) or epg-7 (L). (M and N) In epg-8 mutants, ATG-9 accumulates into aggregates (M) that are suppressed by loss of lgg-1 activity (N). (O) epg-7 mutants exhibit the same distribution pattern of ATG-9::GFP as wild-type embryos. (P) Percentage of ATG-9::GFP puncta colocalized with EPG-7 and SQST-1 aggregates in indicated autophagy mutants.


( Q - T ) The ATG - 9 : : GFP puncta in epg - 6 ( Q ) , epg - 8 ( R ) , unc - 51 ( S ) , and atg - 18 ( T ) mutants colocalize with EPG - 7 aggregates .

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

(Q-T) The ATG-9::GFP puncta in epg-6 (Q), epg-8 (R), unc-51 (S), and atg-18 (T) mutants colocalize with EPG-7 aggregates.


( U and V ) ATG - 9 : : GFP puncta in epg - 6 ( U ) and epg - 8 ( V ) mutants colocalize with SQST - 1 aggregates . ( W and X ) In unc - 51 ( W ) and atg - 18 ( X ) mutants , ATG - 9 : : GFP partially colocalizes with SQST - 1 aggregates .

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

(U and V) ATG-9::GFP puncta in epg-6 (U) and epg-8 (V) mutants colocalize with SQST-1 aggregates. (W and X) In unc-51 (W) and atg-18 (X) mutants, ATG-9::GFP partially colocalizes with SQST-1 aggregates.


( A2 ) Levels of W07G4 . 5 : : GFP in wild type , epg - 7 mutants , and atg - 3 L1 larvae before and after 6 h starvation treatment . ( B2 ) Model for the role of epg - 7 in degradation of SQST - 1 aggregates . SQST - 1 directly interacts with EPG - 7 and is recruited into EPG - 7 aggregates . EPG - 7 also associates with multiple Atg proteins , which may be recruited to the SQST - 1 - EPG - 7 complex in a hierarchical order . IM , isolation membrane . ( C2 ) Hierarchical relationship of the scaffold proteins EPG - 2 and EPG - 7 and other essential ATG and EPG proteins in the aggrephagy

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

(A2) Levels of W07G4.5::GFP in wild type, epg-7 mutants, and atg-3 L1 larvae before and after 6 h starvation treatment. (B2) Model for the role of epg-7 in degradation of SQST-1 aggregates. SQST-1 directly interacts with EPG-7 and is recruited into EPG-7 aggregates. EPG-7 also associates with multiple Atg proteins, which may be recruited to the SQST-1-EPG-7 complex in a hierarchical order. IM, isolation membrane. (C2) Hierarchical relationship of the scaffold proteins EPG-2 and EPG-7 and other essential ATG and EPG proteins in the aggrephagy pathway


( A and B ) C17E4 . 2 : : GFP is weakly expressed and diffusely localized in a wild - type embryo . ( A ) DIC image of the embryo shown in B . ( C and D ) C17E4 . 2 : : GFP is expressed at dramatically elevated levels and accumulates into many aggregates in lgg - 1 ( C ) and epg - 7 ( D ) mutant embryos .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 and B) C17E4.2::GFP is weakly expressed and diffusely localized in a wild-type embryo. (A) DIC image of the embryo shown in B. (C and D) C17E4.2::GFP is expressed at dramatically elevated levels and accumulates into many aggregates in lgg-1 (C) and epg-7 (D) mutant embryos.


( E and F ) W07G4 . 5 : : GFP is expressed in the intestine and a few aggregates are formed in a wild - type embryo at the fourfold stage . ( E ) DIC image of the embryo shown in F . ( G and H ) W07G4 . 5 : : GFP is expressed at dramatically elevated levels and accumulates into a large number of aggregates in atg - 3 ( G ) and epg - 7 ( H ) mutants .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 and F) W07G4.5::GFP is expressed in the intestine and a few aggregates are formed in a wild-type embryo at the fourfold stage. (E) DIC image of the embryo shown in F. (G and H) W07G4.5::GFP is expressed at dramatically elevated levels and accumulates into a large number of aggregates in atg-3 (G) and epg-7 (H) mutants.


( I ) Levels of C33D9 . 6 : : GFP and W07G4 . 5 : : GFP are dramatically elevated in atg - 3 and epg - 7 mutant embryos .

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

(I) Levels of C33D9.6::GFP and W07G4.5::GFP are dramatically elevated in atg-3 and epg-7 mutant embryos.


( J and K ) C17E4 . 2 aggregates in atg - 3 mutants overlap with EPG - 7 aggregates ( J ) , but are separable from SQST - 1 aggregates ( K ) . Insets show a magnified view . ( L ) Colocalization frequency of C17E4 . 2 , C33D9 . 6 , and W07G4 . 5 aggregates with EPG - 7 and SQST - 1 aggregates in atg - 3 mutants .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 and K) C17E4.2 aggregates in atg-3 mutants overlap with EPG-7 aggregates (J), but are separable from SQST-1 aggregates (K). Insets show a magnified view. (L) Colocalization frequency of C17E4.2, C33D9.6, and W07G4.5 aggregates with EPG-7 and SQST-1 aggregates in atg-3 mutants.


( M ) SQST - 1 : : GFP accumulates in hypodermal cells in epg - 7 mutants at the larval stage . ( N ) SQST - 1 : : GFP aggregates disappear in rheb - 1 ( RNAi ) ; epg - 7 mutant larvae . ( O ) Dramatic accumulation of SQST - 1 : : GFP aggregates in hypodermal cells in atg - 3 mutant larvae . ( P ) SQST - 1 : : GFP aggregates persist in rheb - 1 ( RNAi ) ; atg - 3 mutant larvae .

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

(M) SQST-1::GFP accumulates in hypodermal cells in epg-7 mutants at the larval stage. (N) SQST-1::GFP aggregates disappear in rheb-1(RNAi);epg-7 mutant larvae. (O) Dramatic accumulation of SQST-1::GFP aggregates in hypodermal cells in atg-3 mutant larvae. (P) SQST-1::GFP aggregates persist in rheb-1(RNAi);atg-3 mutant larvae.


( Q - T ) At larval stages , W07G4 . 5 : : GFP forms aggregates in intestinal cells ( Q and R ) . These aggregates disappear upon RNAi knockdown of let - 363 ( S and T ) . ( U - X ) W07G4 . 5 : : GFP aggregates accumulate in intestinal cells in epg - 7 mutant larvae ( U and V ) . These aggregates disappear when let - 363 is simultaneously depleted ( W and X ) . ( Y and Z ) In atg - 3 mutants , W07G4 . 5 : : GFP aggregates accumulate in intestinal cells ( Y ) and persist after RNAi knockdown of let - 363 ( Z ) .

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

(Q-T) At larval stages, W07G4.5::GFP forms aggregates in intestinal cells (Q and R). These aggregates disappear upon RNAi knockdown of let-363 (S and T). (U-X) W07G4.5::GFP aggregates accumulate in intestinal cells in epg-7 mutant larvae (U and V). These aggregates disappear when let-363 is simultaneously depleted (W and X). (Y and Z) In atg-3 mutants, W07G4.5::GFP aggregates accumulate in intestinal cells (Y) and persist after RNAi knockdown of let-363 (Z).


A , B Regression plot showing the association between baseline AV45 PET ( x - axis ) and annual AV45 PET change ( y - axis ) across the entire sample ( N = 300 ) . The vertical dashed line represents the Aβ - positivity threshold of AV45 PET SUVR > 0 . 079 ( A ) . Regression plot for the association between baseline AV45 PET and annual AV45 PET change stratified by baseline CSF sTREM2 levels ( B ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, B Regression plot showing the association between baseline AV45 PET (x-axis) and annual AV45 PET change (y-axis) across the entire sample (N=300). The vertical dashed line represents the Aβ-positivity threshold of AV45 PET SUVR > 0.079 (A). Regression plot for the association between baseline AV45 PET and annual AV45 PET change stratified by baseline CSF sTREM2 levels (B).


Boxplots ( central band = median , boxes = Quartiles , whiskers = 1 . 5 * interquartile range ) , illustrating the differences in GE180 PET ( A ) compared between wild type C57BL / 6 mice ( n = 43 ) and APPNL - G - G ( n = 15 ) .

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

Boxplots (central band = median, boxes = Quartiles, whiskers = 1.5 * interquartile range), illustrating the differences in GE180 PET (A) compared between wild type C57BL/6 mice (n=43) and APPNL-G-G (n=15).


B Boxplots ( central band = median , boxes = Quartiles , whiskers = 1 . 5 * interquartile range ) , illustrating the differences in the rate of change in Florbetaben PET ( B ) compared between wild type C57BL / 6 mice ( n = 43 ) and APPNL - G - G ( n = 15 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Boxplots (central band = median, boxes = Quartiles, whiskers = 1.5 * interquartile range), illustrating the differences in the rate of change in Florbetaben PET (B) compared between wild type C57BL/6 mice (n=43) and APPNL-G-G (n=15).


C The regression plot shows lower rate of change in Florbetaben PET to be associated with lower GE180 PET at baseline , i . e . at 5 months of age ( C ) . The shaded area corresponds to the 95 % CI of the regression line .

O O O O O O O O O O O O O O O O O O O O O O O O O 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 The regression plot shows lower rate of change in Florbetaben PET to be associated with lower GE180 PET at baseline, i.e. at 5 months of age (C). The shaded area corresponds to the 95% CI of the regression line.


A - C Coronal ( top row ) and axial ( bottom row ) slices of average 18 - F GE - 180 TSPO ( microglia ) tracer and 18F - florbetaben amyloid - PET split up by group ( A - C ) : C57BL / 6 ( A ) , APPNL - G - F mice subgroup with a low ( < median ) GE - 180 PET ( B ) , and the APPNL - G - F mice subgroup with a high ( > median ) GE - 180 PET ( C ) . APPNL - G - F mice with low baseline GE180 PET levels showed faster increase in Florbetaben PET between 5 and 10 months ( B ) compared to those at high baseline level of GE - 180 PET ( C ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-C Coronal (top row) and axial (bottom row) slices of average 18-F GE-180 TSPO (microglia) tracer and 18F-florbetaben amyloid-PET split up by group (A - C): C57BL/6 (A), APPNL-G-F mice subgroup with a low (< median) GE-180 PET (B), and the APPNL-G-F mice subgroup with a high (> median) GE-180 PET (C). APPNL-G-F mice with low baseline GE180 PET levels showed faster increase in Florbetaben PET between 5 and 10 months (B) compared to those at high baseline level of GE-180 PET (C).


Effects of sTREM2 on AV1451 - PET - assessed tau pathology stratified by Braak - stages shown as boxplots ( central band = median , boxes = Quartiles , whiskers = 1 . 5 * interquartile range ) .

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

Effects of sTREM2 on AV1451-PET-assessed tau pathology stratified by Braak-stages shown as boxplots (central band = median, boxes = Quartiles, whiskers = 1.5 * interquartile range).


( a ) 30 and 150 days post fertilization ( dpf ) zebrafish ventricles were isolated and relative protein levels determined ( n = 3 ) .

O O O 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) 30 and 150 days post fertilization (dpf) zebrafish ventricles were isolated and relative protein levels determined (n = 3).


( c ) qPCR analysis of mRNA levels of glycolytic enzyme genes in 5 dpci remote and wound border cardiac tissue ( n = 2 - 3 technical replicates using pooled cDNA from 10 ventricles for each condition ( n = 2 for pkmb and pdk4 , and n = 3 for the other genes ) ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) qPCR analysis of mRNA levels of glycolytic enzyme genes in 5 dpci remote and wound border cardiac tissue (n = 2-3 technical replicates using pooled cDNA from 10 ventricles for each condition (n = 2 for pkmb and pdk4, and n = 3 for the other genes)).


d , Immunostaining of heart sections for PCNA and MEF2 in 5 dpci animals treated with PBS , 2 - DG or DCA ; magnified view of area in white boxes shown below ; white dashed lines mark the wound border ; arrowheads point to PCNA + CMs ; percentage of PCNA + CMs in the border zone shown on the right ( n = 4 - 5 ventricles ) .

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

d, Immunostaining of heart sections for PCNA and MEF2 in 5 dpci animals treated with PBS, 2-DG or DCA; magnified view of area in white boxes shown below; white dashed lines mark the wound border; arrowheads point to PCNA+ CMs; percentage of PCNA+ CMs in the border zone shown on the right (n = 4-5 ventricles).


e ) Immunostaining of heart sections for MEF2 N2 . 261 and MEF2 in 5 dpci animals treated with PBS , 2 - DG or DCA ; magnified view of area in white boxes shown below ; white dashed lines mark the wound border ; arrowheads point to N2 . 261 + CMs ; percentage of N2 . 261 + CMs in the border zone shown on the right ( n = 4 - 5 ventricles ) .

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

e) Immunostaining of heart sections for MEF2 N2.261 and MEF2 in 5 dpci animals treated with PBS, 2-DG or DCA; magnified view of area in white boxes shown below; white dashed lines mark the wound border; arrowheads point to N2.261+ CMs; percentage of N2.261+ CMs in the border zone shown on the right (n = 4-5 ventricles).


( a Immunostaining of heart sections for PCNA and MEF2 in 5 dpci pkma2 + / - ; pkmb + / - and pkma2 - / - ; pkmb + / - animals ; magnified view of area in white boxes shown below ; arrowheads point to PCNA + CMs ; white dashed lines mark the wound border ; percentage of PCNA + CMs in the border zone shown on the right ( n = 4 - 6 ventricles of each genotype ) .

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

(a Immunostaining of heart sections for PCNA and MEF2 in 5 dpci pkma2+/-; pkmb+/- and pkma2-/-; pkmb+/- animals; magnified view of area in white boxes shown below; arrowheads point to PCNA+ CMs; white dashed lines mark the wound border; percentage of PCNA+ CMs in the border zone shown on the right (n = 4-6 ventricles of each genotype).


Immunostaining of heart sections for N2 . 261 and MEF2 in 5 dpci pkma2 + / - ; pkmb + / - and pkma2 - / - ; pkmb + / - animals ; magnified view of area in white boxes shown below ; arrowheads point to N2 . 261 + CMs ; white dashed lines mark the wound border ; percentage of N2 . 261 + in the border zone shown on the right ( n = 4 - 6 ventricles of each genotype ) .

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

Immunostaining of heart sections for N2.261 and MEF2 in 5 dpci pkma2+/-; pkmb+/- and pkma2-/-; pkmb+/- animals; magnified view of area in white boxes shown below; arrowheads point to N2.261+ CMs; white dashed lines mark the wound border; percentage of N2.261+ in the border zone shown on the right (n = 4-6 ventricles of each genotype).


Immunostaining of heart sections for PCNA and MEF2 in 5 dpci ppargc1a + / + and ppargc1a - / - animals ; magnified view of area in white boxes shown below ; arrowheads point to PCNA + CMs ; white dashed lines mark the wound border ; percentage of PCNA + CMs in the border zone shown on the right ( n = 4 - 6 ventricles of each genotype ) .

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

Immunostaining of heart sections for PCNA and MEF2 in 5 dpci ppargc1a+/+ and ppargc1a-/- animals; magnified view of area in white boxes shown below; arrowheads point to PCNA+ CMs; white dashed lines mark the wound border; percentage of PCNA+ CMs in the border zone shown on the right (n = 4-6 ventricles of each genotype).


d ) Immunostaining of heart sections for N2 . 261 and MEF2 in 5 dpci ppargc1a + / + and ppargc1a - / - animals ; magnified view of area in white boxes shown below ; arrowheads point to N2 . 261 + CMs ; white dashed lines mark the wound border ; percentage of N2 . 261 + CMs in the border zone shown on the right ( n = 4 - 6 ventricles of each genotype ) .

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

d) Immunostaining of heart sections for N2.261 and MEF2 in 5 dpci ppargc1a+/+ and ppargc1a-/- animals; magnified view of area in white boxes shown below; arrowheads point to N2.261+ CMs; white dashed lines mark the wound border; percentage of N2.261+ CMs in the border zone shown on the right (n = 4-6 ventricles of each genotype).


( e AFOG staining of heart sections from 60 dpci pkma2 + / - ; pkmb + / - and pkma2 - / - ; pkmb + / - animals ; black dashed lines outline the scar area ; scar area measured on the right ( n = 3 - 5 ventricles of each genotype ) .

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

(e AFOG staining of heart sections from 60 dpci pkma2+/-; pkmb+/- and pkma2-/-; pkmb+/- animals; black dashed lines outline the scar area; scar area measured on the right (n = 3-5 ventricles of each genotype).


f ) AFOG staining of heart sections from 45 dpci ppargc1a + / + and ppargc1a - / - animals ; black dashed lines outline the scar area ; scar area measured on the right ( n = 3 - 5 ventricles of each genotype ) .

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

f) AFOG staining of heart sections from 45 dpci ppargc1a+/+ and ppargc1a-/- animals; black dashed lines outline the scar area; scar area measured on the right (n = 3-5 ventricles of each genotype).


Immunostaining of heart sections for PCNA and MEF2 in 5 dpci animals ( Tg ( hsp70l : LOXP - STOP - LOXP - pdha1aSTA - T2A - mCherry ) and Tg ( hsp70l : LOXP - STOP - LOXP - pdk3b - T2A - mCherry ) alone ( control ) or in combination with Tg ( myl7 : Cre - ERT2 ) , all after tamoxifen and heat shock treatments ) ; magnified view of area in white boxes shown below ; arrowheads point to PCNA + CMs ; white dashed lines mark the wound border ; percentage of PCNA + CMs in the border zone shown on the right ( n = 4 - 5 ventricles of each genotype ) .

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

Immunostaining of heart sections for PCNA and MEF2 in 5 dpci animals (Tg(hsp70l:LOXP-STOP-LOXP-pdha1aSTA-T2A-mCherry) and Tg(hsp70l:LOXP-STOP-LOXP-pdk3b-T2A-mCherry) alone (control) or in combination with Tg(myl7:Cre-ERT2), all after tamoxifen and heat shock treatments); magnified view of area in white boxes shown below; arrowheads point to PCNA+ CMs; white dashed lines mark the wound border; percentage of PCNA+ CMs in the border zone shown on the right (n = 4-5 ventricles of each genotype).


Immunostaining of heart sections for N2 . 261 and MEF2 in 5 dpci animals ( Tg ( hsp70l : LOXP - STOP - LOXP - pdha1aSTA - T2A - mCherry ) and Tg ( hsp70l : LOXP - STOP - LOXP - pdk3b - T2A - mCherry ) alone ( control ) or in combination with Tg ( myl7 : Cre - ERT2 ) , all after tamoxifen and heat shock treatments ) ; magnified view of area in white boxes shown below ; arrowheads point to N2 . 261 + CMs ; white dashed lines mark the wound border ; percentage of N2 . 261 + CMs in the border zone shown on the right ( n = 4 - 5 ventricles of each genotype ) .

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

Immunostaining of heart sections for N2.261 and MEF2 in 5 dpci animals (Tg(hsp70l:LOXP-STOP-LOXP-pdha1aSTA-T2A-mCherry) and Tg(hsp70l:LOXP-STOP-LOXP-pdk3b-T2A-mCherry) alone (control) or in combination with Tg(myl7:Cre-ERT2), all after tamoxifen and heat shock treatments); magnified view of area in white boxes shown below; arrowheads point to N2.261+ CMs; white dashed lines mark the wound border; percentage of N2.261+ CMs in the border zone shown on the right (n = 4-5 ventricles of each genotype).


( f AFOG staining of heart sections from 60 dpci animals ( Tg ( hsp70l : LOXP - STOP - LOXP - pdha1aSTA - T2A - mCherry ) alone ( control ) or in combination with Tg ( myl7 : Cre - ERT2 ) , all after tamoxifen and heat shock treatments ) ; black dashed lines outline the scar area ; scar area measured on the right ( n = 4 - 5 ventricles of each genotype ) .

(f AFOG staining of heart sections from 60 dpci animals (Tg(hsp70l:LOXP-STOP-LOXP-pdha1aSTA-T2A-mCherry) alone (control) or in combination with Tg(myl7:Cre-ERT2), all after tamoxifen and heat shock treatments); black dashed lines outline the scar area; scar area measured on the right (n = 4-5 ventricles of each genotype).


g ) AFOG staining of heart sections from 60 dpci animals and Tg ( hsp70l : LOXP - STOP - LOXP - pdk3b - T2A - mCherry ) alone ( control ) or in combination with Tg ( myl7 : Cre - ERT2 ) , all after tamoxifen and heat shock treatments ) ; black dashed lines outline the scar area ; scar area measured on the right ( n = 4 - 5 ventricles of each genotype ) .

g) AFOG staining of heart sections from 60 dpci animals and Tg(hsp70l:LOXP-STOP-LOXP-pdk3b-T2A-mCherry) alone (control) or in combination with Tg(myl7:Cre-ERT2), all after tamoxifen and heat shock treatments); black dashed lines outline the scar area; scar area measured on the right (n = 4-5 ventricles of each genotype).


( a , b ) Staining for Ki67 , CTNI and DNA ( DAPI ) in control , PDHA1STA OE or PDK3 OE RNCMs cultured in growth ( a ) or non - growth ( b ) medium ; arrowheads point to Ki67 + RNCMs ; percentage of Ki67 + RNCMs shown on the right ( n = 4 biological replicates for each condition ) .

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

(a, b) Staining for Ki67, CTNI and DNA (DAPI) in control, PDHA1STA OE or PDK3 OE RNCMs cultured in growth (a) or non-growth (b) medium; arrowheads point to Ki67+ RNCMs; percentage of Ki67+ RNCMs shown on the right (n = 4 biological replicates for each condition).


( c ) Staining for CTNI and DNA ( DAPI ) in scratch assay using control , PDHA1STA OE or PDK3 OE RNCMs immediately or at 3 days after generating the scratch ; magnified view of area in white boxes shown below ; arrowheads point to membrane protrusions of RNCMs ; quantification of CM membrane protrusions shown on the right ( n = 4 biological replicates for each condition ) .

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

(c) Staining for CTNI and DNA (DAPI) in scratch assay using control, PDHA1STA OE or PDK3 OE RNCMs immediately or at 3 days after generating the scratch; magnified view of area in white boxes shown below; arrowheads point to membrane protrusions of RNCMs; quantification of CM membrane protrusions shown on the right (n = 4 biological replicates for each condition).


( d ) Reactome over - representation analysis of differentially regulated genes in PDK3 OE RNCMs compared to control .

(d) Reactome over-representation analysis of differentially regulated genes in PDK3 OE RNCMs compared to control.


( e Heat map of gene expression for key regulators of cell cycle in PDK3 OE vs control RNCMs .

(e Heat map of gene expression for key regulators of cell cycle in PDK3 OE vs control RNCMs.


f ) Heat map of gene expression for key regulators of DNA replication in PDK3 OE vs control RNCMs .

f) Heat map of gene expression for key regulators of DNA replication in PDK3 OE vs control RNCMs.


A : Morphology on phase contrast microscopy for parental fibroblast lines ( upper panel ) and iPSCs ( lower panel ) from control Detroit 551 , WS5A and CP2A POLG patients ( scale bars , 50 µm ) .

A: Morphology on phase contrast microscopy for parental fibroblast lines (upper panel) and iPSCs (lower panel) from control Detroit 551, WS5A and CP2A POLG patients (scale bars, 50 µm).


B : Immunofluorescence staining of stem cell markers POU5F1 ( green ) and SSEA4 ( red ) : upper panel - Detroit 551 control iPSCs , middle panel - WS5A iPSCs and lower panel - CP2A iPSCs ( Scale bar , 100 µm ) . Nuclei are stained with DAPI ( blue ) .

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

B: Immunofluorescence staining of stem cell markers POU5F1 (green) and SSEA4 (red): upper panel - Detroit 551 control iPSCs, middle panel - WS5A iPSCs and lower panel - CP2A iPSCs (Scale bar, 100 µm). Nuclei are stained with DAPI (blue).


C : RT - qPCR quantification of gene expression for LIN28A , NANOG , and POU5F1 for all iPSCs from control Detroit 551 , WS5A and CP2A POLG patients ( n = 7 , technical replicates per line for ESCs ; n = 4 , technical replicates per clone for control , WS5A and CP2A iPSCs ) . The gene expression of the individual clones is assessed with fold change using the comparative ΔΔCt method by normalizing iPSCs to ESC1 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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: RT-qPCR quantification of gene expression for LIN28A, NANOG, and POU5F1 for all iPSCs from control Detroit 551, WS5A and CP2A POLG patients (n=7, technical replicates per line for ESCs; n=4, technical replicates per clone for control, WS5A and CP2A iPSCs). The gene expression of the individual clones is assessed with fold change using the comparative ΔΔCt method by normalizing iPSCs to ESC1.


D , E : Flow cytometric quantification of expression level of SSEA4 ( D , n = 9 , technical replicates per line for ESCs ; n = 5 , technical replicates per clone for control iPSCs ; n = 3 , technical replicates per clone for WS5A iPSCs , n = 8 , technical replicates per clone for CP2A iPSCs ) and POU5F1 ( E , n = 9 , technical replicates per line for ESCs ; n = 5 , technical replicates per clone for control iPSCs ; n = 3 , technical replicates per clone for WS5A iPSCs , n = 8 , technical replicates per clone for CP2A iPSCs ) for ESCs and iPSCs for both ESC control lines and iPSCs generated from Detroit 551 control , WS5A and CP2A fibroblasts .

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

D, E: Flow cytometric quantification of expression level of SSEA4 (D, n=9, technical replicates per line for ESCs; n=5, technical replicates per clone for control iPSCs; n=3, technical replicates per clone for WS5A iPSCs, n=8, technical replicates per clone for CP2A iPSCs) and POU5F1 (E, n=9, technical replicates per line for ESCs; n=5, technical replicates per clone for control iPSCs; n=3, technical replicates per clone for WS5A iPSCs, n=8, technical replicates per clone for CP2A iPSCs) for ESCs and iPSCs for both ESC control lines and iPSCs generated from Detroit 551 control, WS5A and CP2A fibroblasts.


A : Representative confocal images of iPSC lineage specific differentiation into germ layers of endoderm derived hepatocytes with positive expression of ALBUMIN ( red ) and HNF4A ( green ) ( a ) ( scale bar , 100 µm ) , mesodermal derived cardiomyocytes with positive expression of TNNT2 ( red ) ( b ) ( scale bar , 100 µm ) and ectodermal derived dopaminergic neurons with positive expression of TH ( green ) and MAP2 ( red ) ( c ) ( scale bar , 10 µm ) . Nuclei are stained with DAPI ( blue ) .

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

A: Representative confocal images of iPSC lineage specific differentiation into germ layers of endoderm derived hepatocytes with positive expression of ALBUMIN (red) and HNF4A (green) (a) (scale bar, 100 µm), mesodermal derived cardiomyocytes with positive expression of TNNT2 (red) (b) (scale bar, 100 µm) and ectodermal derived dopaminergic neurons with positive expression of TH (green) and MAP2 (red) (c) (scale bar, 10 µm). Nuclei are stained with DAPI (blue).


B : Confocal images of mitochondrial morphology for iPSC lines with co - staining of MTG ( upper panel ) and TMRE ( lower panel ) ( scale bars , 25 µm ) . Nuclei are stained with DAPI ( blue ) .

O O O O O O O O O O O O O O O O O O O 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: Confocal images of mitochondrial morphology for iPSC lines with co-staining of MTG (upper panel) and TMRE (lower panel) (scale bars, 25 µm). Nuclei are stained with DAPI (blue).


Flow cytometric analysis of iPSCs generated from Detroit 551 , WS5A and CP2A fibroblasts for mitochondrial volume ( MTG ) ( C , n = 6 , technical replicates per clone for control and CP2A ; n = 5 , technical replicates per clone for WS5A )

Flow cytometric analysis of iPSCs generated from Detroit 551, WS5A and CP2A fibroblasts for mitochondrial volume (MTG) (C, n=6, technical replicates per clone for control and CP2A; n=5, technical replicates per clone for WS5A)


Flow cytometric analysis of iPSCs generated from Detroit 551 , WS5A and CP2A fibroblasts for total MMP ( TMRE ) ( D , n = 6 , technical replicates per clone for control and CP2A ; n = 5 , technical replicates per clone for WS5A ) and specific MMP ( E , n = 6 , technical replicates per clone for control and CP2A ; n = 5 , technical replicates per clone for WS5A ) calculated by dividing median fluorescence intensity ( MFI ) for total TMRE expression by MTG .

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

Flow cytometric analysis of iPSCs generated from Detroit 551, WS5A and CP2A fibroblasts for total MMP (TMRE) (D, n=6, technical replicates per clone for control and CP2A; n=5, technical replicates per clone for WS5A) and specific MMP (E, n=6, technical replicates per clone for control and CP2A; n=5, technical replicates per clone for WS5A) calculated by dividing median fluorescence intensity (MFI) for total TMRE expression by MTG.


F : Intracellular ATP production in iPSCs generated from Detroit 551 , WS5A and CP2A fibroblasts ( n = 3 , technical replicates per clone for control and WS5A ; n = 4 , technical replicates per clone for CP2A ) .

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F: Intracellular ATP production in iPSCs generated from Detroit 551, WS5A and CP2A fibroblasts (n=3, technical replicates per clone for control and WS5A; n=4, technical replicates per clone for CP2A).


G - H : Flow cytometric analysis of TOMM20 expression level ( G , n = 4 , technical replicates per clone ) and specific TFAM level ( total TFAM / TOMM20 ) ( H , n = 4 , technical replicates per clone ) .

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

G-H: Flow cytometric analysis of TOMM20 expression level (G, n=4, technical replicates per clone) and specific TFAM level (total TFAM/TOMM20) (H, n=4, technical replicates per clone).


I : Relative mtDNA copy number in Detroit 551 , WS5A and CP2A iPSCs by RT - qPCR analysis using ND1 and APP ( n = 5 , technical replicates per clone for control ; n = 5 , technical replicates per clone for WS5A and CP2A ) . Values are presented as Log2 of the ratio between the expression values of ND1 in relation to APP .

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

I: Relative mtDNA copy number in Detroit 551, WS5A and CP2A iPSCs by RT-qPCR analysis using ND1 and APP (n=5, technical replicates per clone for control; n=5, technical replicates per clone for WS5A and CP2A). Values are presented as Log2 of the ratio between the expression values of ND1 in relation to APP.


B : Representative phase contrast images ( upper panel ) and immunostaining for specific stages during neural induction from iPSCs to NSCs . Upper panel displays the morphology in culture of different cell types during neural induction to NSCs from iPSCs including iPSCs ( a ) ; neuroepithelium with rosette - like structures ( b ) ; neurospheres with defined round shapes in suspension culture ( c ) and NSCs in monolayers ( d ) ( scale bars , 50 µm ) . Lower panel demonstrates the representative phase contrast images for the immunostaining corresponding to the specific stages in the upper panel : iPSCs with positive staining of SSEA4 ( red ) and POU5F1 ( green ) ( e ) ( scale bar , 50 µm ) ; neuroepithelium with rosette - like structures with positive staining of PAX6 ( green ) and NESTIN ( red ) ( f ) ( scale bar , 50 µm ) ; neurospheres with positive staining of NESTIN ( red ) ( g ) ( scale bar , 100 µm ) ; NSCs with positive staining of PAX6 ( green ) ( h ) ( scale bar , 50 µm ) .

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

B: Representative phase contrast images (upper panel) and immunostaining for specific stages during neural induction from iPSCs to NSCs. Upper panel displays the morphology in culture of different cell types during neural induction to NSCs from iPSCs including iPSCs (a); neuroepithelium with rosette-like structures (b); neurospheres with defined round shapes in suspension culture (c) and NSCs in monolayers (d) (scale bars, 50 µm). Lower panel demonstrates the representative phase contrast images for the immunostaining corresponding to the specific stages in the upper panel: iPSCs with positive staining of SSEA4 (red) and POU5F1 (green) (e) (scale bar, 50 µm); neuroepithelium with rosette-like structures with positive staining of PAX6 (green) and NESTIN (red) (f) (scale bar, 50 µm); neurospheres with positive staining of NESTIN (red) (g) (scale bar, 100 µm); NSCs with positive staining of PAX6 (green) (h) (scale bar, 50 µm).


C : Flow cytometric assessment of stemness marker POU5F1 and NSC markers NESTIN and PAX6 during neural induction from day 0 to day 5 ( n = 3 , technical replicates ) .

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

C: Flow cytometric assessment of stemness marker POU5F1 and NSC markers NESTIN and PAX6 during neural induction from day 0 to day 5 (n=3, technical replicates).


D : Representative images of the immunofluorescent labeling for NSC markers SOX2 ( green ) and NESTIN ( red ) ( scale bar , 25 µm ) from Detroit 551 control iPSC - derived NSCs . Nuclei are stained with DAPI ( blue ) .

O O O O O O O O O O O O O O O O O O O O O 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: Representative images of the immunofluorescent labeling for NSC markers SOX2 (green) and NESTIN (red) (scale bar, 25 µm) from Detroit 551 control iPSC-derived NSCs. Nuclei are stained with DAPI (blue).


A : Quantification of gene expression for NSC markers PAX6 , NESTIN and SOX2 for all NSCs from RT - qPCR analysis . The expression of the neural stem cell markers is assessed with fold change using the comparative ΔΔCt method by normalizing NSCs to iPSCs ( n = 3 , technical replicates per ESC line or iPSC clone ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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: Quantification of gene expression for NSC markers PAX6, NESTIN and SOX2 for all NSCs from RT-qPCR analysis. The expression of the neural stem cell markers is assessed with fold change using the comparative ΔΔCt method by normalizing NSCs to iPSCs (n=3, technical replicates per ESC line or iPSC clone).


B , C : Quantification of protein expression level for PAX6 ( B , n = 6 , technical replicates per line for ESCs ; n = 7 , technical replicates per clone for control ; n = 8 , technical replicates per clone for WS5A ; n = 9 , technical replicates per clone for CP2A ) and NESTIN ( C , n = 6 , technical replicates per line for ESCs ; n = 7 , technical replicates per clone for control ; n = 8 , technical replicates per clone for WS5A ; n = 9 , technical replicates per clone for CP2A ) for iPSC - derived NSCs using flow cytometry .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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: Quantification of protein expression level for PAX6 (B, n=6, technical replicates per line for ESCs; n=7, technical replicates per clone for control; n=8, technical replicates per clone for WS5A; n=9, technical replicates per clone for CP2A) and NESTIN (C, n=6, technical replicates per line for ESCs; n=7, technical replicates per clone for control; n=8, technical replicates per clone for WS5A; n=9, technical replicates per clone for CP2A) for iPSC-derived NSCs using flow cytometry.


D : Sequencing chromatogram showing the homozygous c . 2243G > C variation in POLG in WS5A iPSC - derived NSCs and the heterozygous c . 1399G > A and c . 2243G > C variation in POLG in CP2A iPSC - derived NSCs .

D: Sequencing chromatogram showing the homozygous c.2243G>C variation in POLG in WS5A iPSC-derived NSCs and the heterozygous c.1399G>A and c.2243G>C variation in POLG in CP2A iPSC-derived NSCs.


E : Representative confocal images showing glial and neuronal lineages derived from NSCs . a , Immunostaining of NSC - derived astrocytes with GFAP ( red ) staining ( scale bar , 50 µm ) . b , Immunostaining of oligodendrocytes showing GALC ( red ) positive labeling ( scale bar , 50 µm ) . c , Dopaminergic neurons showing TH ( green ) and TUJ1 ( red ) positive staining ( scale bar , 25 µm ) . Nuclei are stained with DAPI ( blue ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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: Representative confocal images showing glial and neuronal lineages derived from NSCs. a, Immunostaining of NSC-derived astrocytes with GFAP (red) staining (scale bar, 50 µm). b, Immunostaining of oligodendrocytes showing GALC (red) positive labeling (scale bar, 50 µm). c, Dopaminergic neurons showing TH (green) and TUJ1 (red) positive staining (scale bar, 25 µm). Nuclei are stained with DAPI (blue).


A : Representative transmission electron microscopy images of mitochondrial structures in Detroit 551 control , WS5A and CP2A NSCs ( scale bar , 400 nm ) .

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A: Representative transmission electron microscopy images of mitochondrial structures in Detroit 551 control, WS5A and CP2A NSCs (scale bar, 400 nm).


Flow cytometric analysis of mitochondrial volume ( MTG ) ( B , n = 5 , technical replicates per clone ) , total MMP ( TMRE ) ( C , n = 5 , technical replicates per clone ) calculated by dividing median fluorescence intensity ( MFI ) for total TMRE expression by MTG in NSCs generated from control lines , WS5A and CP2A iPSCs .

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

Flow cytometric analysis of mitochondrial volume (MTG) (B, n=5, technical replicates per clone), total MMP (TMRE) (C, n=5, technical replicates per clone) calculated by dividing median fluorescence intensity (MFI) for total TMRE expression by MTG in NSCs generated from control lines, WS5A and CP2A iPSCs.


Flow cytometric analysis of specific MMP ( D , n = 5 , technical replicates per clone ) , calculated by dividing median fluorescence intensity ( MFI ) in NSCs generated from control lines , WS5A and CP2A iPSCs .

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

Flow cytometric analysis of specific MMP (D, n=5, technical replicates per clone), calculated by dividing median fluorescence intensity (MFI) in NSCs generated from control lines, WS5A and CP2A iPSCs.


E : Intracellular ATP production in NSCs ( n = 3 , technical replicates per clone ) .

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E: Intracellular ATP production in NSCs (n=3, technical replicates per clone).


Flow cytometric analysis of mitochondrial volume ( MTG ) ( F , n = 6 , technical replicates for control ; n = 5 , technical replicates for WS5A and CP2A ) , total MMP ( TMRE ) ( G , n = 5 , technical replicates for control ; n = 4 , technical replicates for WS5A and CP2A ) in Detroit 551 , WS5A and CP2A fibroblasts .

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

Flow cytometric analysis of mitochondrial volume (MTG) (F, n=6, technical replicates for control; n=5, technical replicates for WS5A and CP2A), total MMP (TMRE) (G, n=5, technical replicates for control; n=4, technical replicates for WS5A and CP2A) in Detroit 551, WS5A and CP2A fibroblasts.


Flow cytometric analysis of specific MMP ( TMRE / MTG ) ( H , n = 5 , technical replicates for control ; n = 4 , technical replicates for WS5A and CP2A ) in Detroit 551 , WS5A and CP2A fibroblasts .

Flow cytometric analysis of specific MMP (TMRE/MTG) (H, n=5, technical replicates for control; n=4, technical replicates for WS5A and CP2A) in Detroit 551, WS5A and CP2A fibroblasts.


I : Intracellular ATP production in Detroit 551 , WS5A and CP2A fibroblasts ( n = 6 , technical replicates for control ; n = 3 , technical replicates for WS5A and CP2A ) .

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

I: Intracellular ATP production in Detroit 551, WS5A and CP2A fibroblasts (n=6, technical replicates for control; n=3, technical replicates for WS5A and CP2A).


A , B : Flow cytometric analysis of TOMM20 ( A , n = 3 , technical replicates per clone for control ; n = 4 , technical replicates per clone for WS5A and CP2A ) and TFAM protein expression ( B , n = 3 , technical replicates per clone for control and CP2A ; n = 5 , technical replicates per clone for WS5A ) in NSCs . Expressed as specific TFAM level ( total TFAM / TOMM20 ) .

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A, B: Flow cytometric analysis of TOMM20 (A, n=3, technical replicates per clone for control; n=4, technical replicates per clone for WS5A and CP2A) and TFAM protein expression (B, n=3, technical replicates per clone for control and CP2A; n=5, technical replicates per clone for WS5A) in NSCs. Expressed as specific TFAM level (total TFAM/TOMM20).


C : Relative mtDNA copy number analyzed by RT - qPCR using primers and fluorogenic probes for regions of ND1 and nuclear gene APP in all NSCs ( n = 4 , technical replicates per clone for control ; n = 5 , technical replicates per clone for WS5A ; n = 7 , technical replicates per clone for CP2A ) .

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C: Relative mtDNA copy number analyzed by RT-qPCR using primers and fluorogenic probes for regions of ND1 and nuclear gene APP in all NSCs (n=4, technical replicates per clone for control; n=5, technical replicates per clone for WS5A; n=7, technical replicates per clone for CP2A).


D , E : Flow cytometric analysis of TOMM20 expression level ( D , n = 4 , technical replicates per clone ) and TFAM protein expression ( E , n = 4 , technical replicates for control and WS5A ; n = 5 , technical replicates for CP2A ) in fibroblasts . Expressed as specific TFAM level ( total TFAM / TOMM20 ) .

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D, E: Flow cytometric analysis of TOMM20 expression level (D, n=4, technical replicates per clone) and TFAM protein expression (E, n=4, technical replicates for control and WS5A; n=5, technical replicates for CP2A) in fibroblasts. Expressed as specific TFAM level (total TFAM/TOMM20).


F : Relative mtDNA copy number analyzed by RT - qPCR for regions of ND1 and nuclear gene APP in fibroblasts ( n = 3 , technical replicates for control ; n = 4 , technical replicates for WS5A and CP2A ) .

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F: Relative mtDNA copy number analyzed by RT-qPCR for regions of ND1 and nuclear gene APP in fibroblasts (n=3, technical replicates for control; n=4, technical replicates for WS5A and CP2A).


G : Long - PCR for detection of mtDNA deletions in NSCs and their parental fibroblasts from WS5A and CP2A patients .

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G: Long-PCR for detection of mtDNA deletions in NSCs and their parental fibroblasts from WS5A and CP2A patients.


H : Representative images of confocal microscopy with immunofluorescence labeling of DA neuron - specific marker TH ( green ) and neuron - specific marker MAP2 ( red ) for iPSC - derived DA neurons ( scale bars , 25 µm ) . Nuclei are stained with DAPI ( blue ) .

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H: Representative images of confocal microscopy with immunofluorescence labeling of DA neuron-specific marker TH (green) and neuron-specific marker MAP2 (red) for iPSC-derived DA neurons (scale bars, 25 µm). Nuclei are stained with DAPI (blue).


I : Histogram of the positive cell population stained with DA marker TH in iPSC - derived DA neurons using flow cytometric analysis .

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I: Histogram of the positive cell population stained with DA marker TH in iPSC-derived DA neurons using flow cytometric analysis.


J : Flow cytometric measurement of specific TFAM level ( total TFAM / TOMM20 ) in iPSC - derived DA neurons from Detroit 551 control , WS5A and CP2A POLG patients ( n = 8 , technical replicates per clone for control ; n = 5 , technical replicates per clone for WS5A ; n = 4 , technical replicates per clone for CP2A ) .

J: Flow cytometric measurement of specific TFAM level (total TFAM/TOMM20) in iPSC-derived DA neurons from Detroit 551 control, WS5A and CP2A POLG patients (n=8, technical replicates per clone for control; n=5, technical replicates per clone for WS5A; n=4, technical replicates per clone for CP2A).


K : Relative mtDNA copy number analyzed by RT - qPCR for regions of ND1 and nuclear gene APP in all iPSC - derived DA neurons ( n = 3 , technical replicates per clone ) .

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K: Relative mtDNA copy number analyzed by RT-qPCR for regions of ND1 and nuclear gene APP in all iPSC-derived DA neurons (n=3, technical replicates per clone).


A : Complex I immunohistochemistry in the occipital cortex of a neurologically healthy control ( a ) and two patients with POLG - disease ( b and c ) ( Scale bar , 20 µm ) . Patients have numerous complex - I negative neurons ( examples marked by arrows ) . ( d ) mtDNA relative quantification in microdissected neurons from the occipital cortex of patients with POLG diseases ( n = 5 ) and neurologically healthy controls ( n = 5 ) . Each point represents the mean value of three technical replicates from a pooled sample of 10 neurons . For the purposes of comparison , a control sample has been arbitrarily set to one . The medians of the two groups are compared by Mann - Whitney U test . Data are presented as mean ( horizontal bars ) ± SEM ( vertical bars ) .

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A: Complex I immunohistochemistry in the occipital cortex of a neurologically healthy control (a) and two patients with POLG-disease (b and c) (Scale bar, 20 µm). Patients have numerous complex-I negative neurons (examples marked by arrows). (d) mtDNA relative quantification in microdissected neurons from the occipital cortex of patients with POLG diseases (n=5) and neurologically healthy controls (n=5). Each point represents the mean value of three technical replicates from a pooled sample of 10 neurons. For the purposes of comparison, a control sample has been arbitrarily set to one. The medians of the two groups are compared by Mann-Whitney U test. Data are presented as mean (horizontal bars) ± SEM (vertical bars).


B : Representative confocal images of immunostaining for mitocondrial complex I subunit NDUFB10 ( green ) and TOMM20 ( red ) in control , WS5A and CP2A NSCs ( scale bars , 50 µm ) . Nuclei are stained with DAPI ( blue ) .

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B: Representative confocal images of immunostaining for mitocondrial complex I subunit NDUFB10 (green) and TOMM20 (red) in control, WS5A and CP2A NSCs (scale bars, 50 µm). Nuclei are stained with DAPI (blue).


C - E : Flow cytometric measurements of mitochondrial complex I ( C , n = 4 , technical replicates per clone ) , II ( D , n = 3 , technical replicates per clone for control ; n = 4 , technical replicates per clone for WS5A and CP2A ) and IV ( E , n = 3 , technical replicates per clone for control and CP2A ; n = 4 , technical replicates per clone for WS5A ) protein level in iPSC - derived NSCs . Expressed as specific complex I , II and IV level ( total complex I , II , IV level / TOMM20 ) .

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C-E: Flow cytometric measurements of mitochondrial complex I (C, n=4, technical replicates per clone), II (D, n=3, technical replicates per clone for control; n=4, technical replicates per clone for WS5A and CP2A) and IV (E, n=3, technical replicates per clone for control and CP2A; n=4, technical replicates per clone for WS5A) protein level in iPSC-derived NSCs. Expressed as specific complex I, II and IV level (total complex I, II, IV level/TOMM20).


F - H : Flow cytometric measurements of mitochondrial complex I ( F , n = 4 , technical replicates per clone ) , II ( G , n = 4 , technical replicates per clone ) and IV ( H , n = 4 , technical replicates per clone ) protein level in iPSCs . Expressed as specific complex I , II and IV level .

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F-H: Flow cytometric measurements of mitochondrial complex I (F, n=4, technical replicates per clone), II (G, n=4, technical replicates per clone) and IV (H, n=4, technical replicates per clone) protein level in iPSCs. Expressed as specific complex I, II and IV level.


I - K : Flow cytometric measurements of mitochondrial complex I ( I , n = 4 , technical replicates ) , II ( J , n = 4 , technical replicates ) and IV ( K , n = 4 , technical replicates ) protein level in parental fibroblasts expressed as specific complex I , II and IV level .

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I-K: Flow cytometric measurements of mitochondrial complex I (I, n=4, technical replicates), II (J, n=4, technical replicates) and IV (K, n=4, technical replicates) protein level in parental fibroblasts expressed as specific complex I, II and IV level.


A - C : LCMS - based metabolomics for quantitative measurements of NAD + / NADH ratio ( A , n = 3 , technical replicates per clone ) , NAD + ( B , n = 3 , technical replicates per clone ) and NADH ( C , n = 3 , technical replicates per clone ) level in NSCs .

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A-C: LCMS-based metabolomics for quantitative measurements of NAD+/NADH ratio (A, n=3, technical replicates per clone), NAD+ (B, n=3, technical replicates per clone) and NADH (C, n=3, technical replicates per clone) level in NSCs.


D - F : LCMS - based metabolomics for quantitative measurement of NAD + / NADH ratio ( D , n = 3 , technical replicates per clone ) , NAD + ( E , n = 3 , technical replicates per clone ) and NADH ( F , n = 3 , technical replicates per clone ) level in iPSCs .

D-F: LCMS-based metabolomics for quantitative measurement of NAD+/NADH ratio (D, n=3, technical replicates per clone), NAD+ (E, n=3, technical replicates per clone) and NADH (F, n=3, technical replicates per clone) level in iPSCs.


G : Intracellular ROS production measurements of the specific ROS level calculated by total ROS / MTDR in control , WS5A and CP2A iPSC - derived NSCs using DCFDA and MTDR ( n = 6 , technical replicates per clone for control ; n = 5 , technical replicates per clone for WS5A ; n = 3 , technical replicates per clone for CP2A ) .

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G: Intracellular ROS production measurements of the specific ROS level calculated by total ROS/MTDR in control, WS5A and CP2A iPSC-derived NSCs using DCFDA and MTDR (n=6, technical replicates per clone for control; n=5, technical replicates per clone for WS5A; n=3, technical replicates per clone for CP2A).


H : Flow cytometric measurements of mitochondrial ROS production at the specific ROS level in Detroit 551 , WS5A and CP2A iPSC - derived NSCs calculated by total ROS ( MitoSox Red ) / MTG ( n = 4 , technical replicates per clone for control and CP2A ; n = 3 , technical replicates per clone for WS5A ) .

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H: Flow cytometric measurements of mitochondrial ROS production at the specific ROS level in Detroit 551, WS5A and CP2A iPSC-derived NSCs calculated by total ROS (MitoSox Red)/MTG (n=4, technical replicates per clone for control and CP2A; n=3, technical replicates per clone for WS5A).


I - J : Representative images of senescence β - galactosidase staining ( scale bars , 20 µm ) ( I ) and quantification by calculating the percentage of positively stained cells by division of the negative cells from I ( J , n = 3 , technical replicates per clone ) .

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I-J: Representative images of senescence β-galactosidase staining (scale bars, 20 µm) (I) and quantification by calculating the percentage of positively stained cells by division of the negative cells from I (J, n=3, technical replicates per clone).


K - L : Representative images ( K ) and quantification ( L ) for western blotting with Phospho - SirT1 ( Ser47 ) , LC3B , UCP2 , PINK1 , Parkin , BNIP3 and β - ACTIN . Three independent experiments are included .

K-L: Representative images (K) and quantification (L) for western blotting with Phospho-SirT1 (Ser47), LC3B, UCP2, PINK1, Parkin, BNIP3 and β-ACTIN. Three independent experiments are included.


C - E . Full length GFP : Mud shows the wild type pattern of localization in mud3 / mud4 mitotic follicle cells ( C ) and rescues spindle orientation ( D E ) . GFP : Mud lacking the Pins binding domain localizes only at spindle poles ( C ' ) in mud3 / mud4 mitotic follicle cells and fails to rescue spindle orientation ( D E ) . Statistical significance in spindle orientation was determined using the Mann - Whitney test .

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C-E. Full length GFP:Mud shows the wild type pattern of localization in mud3/mud4 mitotic follicle cells (C) and rescues spindle orientation (D E). GFP:Mud lacking the Pins binding domain localizes only at spindle poles (C') in mud3/mud4 mitotic follicle cells and fails to rescue spindle orientation (D E). Statistical significance in spindle orientation was determined using the Mann-Whitney test.


C , D . Pins : Tom and GFP : Mud have overlapping but not identical localization patterns in mitotic follicle cells . Representative pictures ( C ) and quantification from 9 cells ( D ) are shown . Scale bars = 5 microns .

C, D. Pins:Tom and GFP:Mud have overlapping but not identical localization patterns in mitotic follicle cells. Representative pictures (C) and quantification from 9 cells (D) are shown. Scale bars = 5 microns.


A - C . Pins - myr - GFP and myr - RFP show a similar localization pattern to Pins : Tom during mitosis . Representative pictures are shown ( A , B ) ; a heatmap lookup table ( A ' , B ' ) emphasizes the relative enrichments of Pins - myr - GFP and myr - RFP along the cortex . Quantification ( Pins - myr : 4 cells , myr - RFP : 2 cells ) ( C ) .

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A-C. Pins-myr-GFP and myr-RFP show a similar localization pattern to Pins:Tom during mitosis. Representative pictures are shown (A, B); a heatmap lookup table (A', B') emphasizes the relative enrichments of Pins-myr-GFP and myr-RFP along the cortex. Quantification (Pins-myr: 4 cells, myr-RFP: 2 cells) (C).


D - F . Random spindle orientation in pinsp62 / pinsp62 null mutant follicle cells is rescued by the expression of full - length Pins or Pins - myr - GFP . Representative pictures ( D , E ) and quantification ( F ) are shown . Scale bars = 5 microns . Statistical significance in spindle orientation was determined using the Mann - Whitney test . * * * * p < 0 . 0001 .

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D-F. Random spindle orientation in pinsp62/pinsp62 null mutant follicle cells is rescued by the expression of full-length Pins or Pins-myr-GFP. Representative pictures (D, E) and quantification (F) are shown. Scale bars = 5 microns. Statistical significance in spindle orientation was determined using the Mann-Whitney test. **** p<0.0001.


A . Spindle orientation is randomized in Dlg1P20 / Dlg1P20 and Dlg1P20 / Dlg2 mutant follicle cells . B . Random spindle orientation in pinsp62 / pinsp62 null mutant follicle cells is rescued by the expression of full length Pins or phosphomimetic ( S436D ) Pins . Unphosphorylatable ( S436A ) Pins fails to rescue . C . Quantification of spindle orientation phenotypes . Statistical significance in spindle orientation was determined using the Mann - Whitney test . * * * * p < 0 . 0001 , * * * p < 0 . 001 . D .

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A. Spindle orientation is randomized in Dlg1P20/Dlg1P20 and Dlg1P20/Dlg2 mutant follicle cells. B. Random spindle orientation in pinsp62/pinsp62 null mutant follicle cells is rescued by the expression of full length Pins or phosphomimetic (S436D) Pins. Unphosphorylatable (S436A) Pins fails to rescue. C. Quantification of spindle orientation phenotypes. Statistical significance in spindle orientation was determined using the Mann-Whitney test. **** p<0.0001, *** p<0.001. D.


H . Quantification of spindle orientation phenotypes in the Stage 9 / 10 embryonic ectoderm . n = 17 cells from 2 embryos in w1118 and n = 18 cells from 2 embryos in Dlg1P20 / Dlg2 embryos . Statistical significance in spindle orientation was determined using the Mann - Whitney test . * * p = 0 . 0015 .

H. Quantification of spindle orientation phenotypes in the Stage 9/10 embryonic ectoderm. n = 17 cells from 2 embryos in w1118 and n = 18 cells from 2 embryos in Dlg1P20/Dlg2 embryos. Statistical significance in spindle orientation was determined using the Mann-Whitney test. ** p=0.0015.


A - C . Multiple genetic approaches were used to test a role for either Khc73 or GUKHolder in metaphase spindle orientation . Representative pictures ( A - B ) and quantification ( C ) are shown . Scale bars = 5 microns . Spindle angle randomization in pinsp62 / pinsp62 null mutant follicle cells is included as a positive control for comparison .

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A-C. Multiple genetic approaches were used to test a role for either Khc73 or GUKHolder in metaphase spindle orientation. Representative pictures (A-B) and quantification (C) are shown. Scale bars = 5 microns. Spindle angle randomization in pinsp62/pinsp62 null mutant follicle cells is included as a positive control for comparison.


A - D . Two distinct patterns of Inscuteable localization are observed in the follicular epithelium when UAS - Inscuteable is driven by Traffic jam - GAL4 . ( A , C ) In the first pattern ( InscA ) , Inscuteable is highly enriched at the apical cell surface . ( B , D ) In the second pattern ( InscB ) , Inscuteable is observed at both the lateral and apical cortex . Mud and Pins localizations are also shown . Representative pictures ( A , B ) and quantifications ( InscA : average of 5 cells , InscB : average of 7 cells ) ( C , D ) are shown .

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A-D. Two distinct patterns of Inscuteable localization are observed in the follicular epithelium when UAS-Inscuteable is driven by Traffic jam-GAL4. (A, C) In the first pattern (InscA), Inscuteable is highly enriched at the apical cell surface. (B, D) In the second pattern (InscB), Inscuteable is observed at both the lateral and apical cortex. Mud and Pins localizations are also shown. Representative pictures (A, B) and quantifications (InscA: average of 5 cells, InscB: average of 7 cells) (C, D) are shown.


E . aPKC localization in InscA and InscB follicle cells . F . Quantification of spindle orientation phenotypes shows that mitotic spindles are reoriented in InscA cells , but not InscB cells . Reorientation relies on Mud but not on the interaction between Pins and Dlg .

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E. aPKC localization in InscA and InscB follicle cells. F. Quantification of spindle orientation phenotypes shows that mitotic spindles are reoriented in InscA cells, but not InscB cells. Reorientation relies on Mud but not on the interaction between Pins and Dlg. G


A ) Summary of computationally identified circuit using a constraint of maximum three inputs . Shown are input miRNAs and predicted circuit output levels . Expression levels of identified miRNAs inputs are given as fold change over the pre - set input abundance threshold ( t ) of the total miRNA pool ( where t = 1 % ) ( left ) . Calculated circuit output levels are given as mol / cell ( middle ) and logic connectivity of the identified miRNA is depicted ( right ) . EB : embryoid bodies , MPC : mesenchymal progenitor cells , NPC : neural progenitor cells , R - NSC : neural rosettes .

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A) Summary of computationally identified circuit using a constraint of maximum three inputs. Shown are input miRNAs and predicted circuit output levels. Expression levels of identified miRNAs inputs are given as fold change over the pre-set input abundance threshold (t) of the total miRNA pool (where t = 1%) (left). Calculated circuit output levels are given as mol / cell (middle) and logic connectivity of the identified miRNA is depicted (right). EB: embryoid bodies, MPC: mesenchymal progenitor cells, NPC: neural progenitor cells, R-NSC: neural rosettes.


C ) Bar chart showing relative DsRed expression of sensors containing the indicated miRNA target sites as four fully complementary repeats ( discrete miRNA sensing ) . The control vector ( n . t . ) does not contain any target sites . Two - tailed unpaired t - tests were performed to compare the definitive endoderm derived from H1 ( H1 DE ) to H1 and the control vectors . For comparison to H1 , data were normalized with the control vector ( nt ) before performing the t - test . * Pvalue = 0 . 011 and * * P - value = 0 . 006 are both considered very significant . Representative scatterplots of HES - 2 are shown on top . Data information : Each bar in C corresponds to mean + / - s . d . from at least three biological replicates .

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C) Bar chart showing relative DsRed expression of sensors containing the indicated miRNA target sites as four fully complementary repeats (discrete miRNA sensing). The control vector (n.t.) does not contain any target sites. Two-tailed unpaired t-tests were performed to compare the definitive endoderm derived from H1 (H1 DE) to H1 and the control vectors. For comparison to H1, data were normalized with the control vector (nt) before performing the t-test. *Pvalue = 0.011 and ** P- value = 0.006 are both considered very significant. Representative scatterplots of HES-2 are shown on top. Data information: Each bar in C corresponds to mean +/- s.d. from at least three biological replicates.


D ) Bar chart showing relative DsRed expression of sensors containing different mutated target sequence of miR - 17 Additionally , a sensor containing no target ( n . t . ) and two wildtype ( WT ) T17 target sites with 1x and 4x repeats were tested . Two - tailed unpaired t - tests were performed to compare H1 and HES - 2 for each sensor . * P - values < 0 . 01 , were considered very significant ( * * ) , < 0 . 02 significant ( * ) and > 0 . 5 not significant . . Representative scatterplots of HES - 2 are shown on top . Data information : Each bar in D corresponds to mean + / - s . d . from at least three biological replicates .

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D) Bar chart showing relative DsRed expression of sensors containing different mutated target sequence of miR-17 Additionally, a sensor containing no target (n.t.) and two wildtype (WT) T17 target sites with 1x and 4x repeats were tested. Two-tailed unpaired t-tests were performed to compare H1 and HES-2 for each sensor. *P-values < 0.01, were considered very significant (**), < 0.02 significant (*) and > 0.5 not significant.. Representative scatterplots of HES-2 are shown on top. Data information: Each bar in D corresponds to mean +/- s.d. from at least three biological replicates.


C ) Combinatorial screen . Computational combinatorial screening of Act1MAX and Act2MAX using the model depicted in A ( left ) . Experimental combinatorial screening in H1 ( middle ) with circuit schematic on top . Two different Act1 - Act2 circuit configurations are depicted : PIT2 - tTA ( left ) and rtTA - PIT2 ( right ) . Calculated FF4 and Output levels are shown for On , Off and Off / On ( FF4 ) or On / Off ( Output ) . Color plots show normalized mCitrine ( left ) and mCherry ( right ) expression levels for each Act1 - Act2 combination . Color bars show lowest and highest expression range on a linear scale with median as the midpoint D ) Correlation plots with calculated values on X axis ( Model ) and experimental data on Y axis ( Data ) . A linear regression model ( y = a + b * x ) was used to compare experimental data with model predictions . Linear fit and coefficient of determination ( R2 ) as indicated . PIT2 - tTA circuit ( black ) , rtTA - PIT2 circuit ( red ) .

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C) Combinatorial screen. Computational combinatorial screening of Act1MAX and Act2MAX using the model depicted in A (left). Experimental combinatorial screening in H1 (middle) with circuit schematic on top. Two different Act1-Act2 circuit configurations are depicted: PIT2-tTA (left) and rtTA-PIT2 (right). Calculated FF4 and Output levels are shown for On, Off and Off/On (FF4) or On/Off (Output). Color plots show normalized mCitrine (left) and mCherry (right) expression levels for each Act1-Act2 combination. Color bars show lowest and highest expression range on a linear scale with median as the midpoint D) Correlation plots with calculated values on X axis (Model) and experimental data on Y axis (Data). A linear regression model (y = a + b*x) was used to compare experimental data with model predictions. Linear fit and coefficient of determination (R2) as indicated. PIT2-tTA circuit (black), rtTA-PIT2 circuit (red).


C ) Bar chart showing the performance of circuit 1 - 3 in H1 , HES - 2 and HEK293 . mCitrine ( top ) and mCherry ( bottom ) expression was normalized to internal Ef1a - iRFP control ( rel . U . ) ( left ) and further normalized to maximal mCitrine ( circuit 2 ) ( top ) or maximal mCherry ( bottom ) ( rel . U . / Ctrl ) ( right ) . Samples below detection limit are labelled with b . d . Data information : Each bar show mean ± s . d . of at least three biological replicates .

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C) Bar chart showing the performance of circuit 1 - 3 in H1, HES-2 and HEK293. mCitrine (top) and mCherry (bottom) expression was normalized to internal Ef1a-iRFP control (rel.U.) (left) and further normalized to maximal mCitrine (circuit 2) (top) or maximal mCherry (bottom) (rel.U./Ctrl) (right). Samples below detection limit are labelled with b.d. Data information: Each bar show mean ± s.d. of at least three biological replicates.


D ) Bar chart showing performance of circuit 1 in response to administration of miRNA mimics in H1 ( left ) and HEK293 ( right ) . First sample ( with profile + + - - - for H1 and - - - - - for HEK292 ) has no miRNA administered . For the other samples , miRNA dose with highest change in response has been selected from miRNA titration experiments Maximal mCitrine and mCherry levels ( as calculated in B ) are depicted as yellow and red dashed lines , respectively . Chart is shown in a base 10 logarithmic scale . Data information : Each bar show mean ± s . d . of at least three biological replicates .

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D) Bar chart showing performance of circuit 1 in response to administration of miRNA mimics in H1 (left) and HEK293 (right). First sample (with profile + + - - - for H1 and - - - - - for HEK292) has no miRNA administered. For the other samples, miRNA dose with highest change in response has been selected from miRNA titration experiments Maximal mCitrine and mCherry levels (as calculated in B) are depicted as yellow and red dashed lines, respectively. Chart is shown in a base 10 logarithmic scale. Data information: Each bar show mean ± s.d. of at least three biological replicates.


E ) Bar chart showing fine tuning of output using miSFITs library . Shown are FF4 / mCitrine ( top ) and Output / mCherry ( bottom ) of circuit 1 and 3 for different T17 variants . Data were normalized to control circuits . Data information : Each bar show mean ± s . d . of at least three biological replicates .

E) Bar chart showing fine tuning of output using miSFITs library. Shown are FF4 / mCitrine (top) and Output / mCherry (bottom) of circuit 1 and 3 for different T17 variants. Data were normalized to control circuits. Data information: Each bar show mean ± s.d. of at least three biological replicates.


A ) Circuit schematics ( left ) . Chart shows normalized Output production in response to changing amounts of plasmids encoding Act2 , tTA ( left ) and PIT2 ( right ) . Two transactivator constructs have been tested , onecontains miR302a / b target sites ( Off ) , the other by mock miRFF3 / FF6 target sites ( ON ) . Data information : Charts show mean ± s . d . of at least three biological replicates .

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A) Circuit schematics (left). Chart shows normalized Output production in response to changing amounts of plasmids encoding Act2, tTA (left) and PIT2 (right). Two transactivator constructs have been tested, onecontains miR302a/b target sites (Off), the other by mock miRFF3/FF6 target sites (ON). Data information: Charts show mean ± s.d. of at least three biological replicates.


B ) Discrete circuit - mediated control of BMP4 by endogenous miRNAs ( On ) . B ) Micropatterned RUES2 have been transfected with On and Offcircuits . Bar chart shows BMP4 concentration as determined by ELISA and average iRFP expression as measured by Flow Cytometry ( left ) . Corresponding bar chart showing the average fraction of cells expressing a given germ - layer marker ( right ) . Data information : Each bar show mean ± s . d . from at least three biological replicates for BMP4 and iRFP data and from at least 20 colonies for germ - layer data . Scale bar represents 200 μm .

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B) Discrete circuit-mediated control of BMP4 by endogenous miRNAs (On).B) Micropatterned RUES2 have been transfected with On and Offcircuits. Bar chart shows BMP4 concentration as determined by ELISA and average iRFP expression as measured by Flow Cytometry (left). Corresponding bar chart showing the average fraction of cells expressing a given germ-layer marker (right). Data information: Each bar show mean ± s.d. from at least three biological replicates for BMP4 and iRFP data and from at least 20 colonies for germ-layer data. Scale bar represents 200 μm.


D , Circuit - mediated tuning of BMP4 = using miSFITs library . D ) . Bar chart on the left shows BMP4 concentration for each circuit variant determined from culture media by ELISA . Bar chart on the right shows the same secreted BMP4 concentration along cell internal iRFP expression signal , measured by Flow Cytometry in comparison to untransfected controls in the absence ( - BMP4 ) and presence ( + BMP4 ) of BMP4 ( right ) . E Data information : Each bar show mean ± s . d . from at least three biological replicates for BMP4 and iRFP data and from at least 20 colonies for germ - layer data . Scale bar represents 200 μm .

D, Circuit-mediated tuning of BMP4 =using miSFITs library. D). Bar chart on the left shows BMP4 concentration for each circuit variant determined from culture media by ELISA. Bar chart on the right shows the same secreted BMP4 concentration along cell internal iRFP expression signal, measured by Flow Cytometry in comparison to untransfected controls in the absence (-BMP4) and presence (+ BMP4) of BMP4 (right).E Data information: Each bar show mean ± s.d. from at least three biological replicates for BMP4 and iRFP data and from at least 20 colonies for germ-layer data. Scale bar represents 200 μm.


E , Circuit - mediated tuning of BMP4 = using miSFITs library . ( right ) . E ) Bar chart showing the relative fraction of cells expressing a given germ - layer marker as analyzed from confocal microscopy images ( right ) . Dose response chart of the marker fraction in response to measured BMP4 concentration ( left ) . Data information : Each bar show mean ± s . d . from at least three biological replicates for BMP4 and from at least 20 colonies for germ - layer data .

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E, Circuit-mediated tuning of BMP4 =using miSFITs library. (right).E) Bar chart showing the relative fraction of cells expressing a given germ-layer marker as analyzed from confocal microscopy images (right). Dose response chart of the marker fraction in response to measured BMP4 concentration (left). Data information: Each bar show mean ± s.d. from at least three biological replicates for BMP4 and from at least 20 colonies for germ-layer data.


F ) Circuit - mediated tuning of BMP4 = using miSFITs library . EV5C . F ) Radial marker profiles of each germ - layer marker analyzed from confocal microscopy images . Charts on the left show normalized intensity of marker expression at a given colony position , where x = 0 is the center of the colony and x = 1 is the edge of the colony . Representative microscopy image of a single colony ( top ) showing composite of Sox2 / mCitrine ( yellow ) , Sox17 / mCherry ( red ) and TBXT / AF647 ( blue ) . Data information : Each bar show mean ± s . d . from at least three biological replicates for BMP4 and from at least 20 colonies for germ - layer data . Scale bar represents 200 μm .

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F) Circuit-mediated tuning of BMP4 =using miSFITs library. EV5C.F) Radial marker profiles of each germ-layer marker analyzed from confocal microscopy images. Charts on the left show normalized intensity of marker expression at a given colony position, where x=0 is the center of the colony and x=1 is the edge of the colony.Representative microscopy image of a single colony (top) showing composite of Sox2/mCitrine (yellow), Sox17/mCherry (red) and TBXT/AF647 (blue). Data information: Each bar show mean ± s.d. from at least three biological replicates for BMP4 and from at least 20 colonies for germ-layer data. Scale bar represents 200 μm.


Quantification of human EGFL7 by qRT - PCR in primary GBM biopsies and corresponding PDX xenografts upon implantation of GBM - derived organotypic spheroids ( n = 3 ; Mann - Whitney U - test ; mean ± SEM ) ; B - GBM biopsy ; G - generation .

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Quantification of human EGFL7 by qRT-PCR in primary GBM biopsies and corresponding PDX xenografts upon implantation of GBM-derived organotypic spheroids (n = 3; Mann-Whitney U-test; mean ± SEM); B - GBM biopsy; G - generation.


Alignment of EGFL7 expression along with stromal ( VWF , VEGFR1 , VEGFR2 , ACTA2 , CD248 , ITGAX , AIF1 , CD68 , CD4 , and PTPRC ) or tumor markers ( EGFR , PDGFRA , VEGFA , VIM , and NES ) in primary GBM biopsies or GBM - derived organotypic spheroids ( PDX xenografts ) .

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Alignment of EGFL7 expression along with stromal (VWF, VEGFR1, VEGFR2, ACTA2, CD248, ITGAX, AIF1, CD68, CD4, and PTPRC) or tumor markers (EGFR, PDGFRA, VEGFA, VIM, and NES) in primary GBM biopsies or GBM-derived organotypic spheroids (PDX xenografts).


Immunohisto chemical ( IHC ) analyses revealed EGFL7 expression in blood vessels ( arrow ) and neurons ( arrowheads ) of healthy gray matter . Expression in malignant brain tumor specimens was restricted to blood vessels , in particular with a large diameter ( arrows ) . Scale bar represents 50 µm . Quantitative scoring of EGFL7 staining in glioma specimens yielded about 25 - 40 % EGFL7 - positive blood vessels ( n = 13 astrocytoma WHO ° grade II , n = 25 astrocytoma WHO ° grade III , n = 24 GBMs WHO ° grade IV ) .

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Immunohistochemical (IHC) analyses revealed EGFL7 expression in blood vessels (arrow) and neurons (arrowheads) of healthy gray matter. Expression in malignant brain tumor specimens was restricted to blood vessels, in particular with a large diameter (arrows). Scale bar represents 50 µm. Quantitative scoring of EGFL7 staining in glioma specimens yielded about 25-40% EGFL7-positive blood vessels (n = 13 astrocytoma WHO° grade II, n = 25 astrocytoma WHO° grade III, n = 24 GBMs WHO° grade IV).


Overexpression of human EGFL7 ( hE7 ) or murine EGFL7 ( mE7 ) increased tumor growth ( n = 9 ; one - way ANOVA ) .

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Overexpression of human EGFL7 (hE7) or murine EGFL7 (mE7) increased tumor growth (n = 9; one-way ANOVA).


Immunocompetent mice bearing GL261 tumors . Representative MRI images confirmed tumor implantation in all experimental groups ( left ) . Kaplan - Meier curves revealed decreased survival of mice bearing EGFL7 expressing tumors ( right ; 34 d ( control ) vs . 29 . 5 d ( hE7 ) vs . 31 . 5 d ( mE7 ) ; n = 8 ; log - rank test ) . Immunodeficient mice bearing U87 tumors . Representative magnetic resonance images confirmed tumor implantation in all experimental groups ( left ) . Kaplan - Meier curves revealed decreased survival of mice bearing EGFL7 expressing tumors ( right ; 70 d ( control ) vs . 42 . 5 d ( hE7 ) vs . 40 d ( mE7 ) ; n = 8 ; log - rank test ) .

Immunocompetent mice bearing GL261 tumors. Representative MRI images confirmed tumor implantation in all experimental groups (left). Kaplan-Meier curves revealed decreased survival of mice bearing EGFL7 expressing tumors (right; 34 d (control) vs. 29.5 d (hE7) vs. 31.5 d (mE7); n = 8; log-rank test). Immunodeficient mice bearing U87 tumors. Representative magnetic resonance images confirmed tumor implantation in all experimental groups (left). Kaplan-Meier curves revealed decreased survival of mice bearing EGFL7 expressing tumors (right; 70 d (control) vs. 42.5 d (hE7) vs. 40 d (mE7); n = 8; log-rank test).


miR - 126 KO did not affect overall survival of GL261 tumor - bearing mice ( n = 8 ) .

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miR-126 KO did not affect overall survival of GL261 tumor-bearing mice (n = 8).


EGFL7 KO prolonged overall survival of GL261 tumor - bearing mice as compared to wild - type ( WT ) littermates ( 44 d in KO vs . 35 . 5 d in WT ; n = 8 each ; log - rank test ) .

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EGFL7 KO prolonged overall survival of GL261 tumor-bearing mice as compared to wild-type (WT) littermates (44 d in KO vs. 35.5 d in WT; n = 8 each; log-rank test).


Residual EGFL7 expression in BTPC11 glioma cells was reduced by a lentivirus - based approach . Knock - down of EGFL7 ( shE7 _ 1 or shE7 _ 2 ) prolonged the median survival time of glioma - bearing mice ( 68 d or 69 d ) as compared to scrambled control ( shScr ; 56 d ; n = 6 ; log - rank test ) .

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Residual EGFL7 expression in BTPC11 glioma cells was reduced by a lentivirus-based approach. Knock-down of EGFL7 (shE7_1 or shE7_2) prolonged the median survival time of glioma-bearing mice (68 d or 69 d) as compared to scrambled control (shScr; 56 d; n = 6; log-rank test).


The blood vessel - specific and miR126 - independent KO of EGFL7 in EGFL7fl / fl ; Cdh5 - CreERT2 mice prolonged the overall survival of GL261 tumor - bearing mice as compared to WT littermates ( 38 d in KO vs . 33 d in WT ; n = 7 for KO ; n = 5 for WT ; log - rank test ) . All data are presented as mean ± SEM . Scale bars represent 5 mm .

The blood vessel-specific and miR126-independent KO of EGFL7 in EGFL7fl/fl;Cdh5-CreERT2 mice prolonged the overall survival of GL261 tumor-bearing mice as compared to WT littermates (38 d in KO vs. 33 d in WT; n = 7 for KO; n = 5 for WT; log-rank test). All data are presented as mean ± SEM. Scale bars represent 5 mm.


CD31 staining for endothelial cells revealed increased tumor vascularization in mice bearing human EGFL7 ( hE7 ) - or murine EGFL7 ( mE7 ) - positive tumors ( n = 3 ; one - way ANOVA ) .

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CD31 staining for endothelial cells revealed increased tumor vascularization in mice bearing human EGFL7 (hE7)- or murine EGFL7 (mE7)-positive tumors (n = 3; one-way ANOVA).


T2 - weighted magnetic resonance imaging ( MRI ) analysis of these brains confirmed that all mice developed tumors of similar size . T1 - weighted MRI images showed decreased vessel permeability as measured by Gadovist extravasation in tumors expressing hE7 or mE7 ( red arrows ; n = 6 ; one - way ANOVA ) . Scale bar represents 2 . 5 mm .

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T2-weighted magnetic resonance imaging (MRI) analysis of these brains confirmed that all mice developed tumors of similar size. T1-weighted MRI images showed decreased vessel permeability as measured by Gadovist extravasation in tumors expressing hE7 or mE7 (red arrows; n = 6; one-way ANOVA). Scale bar represents 2.5 mm.


EGFL7 promoted density and maturation state of glioma vessels Enhanced maturation of glioma vessels in the presence of hE7 or mE7 was verified by increased co - localization of PDGFRβ ( pericytes ) with CD31 ( n = 3 ; one - way ANOVA ; quantifications normalized to CD31 ) . Data presented as mean ± SEM , AU - arbitrary units . Scale bars represent 60 µm .

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EGFL7 promoted density and maturation state of glioma vessels Enhanced maturation of glioma vessels in the presence of hE7 or mE7 was verified by increased co-localization of PDGFRβ (pericytes) with CD31 (n = 3; one-way ANOVA; quantifications normalized to CD31). Data presented as mean ± SEM, AU-arbitrary units. Scale bars represent 60 µm.


Enhanced maturation of glioma vessels in the presence of hE7 or mE7 was verified by increased co - localization of SMA ( smooth muscle cells ) , with CD31 ( n = 3 ; one - way ANOVA ; quantifications normalized to CD31 ) . Data presented as mean ± SEM , AU - arbitrary units . Scale bars represent 60 µm .

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Enhanced maturation of glioma vessels in the presence of hE7 or mE7 was verified by increased co-localization of SMA (smooth muscle cells), with CD31 (n = 3; one-way ANOVA; quantifications normalized to CD31). Data presented as mean ± SEM, AU-arbitrary units. Scale bars represent 60 µm.


Enhanced maturation of glioma vessels in the presence of hE7 or mE7 was verified by increased co - localization of ( I + J ) Col IV ( basement membrane ) with CD31 ( n = 3 ; one - way ANOVA ; quantifications normalized to CD31 ) . Data presented as mean ± SEM , AU - arbitrary units . Scale bars represent 60 µm .

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Enhanced maturation of glioma vessels in the presence of hE7 or mE7 was verified by increased co-localization of (I+J) Col IV (basement membrane) with CD31 (n = 3; one-way ANOVA; quantifications normalized to CD31). Data presented as mean ± SEM, AU-arbitrary units. Scale bars represent 60 µm.


Structural analysis of recombinant EGFL7 by transmission electron microscopy ( TEM ) showed aggregates with a fibrillar structure of varying sizes . Black scale bar left represents 50 nm . Higher magnifications depict rod - like EGFL7 particles . Black scale bar right represents 15 nm . White scale bar represents 5 nm .

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Structural analysis of recombinant EGFL7 by transmission electron microscopy (TEM) showed aggregates with a fibrillar structure of varying sizes. Black scale bar left represents 50 nm. Higher magnifications depict rod-like EGFL7 particles. Black scale bar right represents 15 nm. White scale bar represents 5 nm.


Angiogenic sprouting of primary human umbilical vein endothelial cell ( HUVEC ) spheroids embedded in a collagen - based matrix was induced by the application of EGFL7 ( E7 ) , fibronectin ( Fn ) , or a combination of both ( upper row ) . Co - application of an integrin α5β1 blocking antibody ( anti - α5β1 ) reduced sprouting in all cases but control ( lower row ) . Scale bar represents 100 µm .

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Angiogenic sprouting of primary human umbilical vein endothelial cell (HUVEC) spheroids embedded in a collagen-based matrix was induced by the application of EGFL7 (E7), fibronectin (Fn), or a combination of both (upper row). Co-application of an integrin α5β1 blocking antibody (anti-α5β1) reduced sprouting in all cases but control (lower row). Scale bar represents 100 µm.


E7 , Fn and a combination of both significantly increased the mean cumulative sprout length per spheroid , an effect blocked by anti - α5β1 ( n = 3 ; one - way ANOVA ( inter - group differences ) and Mann - Whitney U - test ( intra - group differences ) ; mean ± SEM ) .

E7, Fn and a combination of both significantly increased the mean cumulative sprout length per spheroid, an effect blocked by anti-α5β1 (n = 3; one-way ANOVA (inter-group differences) and Mann-Whitney U-test (intra-group differences); mean ± SEM).