( a ) Time - lapse images of MCF10A cell - in - cell structures with an internalized cell that undergoes apoptosis . The arrows mark the recruitment of LC3 around apoptotic fragments ( see Supplementary Movie S7 ) . Scale bar , 10 μm . ( b ) Quantification of LC3 recruitment to apoptotic internalized cells in the presence of ATG5 or FIP200 siRNA ; n , number of cells analysed ; * * * P0 . 0001 , chi - squared test .

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(a) Time-lapse images of MCF10A cell-in-cell structures with an internalized cell that undergoes apoptosis. The arrows mark the recruitment of LC3 around apoptotic fragments (see Supplementary Movie S7). Scale bar, 10 μm. (b) Quantification of LC3 recruitment to apoptotic internalized cells in the presence of ATG5 or FIP200 siRNA; n, number of cells analysed; ***P0.0001, chi-squared test.


( c ) Apoptotic U937 cells expressing H2B - mCherry phagocytized by J774 macrophages expressing GFP - LC3 . Confocal images show LC3 recruitment to an engulfed corpse ( arrow ; see Supplementary Movie S8 ) . Scale bar , 15 μm . ( d ) Quantification of LC3 recruitment to apoptotic phagosomes in control and Atg5 - shRNA - expressing cells ; * * * P0 . 0001 , chi - squared test .

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(c) Apoptotic U937 cells expressing H2B-mCherry phagocytized by J774 macrophages expressing GFP-LC3. Confocal images show LC3 recruitment to an engulfed corpse (arrow; see Supplementary Movie S8). Scale bar, 15 μm. (d) Quantification of LC3 recruitment to apoptotic phagosomes in control and Atg5-shRNA-expressing cells; ***P0.0001, chi-squared test.


( e ) Correlative light - electron microscopy of an apoptotic phagosome with GFP - LC3 recruitment ( top left image , white arrow ) . The LC3 - loaded phagosome consists of a single membrane ( black arrow in right image , which is a higher magnification of the area outlined in the bottom left image ; EM , electron microscopy ; AP , apoptotic cell nucleus ) .

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(e) Correlative light-electron microscopy of an apoptotic phagosome with GFP-LC3 recruitment (top left image, white arrow). The LC3-loaded phagosome consists of a single membrane (black arrow in right image, which is a higher magnification of the area outlined in the bottom left image; EM, electron microscopy; AP, apoptotic cell nucleus).


( f ) Representative images of apoptotic cell phagocytosis and degradation in control ( n = 21 ) and Atg5 shRNA ( n = 21 ) J774 GFP - LC3 cells ; * * * P0 . 0001 , chi - squared test . Scale bar , 10 μm . See also Supplementary Fig . S3 .

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(f) Representative images of apoptotic cell phagocytosis and degradation in control (n=21) and Atg5 shRNA (n=21) J774 GFP-LC3 cells; ***P0.0001, chi-squared test. Scale bar, 10 μm. See also Supplementary Fig. S3.


( a ) Representative images of a J774 macrophage expressing GFP - LC3 incubated in media containing red dextran . The arrow marks LC3 recruitment to a dextran - containing macropinosome . Scale bar , 2 μm . ( b ) Quantification of LC3 recruitment to macropinosomes in control and Atg5 shRNA J774 macrophages ; n , number of macropinosomes analysed ; * * * P0 . 0001 , chi - squared test . See Supplementary Movie S9 .

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(a) Representative images of a J774 macrophage expressing GFP-LC3 incubated in media containing red dextran. The arrow marks LC3 recruitment to a dextran-containing macropinosome. Scale bar, 2 μm. (b) Quantification of LC3 recruitment to macropinosomes in control and Atg5 shRNA J774 macrophages; n, number of macropinosomes analysed; ***P0.0001, chi-squared test. See Supplementary Movie S9.


( c ) Representative image of LC3 recruitment to macropinosomes ( arrows ) in MCF10A cells expressing GFP - LC3 incubated with red dextran . Scale bar , 2 μm . ( d ) Quantification of LC3 recruitment to macropinosomes in MCF10A cells treated with siRNA against FIP200 or ATG5 ; n , number of macropinosomes analysed ; * * * P0 . 0001 , chi - squared test .

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(c) Representative image of LC3 recruitment to macropinosomes (arrows) in MCF10A cells expressing GFP-LC3 incubated with red dextran. Scale bar, 2 μm. (d) Quantification of LC3 recruitment to macropinosomes in MCF10A cells treated with siRNA against FIP200 or ATG5; n, number of macropinosomes analysed; ***P0.0001, chi-squared test.


( e ) J774 GFP - LC3 macrophages were incubated with 3 μm uncoated latex beads and imaged by confocal microscopy for 1 h , followed by the addition of LysoTracker red to mark acidified compartments . A macropinosome recruited LC3 ( arrow ) whereas engulfed latex beads did not ( arrowheads ) . Scale bar , 5 μm . See Supplementary Movie S10 .

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(e) J774 GFP-LC3 macrophages were incubated with 3 μm uncoated latex beads and imaged by confocal microscopy for 1 h, followed by the addition of LysoTracker red to mark acidified compartments. A macropinosome recruited LC3 (arrow) whereas engulfed latex beads did not (arrowheads). Scale bar, 5 μm. See Supplementary Movie S10.


( a ) A C . elegans embryo expressing mCherry : : RAB - 5 and GFP : : LGG - 1 . The arrows point to two apoptotic phagosomes . See Supplementary Movie S11 . Scale bar , 10 μm . ( b ) Cropped time - lapse images showing RAB - 5 and LGG - 1 recruitment to an apoptotic phagosome ( from a , left arrow ) .

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(a) A C. elegans embryo expressing mCherry::RAB-5 and GFP::LGG-1. The arrows point to two apoptotic phagosomes. See Supplementary Movie S11. Scale bar, 10 μm. (b) Cropped time-lapse images showing RAB-5 and LGG-1 recruitment to an apoptotic phagosome (from a, left arrow).


( c ) Representative images of apoptotic phagosomes in embryos from control - RNAi - or bec - 1 - RNAi - fed worms . The insets show higher magnifications of the areas outlined in the main panels . ( d ) Quantification of GFP : : LGG - 1 - and mCherry : : RAB - 5 - positive phagosomes in control - or bec - 1 - RNAi embryos determined by time - lapse imaging . Control , 21 embryos , 52 phagosomes ; bec - 1 , 9 embryos , 25 phagosomes ; * * * P0 . 0001 , chi - squared test .

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(c) Representative images of apoptotic phagosomes in embryos from control-RNAi- or bec-1-RNAi-fed worms. The insets show higher magnifications of the areas outlined in the main panels. (d) Quantification of GFP::LGG-1- and mCherry::RAB-5-positive phagosomes in control- or bec-1-RNAi embryos determined by time-lapse imaging. Control, 21 embryos, 52 phagosomes; bec-1, 9 embryos, 25 phagosomes; ***P0.0001, chi-squared test.


( e ) Representative DIC images from control - and bec - 1 - RNAi embryos at different stages of development ( minutes after first division ) . The yellow arrowheads mark apoptotic corpses . Scale bar , 10 μm . ( f ) Quantification of apoptotic corpses in control - and bec - 1 - RNAi embryos ; data show ± s . d . ; * * * P0 . 0001 .

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(e) Representative DIC images from control- and bec-1-RNAi embryos at different stages of development (minutes after first division). The yellow arrowheads mark apoptotic corpses. Scale bar, 10 μm. (f) Quantification of apoptotic corpses in control- and bec-1-RNAi embryos; data show ± s.d.; ***P0.0001.


( g ) Representative images of CED - 1 : : GFP embryos with control or bec - 1 RNAi . The yellow arrowheads mark engulfed corpses surrounded by CED - 1 : : GFP . Scale bar , 5 μm .

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(g) Representative images of CED-1::GFP embryos with control or bec-1 RNAi. The yellow arrowheads mark engulfed corpses surrounded by CED-1::GFP. Scale bar, 5 μm.


Mitochondria - enriched fractions were isolated from HdhQ7 or HdhQ111 cells . mtHSF1 was examined by WB analysis . VDAC was analyzed as a loading control . ( n = 6 biological replicates ) .

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Mitochondria-enriched fractions were isolated from HdhQ7 or HdhQ111 cells. mtHSF1 was examined by WB analysis. VDAC was analyzed as a loading control. (n=6 biological replicates).


HdhQ7 WT striatal cells were exposed to 5 mM 3 - NP for 2 h . mtHSF1 levels were determined by immunoblotting . VDAC was used as a loading control . ( n = 5 biological replicates ) .

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HdhQ7 WT striatal cells were exposed to 5 mM 3-NP for 2 h. mtHSF1 levels were determined by immunoblotting. VDAC was used as a loading control. (n=5 biological replicates).


HSF1 was measured in the mitochondria - enriched fractions of the HD transgenic YAC128 mice ( 9 months old ) or age - matched littermates . ( n = 5 biological replicates ) .

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

HSF1 was measured in the mitochondria-enriched fractions of the HD transgenic YAC128 mice (9 months old) or age-matched littermates. (n=5 biological replicates).


mtHSF1 was analyzed in HD patient fibroblasts ( GM04208 , GM04222 , and GM21756 ) and fibroblasts from healthy individuals . ( n = 5 for Con1 / GM04208 and Con2 / GM04222 groups ; n = 3 for Con3 / GM21756 group ) .

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mtHSF1 was analyzed in HD patient fibroblasts (GM04208, GM04222, and GM21756) and fibroblasts from healthy individuals. (n = 5 for Con1/ GM04208 and Con2/ GM04222 groups; n = 3 for Con3/ GM21756 group).


Representative immunoblot of HSF1 levels in mitochondria isolated from patient - derived iPSCs and control iPSCs . ( n = 3 biological replicates ) .

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Representative immunoblot of HSF1 levels in mitochondria isolated from patient-derived iPSCs and control iPSCs. (n=3 biological replicates).


Representative images of striatal organoids . Left , immunostaining for the MSN marker DARPP32 ( green ) and the radial glial marker SOX2 ( red ) in striatal organoids at D60 . Middle , immunostaining for the LGE progenitor markers Ctip2 ( green ) and GSH2 ( red ) at D30 . Right panel , immunostaining for the neuronal marker NeuN ( green ) and the proliferation marker Ki67 ( red ) at D30 . The images are representative of 12 independent differentiation experiments of H9 and IMR90 - 4 cells . HO = Hoechst ( blue ) . The scale bar represents 100 µm .

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Representative images of striatal organoids. Left, immunostaining for the MSN marker DARPP32 (green) and the radial glial marker SOX2 (red) in striatal organoids at D60. Middle, immunostaining for the LGE progenitor markers Ctip2 (green) and GSH2 (red) at D30. Right panel, immunostaining for the neuronal marker NeuN (green) and the proliferation marker Ki67 (red) at D30. The images are representative of 12 independent differentiation experiments of H9 and IMR90-4 cells. HO= Hoechst (blue). The scale bar represents 100 µm.


Representative images and scatterplot confirming the increased HSF1 ( green ) translocation to mitochondria ( Tom20 , red ) . The data were obtained from 3 independent biological experiments . Con : n = 15 organoids ; HD : n = 14 organoids . Two - tailed unpaired t - test ; Means ± SEMs . Images were taken using Structured Illumination Microscopy ( SIM ) .

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Representative images and scatterplot confirming the increased HSF1 (green) translocation to mitochondria (Tom20, red). The data were obtained from 3 independent biological experiments. Con: n=15 organoids; HD: n=14 organoids. Two-tailed unpaired t-test; Means ± SEMs. Images were taken using Structured Illumination Microscopy (SIM).


The subcellular localization of HSF1 in HD striatal organoids was observed by immunoelectron microscopy . The arrows ( red ) mark HSF1 in mitochondria . The negative control lacked the HSF1 antibody . The scale bar represents 200 nm .

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The subcellular localization of HSF1 in HD striatal organoids was observed by immunoelectron microscopy. The arrows (red) mark HSF1 in mitochondria. The negative control lacked the HSF1 antibody. The scale bar represents 200 nm.


HTT was knocked down in HdhQ7 or HdhQ111 cells by lentiviral infection . The protein levels of HSF1 were detected in mitochondrial fractions . ( n = 3 biological replicates ) .

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HTT was knocked down in HdhQ7 or HdhQ111 cells by lentiviral infection. The protein levels of HSF1 were detected in mitochondrial fractions. (n=3 biological replicates).


HdhQ7 cells were transfected with Flag - mtHSF1 and stained with anti - Tom20 ( green ) and anti - Flag ( red ) antibodies . Mitochondrial morphology was examined by confocal microscopy . Scatterplot with bar showed the percentage of cells with mitochondrial fragmentation . The scale bar represents 40 µm . ( n = 3 biological replicates , at least 100 cells per group were counted ) .

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HdhQ7 cells were transfected with Flag-mtHSF1 and stained with anti-Tom20 (green) and anti-Flag (red) antibodies. Mitochondrial morphology was examined by confocal microscopy. Scatterplot with bar showed the percentage of cells with mitochondrial fragmentation. The scale bar represents 40 µm. (n=3 biological replicates, at least 100 cells per group were counted).


The total protein levels of p - Drp1 S616 , p - Drp1S637 , Drp1 , OPA1 , MFN2 , Flag - mtHSF1 and actin ( loading control ) were determined by WB analysis . ( n = 3 biological replicates ) .

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The total protein levels of p-Drp1 S616, p-Drp1S637, Drp1, OPA1, MFN2, Flag-mtHSF1 and actin (loading control) were determined by WB analysis. (n=3 biological replicates).


Brain sections were stained with an anti - Tom20 antibody ( red ) , an anti - Flag antibody ( green ) and DAPI ( blue ) . Mitochondrial morphology was analyzed in Flag + or Flag - regions by confocal microscopy . The scale bar presents 2 mm , and the enlarged image scale bar is 40 µm . ( n = 6 mice / group ) .

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Brain sections were stained with an anti-Tom20 antibody (red), an anti-Flag antibody (green) and DAPI (blue). Mitochondrial morphology was analyzed in Flag+ or Flag- regions by confocal microscopy. The scale bar presents 2 mm, and the enlarged image scale bar is 40 µm. (n=6 mice/group).


Striata injected with AAVs were lysed . p - Drp1 S616 was tested by immunoblotting . ( n = 5 mice / group ) .

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Striata injected with AAVs were lysed. p-Drp1 S616 was tested by immunoblotting. (n=5 mice/group).


Mitochondrial morphology was analyzed by electron microscopy ( EM ) in mice injected with AAV - Con or AAV - mtHSF1 . Scatterplot showed the length of mitochondria . The scale bar presents 4 µm , and the enlarged image scale bar is 400 nm . ( n = 3 mice / group ) . AAV - Con group , n = 268 mitochondria were measured ; AAV - Flag - mtHSF1 group , n = 179 mitochondria were measured .

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Mitochondrial morphology was analyzed by electron microscopy (EM) in mice injected with AAV-Con or AAV-mtHSF1. Scatterplot showed the length of mitochondria. The scale bar presents 4 µm, and the enlarged image scale bar is 400 nm. (n=3 mice/group). AAV-Con group, n=268 mitochondria were measured; AAV-Flag-mtHSF1 group, n=179 mitochondria were measured.


Representative images and scatterplot of mitochondrial length ( Tom20 , red ) in Flag + or Flag - cells . The data were obtained from 3 independent biological experiments . Flag - : n = 154 mitochondria ; Flag + : n = 132 mitochondria .

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Representative images and scatterplot of mitochondrial length (Tom20, red) in Flag+ or Flag- cells. The data were obtained from 3 independent biological experiments. Flag-: n=154 mitochondria; Flag+: n=132 mitochondria.


Representative images and scatterplot showing that the number of p - Drp1 S616 puncta in Flag + cells was greater than that in Flag - cells . The data were obtained from 3 independent biological experiments . Flag - : n = 70 cells from 10 organoids ; Flag + : n = 55 cells from 10 organoids . The images were obtained using structured illumination microscopy ( SIM ) .

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Representative images and scatterplot showing that the number of p-Drp1 S616 puncta in Flag+ cells was greater than that in Flag- cells. The data were obtained from 3 independent biological experiments. Flag-: n=70 cells from 10 organoids; Flag+: n=55 cells from 10 organoids. The images were obtained using structured illumination microscopy (SIM).


The mtDNA content was examined with primers of D - loop , mtCO1 , mtCO2 and mtND2 by qPCR in control or Flag - mtHSF1 - expressing striatal cells . Scatterplot with bar showed the relative percentage of mtDNA content . ( n = 3 biological replicates ) .

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The mtDNA content was examined with primers of D-loop, mtCO1, mtCO2 and mtND2 by qPCR in control or Flag-mtHSF1-expressing striatal cells. Scatterplot with bar showed the relative percentage of mtDNA content. (n=3 biological replicates).


Measurement of mitochondrial respiratory activity by Seahorse analysis . ( n = 9 - 10 biological replicates )

Measurement of mitochondrial respiratory activity by Seahorse analysis. (n=9-10 biological replicates)


Cells were treated with EtBr for six days to delete mtDNA . mtDNA repopulation efficiency was measured by qPCR after withdrawal of EtBr . ( n = 3 biological replicates ) .

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Cells were treated with EtBr for six days to delete mtDNA. mtDNA repopulation efficiency was measured by qPCR after withdrawal of EtBr. (n=3 biological replicates).


D - E . Cell death was determined by detection of LDH release into the medium and cleavage of PARP . Scatterplot with bar showed the relative percentage of LDH release . ( n = 3 biological replicates ) .

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D-E. Cell death was determined by detection of LDH release into the medium and cleavage of PARP. Scatterplot with bar showed the relative percentage of LDH release. (n=3 biological replicates).


qPCR analysis of the mtDNA content of AAV - Con - injected mice or AAV - mtHSF1 - injected mice . Scatterplot with bar showed the relative percentage of mtDNA content ( n = 3 mice / group ) .

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qPCR analysis of the mtDNA content of AAV-Con-injected mice or AAV-mtHSF1-injected mice. Scatterplot with bar showed the relative percentage of mtDNA content (n=3 mice/group).


Cleaved - Caspase3 in AAV - Con - or AAV - mtHSF1 - injected mice was detected by WB analysis . ( n = 5 mice / group ) .

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Cleaved-Caspase3 in AAV-Con- or AAV-mtHSF1-injected mice was detected by WB analysis. (n=5 mice/group).


Representative images and scatterplot showing the number of mtDNA ( purple ) colocalized with mitochondria ( Tom20 , green ) in striatal organoids injected with AAV - Con or AAV - mtHSF1 . The data were obtained from 3 independent biological experiments . Con : n = 65 cells from 20 organoids ; Flag - mtHSF1 : n = 70 cells from 20 organoids . Two - tailed unpaired t - test was used . Images were captured by Structured Illumination Microscopy ( SIM ) .

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Representative images and scatterplot showing the number of mtDNA (purple) colocalized with mitochondria (Tom20, green) in striatal organoids injected with AAV-Con or AAV-mtHSF1. The data were obtained from 3 independent biological experiments. Con: n=65 cells from 20 organoids; Flag-mtHSF1: n=70 cells from 20 organoids. Two-tailed unpaired t-test was used. Images were captured by Structured Illumination Microscopy (SIM).


The typical nuclear fragmentation morphology of AAV - Con - ( GFP ) or AAV - mtHSF1 - injected striatal organoids was measured . Scatterplot showed the quantitative result of nuclear fragmentation . The arrows ( yellow ) mark fragmented nuclei . Two - tailed unpaired t - test was used . The data were obtained from 3 independent biological experiments . Con : n = 14 organoids ; Flag - mtHSF1 : n = 15 organoids .

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The typical nuclear fragmentation morphology of AAV-Con- (GFP) or AAV-mtHSF1-injected striatal organoids was measured. Scatterplot showed the quantitative result of nuclear fragmentation. The arrows (yellow) mark fragmented nuclei. Two-tailed unpaired t-test was used. The data were obtained from 3 independent biological experiments. Con: n=14 organoids; Flag-mtHSF1: n=15 organoids.


Total cell lysates were subjected to immunoprecipitation with an anti - HSF1 antibody followed by WB analysis . ( n = 3 biological replicates ) .

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Total cell lysates were subjected to immunoprecipitation with an anti-HSF1 antibody followed by WB analysis. (n=3 biological replicates).


HdhQ7 or HdhQ111 cell extracts were incubated with DSS ( 1 mM ) for 30 min at RT . SSBP1 oligomers were detected by immunoblotting with an anti - SSBP1 antibody . ( n = 3 biological replicates ) .

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HdhQ7 or HdhQ111 cell extracts were incubated with DSS (1 mM) for 30 min at RT. SSBP1 oligomers were detected by immunoblotting with an anti-SSBP1 antibody. (n=3 biological replicates).


SSBP1 oligomers in control and patient fibroblasts ( GM04208 and GM21756 ) were analyzed . ( n = 3 biological replicates ) .

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SSBP1 oligomers in control and patient fibroblasts (GM04208 and GM21756) were analyzed. (n=3 biological replicates).


SSBP1 oligomers were measured in WT mice or YAC128 mice . ( n = 3 mice / group ) .

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SSBP1 oligomers were measured in WT mice or YAC128 mice. (n=3 mice/group).


Cells were transfected with an empty vector or Flag - mtHSF1 for 48 h . The total cell lysates were harvested and crosslinked with DSS . SSBP1 oligomers were tested by immunoblotting . ( n = 4 biological replicates ) .

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Cells were transfected with an empty vector or Flag-mtHSF1 for 48 h. The total cell lysates were harvested and crosslinked with DSS. SSBP1 oligomers were tested by immunoblotting. (n=4 biological replicates).


Flag - mtHSF1 was expressed in the striata of WT mice for three weeks . SSBP1 oligomers were determined by immunoblotting . ( n = 5 mice / group ) .

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Flag-mtHSF1 was expressed in the striata of WT mice for three weeks. SSBP1 oligomers were determined by immunoblotting. (n=5 mice/group).


Flag - mtHSF1 or sh - SSBP1 was delivered into cells via lentiviral infection . The expression of SSBP1 and mtHSF1 was detected by WB analysis . The mtDNA content was examined by qPCR analysis . ( n = 3 biological replicates ) .

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Flag-mtHSF1 or sh-SSBP1 was delivered into cells via lentiviral infection. The expression of SSBP1 and mtHSF1 was detected by WB analysis. The mtDNA content was examined by qPCR analysis. (n=3 biological replicates).


Primary neurons expressing Flag - mtHSF1 were stained with anti - DARPP32 ( green ) and anti - Flag ( red ) antibodies . The arrows ( white ) mark Flag + or Flag - primary neurons . The neurite length of Flag + or Flag - neurons was measured by Image J and showed by scatterplot . The scale bar represents 50 µm . ( n = 155 for Flag - group and n = 154 for Flag + group ) .

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Primary neurons expressing Flag-mtHSF1 were stained with anti-DARPP32 (green) and anti-Flag (red) antibodies. The arrows (white) mark Flag+ or Flag- primary neurons. The neurite length of Flag+ or Flag- neurons was measured by Image J and showed by scatterplot. The scale bar represents 50 µm. (n = 155 for Flag- group and n = 154 for Flag+ group).


Human striatal organoids were injected with AAV - Con ( GFP ) or AAV - mtHSF1 at D42 . Scatterplot showed the percentage of NeuN + cells ( red ) among Flag + or GFP + cells ( green ) after injection for 2 weeks . The data were obtained from 3 independent biological experiments . Con : n = 11 organoids ; Flag - mtHSF1 : n = 13 organoids . The scale bar represents 100 µm .

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Human striatal organoids were injected with AAV-Con (GFP) or AAV-mtHSF1 at D42. Scatterplot showed the percentage of NeuN+ cells (red) among Flag+ or GFP+ cells (green) after injection for 2 weeks. The data were obtained from 3 independent biological experiments. Con: n=11 organoids; Flag-mtHSF1: n=13 organoids. The scale bar represents 100 µm.


Representative images of immunostaining with DARPP32 ( red ) and Flag ( green ) . The DARPP32 fluorescence density was analyzed in Flag + or Flag - sections by confocal microscopy and showed by scatterplot . Thickness : 30 µm . The scale bar is 2 mm , and the enlarged image scale bar is 40 µm . ( n = 12 mice / group ) .

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Representative images of immunostaining with DARPP32 (red) and Flag (green). The DARPP32 fluorescence density was analyzed in Flag+ or Flag- sections by confocal microscopy and showed by scatterplot. Thickness: 30 µm. The scale bar is 2 mm, and the enlarged image scale bar is 40 µm. (n=12 mice/group).


The striatum of AAV - Con - or AAV - mtHSF1 - injected mice were harvested . The DARPP32 protein levels were tested by WB analysis . ( n = 6 mice / group ) .

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The striatum of AAV-Con- or AAV-mtHSF1-injected mice were harvested. The DARPP32 protein levels were tested by WB analysis. (n=6 mice/group).


The percentage of NeuN + cells in Flag + or Flag - sections was determined by confocal microscopy and showed by scatterplot . Thickness : 10 µm . ( n = 9 mice / group ) .

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The percentage of NeuN+ cells in Flag+ or Flag- sections was determined by confocal microscopy and showed by scatterplot. Thickness: 10 µm. (n=9 mice/group).


Mouse weight and motor activity were recorded post AAV injection . ( n = 7 - 13 mice / group ) .

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Mouse weight and motor activity were recorded post AAV injection. (n=7-13 mice/group).


Total lysates of HdhQ7 or HdhQ111 cells were subjected to immunoprecipitation with an anti - HSF1 antibody followed by immunoblotting . ( n = 3 biological replicates ) .

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Total lysates of HdhQ7 or HdhQ111 cells were subjected to immunoprecipitation with an anti-HSF1 antibody followed by immunoblotting. (n=3 biological replicates).


Drp1 and HSF1 binding in vitro was analyzed by WB analysis . ( n = 3 biological replicates ) .

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Drp1 and HSF1 binding in vitro was analyzed by WB analysis. (n=3 biological replicates).


Mitochondrial fractions were isolated from WT or Drp1 - KO MEFs . The protein levels of Drp1 and HSF1 were tested by immunoblotting . ( n = 3 biological replicates ) .

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Mitochondrial fractions were isolated from WT or Drp1-KO MEFs. The protein levels of Drp1 and HSF1 were tested by immunoblotting. (n=3 biological replicates).


GST pulldown assay of Drp1 and HSF1 in the presence or absence of DH1 . ( n = 3 biological replicates ) .

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GST pulldown assay of Drp1 and HSF1 in the presence or absence of DH1. (n=3 biological replicates).


HdhQ7 or HdhQ111 cells ( 1 μM / day , 3 days ) , were treated with DH1 or TAT . mtHSF1 was measured by immunoblotting . ( n = 3 - 5 biological replicates ) .

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HdhQ7 or HdhQ111 cells (1 μM/day, 3 days), were treated with DH1 or TAT. mtHSF1 was measured by immunoblotting. (n=3-5 biological replicates).


H . YAC128 or age - matched littermates ( 3 mg / kg / day , 3 months ) , were treated with DH1 or TAT . mtHSF1 was measured by immunoblotting . ( n = 3 - 5 biological replicates ) .

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H. YAC128 or age-matched littermates (3 mg/kg/day, 3 months), were treated with DH1 or TAT. mtHSF1 was measured by immunoblotting. (n=3-5 biological replicates).


I . striatal organoids differentiated from patient iPSCs or control iPSCs ( 1 μM / day , 7 days ) were treated with DH1 or TAT . mtHSF1 was measured by immunoblotting . ( n = 3 - 5 biological replicates ) .

I. striatal organoids differentiated from patient iPSCs or control iPSCs (1 μM/day, 7 days) were treated with DH1 or TAT. mtHSF1 was measured by immunoblotting. (n=3-5 biological replicates).


J . patient fibroblasts ( 1 μM / day , 5 days ) were treated with DH1 or TAT . mtHSF1 was measured by immunoblotting . ( n = 3 - 5 biological replicates ) .

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J. patient fibroblasts (1 μM/day, 5 days) were treated with DH1 or TAT. mtHSF1 was measured by immunoblotting. (n=3-5 biological replicates).


HdhQ7 or HdhQ111 were treated with DH1 or TAT ( 1 μM / day , 3 days ) . SSBP1 oligomers were analyzed by immunoblotting . ( n = 3 - 6 biological replicates ) .

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HdhQ7 or HdhQ111 were treated with DH1 or TAT (1 μM/day, 3 days). SSBP1 oligomers were analyzed by immunoblotting. (n=3-6 biological replicates).


L . patient fibroblasts were treated with DH1 or TAT ( 1 μM / day , 3 days ) . SSBP1 oligomers were analyzed by immunoblotting . ( n = 3 - 6 biological replicates ) .

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L. patient fibroblasts were treated with DH1 or TAT (1 μM/day, 3 days). SSBP1 oligomers were analyzed by immunoblotting. (n=3-6 biological replicates).


Immunostaining for mitochondria ( Tom20 , green ) and mtDNA ( anti - DNA , red ) in striatal organoids treated with DH1 or TAT at D60 . Scatterplots with bar showed the length of mitochondria and the number of mtDNA colocalized with mitochondria . The data were obtained from 3 independent biological experiments . Left , n = 222 - 311 mitochondria per group ; Right , n = 14 - 20 organoids per group . Images were taken using Structured Illumination Microscopy ( SIM ) . The scale bar represents 10 μm .

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Immunostaining for mitochondria (Tom20, green) and mtDNA (anti-DNA, red) in striatal organoids treated with DH1 or TAT at D60. Scatterplots with bar showed the length of mitochondria and the number of mtDNA colocalized with mitochondria. The data were obtained from 3 independent biological experiments. Left, n=222-311 mitochondria per group; Right, n=14-20 organoids per group. Images were taken using Structured Illumination Microscopy (SIM). The scale bar represents 10 μm.


Immunostaining for mitochondria ( Tom20 , red ) and p - Drp1 S616 ( green ) in striatal treated with DH1 or TAT at D60 . Scatterplot with bar showed the number of p - Drp1 S616 per cell in organoids . The data were obtained from 3 independent biological experiments . n = 29 - 34 cells from at least 10 organoids . The scale bar represents 5 μm .

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Immunostaining for mitochondria (Tom20, red) and p-Drp1 S616 (green) in striatal  treated with DH1 or TAT at D60. Scatterplot with bar showed the number of p-Drp1 S616 per cell in organoids. The data were obtained from 3 independent biological experiments. n=29-34 cells from at least 10 organoids. The scale bar represents 5 μm.


Scatterplot with bar showed the ratio of MSNs ( DARPP32 , green ) in striatal organoids in the presence or absence of DH1 . The data were obtained from 3 independent biological experiments . n = 30 organoids per group .

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Scatterplot with bar showed the ratio of MSNs (DARPP32, green) in striatal organoids in the presence or absence of DH1. The data were obtained from 3 independent biological experiments. n=30 organoids per group.


Representative tracing images of DARPP32 + neurons treated with DH1 or TAT at D45 . Scatterplot with bar showed the results of longest neurites length . The data were obtained from 3 independent biological experiments . n = 45 - 92 neurons per group .

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Representative tracing images of DARPP32+ neurons treated with DH1 or TAT at D45. Scatterplot with bar showed the results of longest neurites length. The data were obtained from 3 independent biological experiments. n=45-92 neurons per group.


Representative images and scatterplot with bar of nuclear fragmentation in striatal organoids treated with DH1 . The arrows ( white ) mark fragmented nuclei . The data were obtained from 3 independent biological experiments . n = 7 - 10 organoids per group . The scale bar represents 20 μm .

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Representative images and scatterplot with bar of nuclear fragmentation in striatal organoids treated with DH1. The arrows (white) mark fragmented nuclei. The data were obtained from 3 independent biological experiments. n=7-10 organoids per group. The scale bar represents 20 μm.


H . PGC1α protein levels were determined by immunoblotting . ( n = 3 biological replicates ) .

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H. PGC1α protein levels were determined by immunoblotting. (n=3 biological replicates).


I . DARPP32 protein levels were determined by immunoblotting . ( n = 3 biological replicates ) .

I. DARPP32 protein levels were determined by immunoblotting. (n=3 biological replicates).


Mouse behavior was tested after DH1 treatment . ( n = 9 - 12 mice / group ) .

Mouse behavior was tested after DH1 treatment. (n=9-12 mice/group).


) Animals harboring CRISPR - generated deletion alleles in y48g1c . 1 ( gg682 or gg683 ) were treated with dpy - 11 RNAi and progeny , grown in the absence of dpy - 11 RNAi , were scored for Dpy . Percentage of Dpy animals is shown . N = 3 , error bar is standard deviation , s . d .

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) Animals harboring CRISPR-generated deletion alleles in y48g1c.1 (gg682 or gg683) were treated with dpy-11 RNAi and progeny, grown in the absence of dpy-11 RNAi, were scored for Dpy. Percentage of Dpy animals is shown. N=3, error bar is standard deviation, s.d.


E ) Fluorescence micrograph of GFP : : ZNFX - 1 and PGL - 1 : : TagRFP fluorescence in animals harboring a CRISPR - generated deletion allele ( gg682 ) in y48g1c . 1 . Scale bar , 5μm .

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E) Fluorescence micrograph of GFP::ZNFX-1 and PGL-1::TagRFP fluorescence in animals harboring a CRISPR-generated deletion allele (gg682) in y48g1c.1. Scale bar, 5μm.


Fluorescence micrograph of gfp : : zsp - 1 at the indicated stages using a Nikon Eclipse Ti microscope equipped with a W1 Yokogawa Spinning disc under a 63X / 1 . 4 Plan Apo oil objective . Scale bar , embryos : 5μm , germline : 10μm . Dashed lines indicate the outline of the adult germline .

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Fluorescence micrograph of gfp::zsp-1 at the indicated stages using a Nikon Eclipse Ti microscope equipped with a W1 Yokogawa Spinning disc under a 63X/1.4 Plan Apo oil objective. Scale bar, embryos: 5μm, germline: 10μm. Dashed lines indicate the outline of the adult germline.


E ) Germlines dissected from animals expressing the indicated proteins were fixed and stained with α - GFP and α - HA or α - FLAG antibodies , followed by secondary antibodies conjugated with Alexa 488 and CF430 , respectively . Pachytene germ - cell nuclei were imaged with a Leica TCS SP8 STED 3X microscope using 592nm to selectively deactivate fluorophores . Bottom panel . GFP : : ZNFX - 1 and HA : : TagRFP : : ZNFX - 1 expressing animals were crossed and germlines of trans - heterozygous progeny from this cross were fixed and stained and imaged by STED as described above . Scale bar , 0 . 5μm .

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E) Germlines dissected from animals expressing the indicated proteins were fixed and stained with α-GFP and α-HA or α-FLAG antibodies, followed by secondary antibodies conjugated with Alexa 488 and CF430, respectively. Pachytene germ-cell nuclei were imaged with a Leica TCS SP8 STED 3X microscope using 592nm to selectively deactivate fluorophores. Bottom panel. GFP::ZNFX-1 and HA::TagRFP::ZNFX-1 expressing animals were crossed and germlines of trans-heterozygous progeny from this cross were fixed and stained and imaged by STED as described above. Scale bar, 0.5μm.


Z - stack confocal images from pachytene germ cells from animals of the indicated genotypes were analysed as described in Fig . EV4B and Materials and Methods to determine ; the size of P granules ( panel B ) Granules from 9 total nuclei from 3 different animals ( 3 cells per animal ) were quantified for these analyses . Black lines indicate mean of all data points in each group . P value , student ' s t test , two tailed .

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Z-stack confocal images from pachytene germ cells from animals of the indicated genotypes were analysed as described in Fig. EV4B and Materials and Methods to determine; the size of P granules (panel B) Granules from 9 total nuclei from 3 different animals (3 cells per animal) were quantified for these analyses. Black lines indicate mean of all data points in each group. P value, student's t test, two tailed.


Z - stack confocal images from pachytene germ cells from animals of the indicated genotypes were analysed as described to determine ; the size of P or Z granules ( panel C ) ; or the number of P or Z granules surrounding a typical nucleus in wild - type and zsp - 1 ( gg682 ) animals ( panel D ) . Granules from 9 total nuclei from 3 different animals ( 3 cells per animal ) were quantified for these analyses . Black lines indicate mean of all data points in each group . P value , student ' s t test , two tailed .

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Z-stack confocal images from pachytene germ cells from animals of the indicated genotypes were analysed as described to determine; the size of P or Z granules (panel C); or the number of P or Z granules surrounding a typical nucleus in wild-type and zsp-1(gg682) animals (panel D). Granules from 9 total nuclei from 3 different animals (3 cells per animal) were quantified for these analyses. Black lines indicate mean of all data points in each group. P value, student's t test, two tailed.


G , H ) GFP : : ZNFX - 1 ( panel G ) fluorescence or PGL - 1 : : TagRFP fluorescence ( panel H ) in the anterior ( A ) or posterior ( P ) section of a one cell embryo . Anterior and posterior regions were determined based on corresponding DIC images . N = 4 embryos for G , N = 3 embryos for H . Error bars are + / - s . d .

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G,H) GFP::ZNFX-1 (panel G) fluorescence or PGL-1::TagRFP fluorescence (panel H) in the anterior (A) or posterior (P) section of a one cell embryo. Anterior and posterior regions were determined based on corresponding DIC images. N=4 embryos for G, N=3 embryos for H. Error bars are +/- s.d.


A ) Heat map showing recovery of ZNFX - 1 fluorescence after photobleaching in Z granules from pachytene stage adult germ cells from wild - type or zsp - 1 ( gg682 ) mutant animals . Scale bar , 0 . 5 μm . B ) Normalized recovery of fluorescence after photobleaching of GFP : : ZNFX - 1 in wild - type or zsp - 1 ( - ) mutant animals over indicated time points . Bleaching event occurred at 0s . Data are represented as ± s . e . m . of n = 13 , and n = 16 individual granules from wild - type and zsp - 1 ( gg682 ) animals , respectively .

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A) Heat map showing recovery of ZNFX-1 fluorescence after photobleaching in Z granules from pachytene stage adult germ cells from wild-type or zsp-1(gg682) mutant animals. Scale bar, 0.5 μm. B) Normalized recovery of fluorescence after photobleaching of GFP::ZNFX-1 in wild-type or zsp-1(-) mutant animals over indicated time points. Bleaching event occurred at 0s. Data are represented as ± s.e.m. of n=13, and n=16 individual granules from wild-type and zsp-1(gg682) animals, respectively.


A ) Lineages of animals of the indicated genotypes were grown at 25 ° C or 20 ° C for the indicated generations . Each generation six animals were isolated in order to maintain the lineage and to determine numbers of progeny . Data are represented as + / - s . e . m . N = 3 for generation 8 or 9 , N = 6 for all other generations .

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A) Lineages of animals of the indicated genotypes were grown at 25°C or 20°C for the indicated generations. Each generation six animals were isolated in order to maintain the lineage and to determine numbers of progeny. Data are represented as +/- s.e.m. N=3 for generation 8 or 9, N=6 for all other generations.


F ) oma - 1 ( zu405 ) is a temperature sensitive ( ts ) lethal ( embryonic arrest at temperatures > 20 ° C ) allele of oma - 1 ( R . Lin 2003 ) . oma - 1 RNAi suppresses oma - 1 ( zu405ts ) lethality . Animals were exposed to oma - 1 RNAi and the number of embryos that hatched was scored at 20 ° C . N = 3 . Error bars are + / - s . d . Animals harboring mutations in genes specifically required for the inheritance of RNAi would respond normally to direct exposure to oma - 1 dsRNA but fail to inherit this silencing ( Wan et al . 2018 ) . zsp - 1 mutants fail to respond immediately to direct exposure to oma - 1 dsRNA , indicating that ZSP - 1 is not specific to inheritance and , therefore , is generally required for RNAi ( see also Fig . EV5B - D ) .

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F) oma-1(zu405) is a temperature sensitive (ts) lethal (embryonic arrest at temperatures > 20°C) allele of oma-1 (R. Lin 2003). oma-1 RNAi suppresses oma-1(zu405ts) lethality. Animals were exposed to oma-1 RNAi and the number of embryos that hatched was scored at 20°C. N=3. Error bars are +/- s.d. Animals harboring mutations in genes specifically required for the inheritance of RNAi would respond normally to direct exposure to oma-1 dsRNA but fail to inherit this silencing (Wan et al. 2018). zsp-1 mutants fail to respond immediately to direct exposure to oma-1 dsRNA, indicating that ZSP-1 is not specific to inheritance and, therefore, is generally required for RNAi (see also Fig. EV5B-D).


Volcano plot highlighting proteins significantly enriched ( open circles , FDR < 0 . 05 ) in eNOS - FLAG immunoprecipitates from growth factor - stimulated human endothelial cells ; n = 3 independent cell batches .

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Volcano plot highlighting proteins significantly enriched (open circles, FDR<0.05) in eNOS-FLAG immunoprecipitates from growth factor-stimulated human endothelial cells; n=3 independent cell batches.


Nitrite in the cell supernatant of HEK293 cells expressing wild - type ( WT ) , Y657F ( YF ) or Y657D ( YD ) eNOS and treated with solvent ( Sol ) or ionomycin ( Io , 1 µmol / L , 15 min ) ; n = 6 independent experiments ( 2 - way ANOVA and Bonferroni ) .

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Nitrite in the cell supernatant of HEK293 cells expressing wild-type (WT), Y657F (YF) or Y657D (YD) eNOS and treated with solvent (Sol) or ionomycin (Io, 1 µmol/L, 15 min); n=6 independent experiments (2-way ANOVA and Bonferroni).


The FMN / FAD ratio measured in eNOS immunoprecipitates from cells expressing WT , YF or YD eNOS ; n = 6 independent experiments ( 1 - way ANOVA and Newman - Keuls ) .

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The FMN/FAD ratio measured in eNOS immunoprecipitates from cells expressing WT, YF or YD eNOS; n=6 independent experiments (1-way ANOVA and Newman-Keuls).


Consequence of mutating Y657 on the growth factor - induced formation of eNOS complexes with Hsp90 and calmodulin ( CaM ) ; n = 6 - 19 independent cell batches ( 1 - way ANOVA and Newman - Keuls ) .

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

Consequence of mutating Y657 on the growth factor-induced formation of eNOS complexes with Hsp90 and calmodulin (CaM); n=6-19 independent cell batches (1-way ANOVA and Newman-Keuls).


Effect of mutating Y657 on the formation of complexes between eNOS , Hsp90 and PKM2 in growth factor - stimulated human endothelial cells . Complex formation was assessed in eNOS - FLAG immunoprecipitates ( E ) , or PKM2 immunoprecipitates ( F ) ; n = 4 - 8 independent cell batches ( 1 - way ANOVA and Newman - Keuls ) .

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

Effect of mutating Y657 on the formation of complexes between eNOS, Hsp90 and PKM2 in growth factor-stimulated human endothelial cells. Complex formation was assessed in eNOS-FLAG immunoprecipitates (E), or PKM2 immunoprecipitates (F); n=4-8 independent cell batches (1-way ANOVA and Newman-Keuls).


Pyruvate kinase ( PK ) activity in FLAG immunoprecipitates from human endothelial cells expressing FLAG - tagged wild - type ( WT ) , Y657F ( YF ) or Y657D ( YD ) eNOS ; n = 6 independent cell batches ( 1 - way ANOVA and Tukey ) .

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

Pyruvate kinase (PK) activity in FLAG immunoprecipitates from human endothelial cells expressing FLAG-tagged wild-type (WT), Y657F (YF) or Y657D (YD) eNOS; n=6 independent cell batches (1-way ANOVA and Tukey).


PK activity in eNOS immunoprecipitates from HEK293 cells expressing WT , YF , YD or S633D ( SD ) eNOS ; n = 5 - 9 independent experiments ( 1 - way ANOVA and Bonferroni ) .

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

PK activity in eNOS immunoprecipitates from HEK293 cells expressing WT, YF, YD or S633D (SD) eNOS; n=5-9 independent experiments (1-way ANOVA and Bonferroni).


S - nitrosation of PKM2 in human endothelial cells expressing FLAG - tagged WT or YF eNOS and treated with H2O2 ( 30 µmol / L ) for 15 min ; n = 8 independent cell batches ( Unpaired Student ' s t test ) . Dithiothreitol ( DTT ) was included to demonstrate specificity of the S - NO signal .

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

S-nitrosation of PKM2 in human endothelial cells expressing FLAG-tagged WT or YF eNOS and treated with H2O2 (30 µmol/L) for 15 min; n=8 independent cell batches (Unpaired Student's t test). Dithiothreitol (DTT) was included to demonstrate specificity of the S-NO signal.


PKM2 S - nitrosation in HEK293 cells expressing YF or YD eNOS and either FLAG - tagged WT PKM2 or a C358S ( CS ) PKM2 mutant . SNO - FLAG - PKM2 was detected with an anti - FLAG antibody after biotin switch technique ; n = 4 independent experiments ( 2 - way ANOVA & Bonferroni ) . DTT treatment was included to demonstrate specificity of the SNO signal .

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

PKM2 S-nitrosation in HEK293 cells expressing YF or YD eNOS and either FLAG-tagged WT PKM2 or a C358S (CS) PKM2 mutant. SNO-FLAG-PKM2 was detected with an anti-FLAG antibody after biotin switch technique; n=4 independent experiments (2-way ANOVA & Bonferroni). DTT treatment was included to demonstrate specificity of the SNO signal.


PK activity in eNOS immunoprecipitates from HEK293 cells co - expressing YF or YD eNOS and either WT PKM2 or the CS PKM2 mutant ; n = 6 independent experiments ( 2 - way ANOVA & Bonferroni ) .

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

PK activity in eNOS immunoprecipitates from HEK293 cells co-expressing YF or YD eNOS and either WT PKM2 or the CS PKM2 mutant; n=6 independent experiments (2-way ANOVA & Bonferroni).


PKM2 enzyme kinetics measured with increasing concentrations of phosphoenolpyruvate ( PEP ) in eNOS immunoprecipitates from HEK293 cells expressing YF or YD eNOS . The data were fit with the Michaelis - Menten equation to determine Vmax and Km ; n = 4 independent experiments ( 2 - way ANOVA & Bonferroni ) .

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

PKM2 enzyme kinetics measured with increasing concentrations of phosphoenolpyruvate (PEP) in eNOS immunoprecipitates from HEK293 cells expressing YF or YD eNOS. The data were fit with the Michaelis-Menten equation to determine Vmax and Km; n=4 independent experiments (2-way ANOVA & Bonferroni).


Quantification of pentose phosphate pathway ( PPP ) intermediates ; gluconate - 6 - P ( G6P ) , ribulose - 5 - P ( Rl5P ) , ribose - 5 - P ( R5P ) , sedoheptulose - 7 - P ( S7P ) , fructose - 6 - P ( F6P ) and erythrose - 4 - P ( E4P ) in human endothelial cells expressing YF or YD eNOS ; n = 6 - 8 independent cell batches ( Unpaired Student ' s t test ) .

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

Quantification of pentose phosphate pathway (PPP) intermediates; gluconate-6-P (G6P), ribulose-5-P (Rl5P), ribose-5-P (R5P), sedoheptulose-7-P (S7P), fructose-6-P (F6P) and erythrose-4-P (E4P) in human endothelial cells expressing YF or YD eNOS; n=6-8 independent cell batches (Unpaired Student's t test).


NADPH / NADP + and GSH / GSSG ratios in human endothelial cells expressing YF or YD eNOS ; n = 6 - 8 independent cell batches ( Unpaired Student ' s t test ) .

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

NADPH/NADP+ and GSH/GSSG ratios in human endothelial cells expressing YF or YD eNOS; n=6-8 independent cell batches (Unpaired Student's t test).


NADPH / NADP + and GSH / GSSG ratios in human endothelial cells treated with solvent ( Sol ) or the PKM2 inhibitor shikonin ( SKN , 1 µmol / L ) for 45 minutes ; n = 4 independent cell batches ( Unpaired Student ' s t test ) .

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

NADPH/NADP+ and GSH/GSSG ratios in human endothelial cells treated with solvent (Sol) or the PKM2 inhibitor shikonin (SKN, 1 µmol/L) for 45 minutes; n=4 independent cell batches (Unpaired Student's t test).


Systolic blood pressure ( SBP ) in wild - type ( WT ) and YF - eNOS ( YF ) mice ; n = 9 animals per group ( Unpaired Student ' s t test ) .

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

Systolic blood pressure (SBP) in wild-type (WT) and YF-eNOS (YF) mice; n=9 animals per group (Unpaired Student's t test).


Phenylephrine ( PE ) - induced contraction of endothelium - intact aortic rings from WT and YF mice . Experiments were performed in the absence and presence of L - NAME ( LN , 300 µmol / L ) ; n = 6 - 10 animals per group ( 2 - way ANOVA and Bonferroni ) .

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

Phenylephrine (PE)-induced contraction of endothelium-intact aortic rings from WT and YF mice. Experiments were performed in the absence and presence of L-NAME (LN, 300 µmol/L); n=6-10 animals per group (2-way ANOVA and Bonferroni).


acetylcholine ( Ach ) - induced relaxation ( C ) of endothelium - intact aortic rings from WT and YF mice . Experiments were performed in the absence and presence of L - NAME ( LN , 300 µmol / L ) ; n = 6 - 10 animals per group ( 2 - way ANOVA and Bonferroni ) .

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

acetylcholine (Ach)-induced relaxation (C) of endothelium-intact aortic rings from WT and YF mice. Experiments were performed in the absence and presence of L-NAME (LN, 300 µmol/L); n=6-10 animals per group (2-way ANOVA and Bonferroni).


Acetylcholine ( ACh ) - induced vasodilatation of the buffer - perfused hindlimb in situ . Experiments were performed in the absence and presence of L - NAME ( LN , 300 µmol / L ) ; n = 12 - 13 animals per group ( 2 - way ANOVA and Bonferroni ) .

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

Acetylcholine (ACh)-induced vasodilatation of the buffer-perfused hindlimb in situ. Experiments were performed in the absence and presence of L-NAME (LN, 300 µmol/L); n=12-13 animals per group (2-way ANOVA and Bonferroni).


PKM2 S - nitrosation in pulmonary endothelial cells from WT and YF mice ; n = 5 - 6 independent cell batches ( Unpaired Student ' s t test ) . Dithiothreitol ( DTT ) treatment was included to demonstrate specificity of the SNO signal . The blots show non adjacent bands cropped from the same membranes .

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

PKM2 S-nitrosation in pulmonary endothelial cells from WT and YF mice; n=5-6 independent cell batches (Unpaired Student's t test). Dithiothreitol (DTT) treatment was included to demonstrate specificity of the SNO signal. The blots show non adjacent bands cropped from the same membranes.


PK activity in eNOS immunoprecipitates from WT and YF pulmonary endothelial cells ; n = 7 - 10 independent cell batches ( Unpaired Student ' s t test ) . PK activity in eNOS immunoprecipitates from hearts from WT and YF mice ; n = 6 mice per group ( Unpaired Student ' s t test ) .

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

PK activity in eNOS immunoprecipitates from WT and YF pulmonary endothelial cells; n=7-10 independent cell batches (Unpaired Student's t test). PK activity in eNOS immunoprecipitates from hearts from WT and YF mice; n=6 mice per group (Unpaired Student's t test).


Representative images of eNOS - PKM2 interaction ( PLA foci , red ) in mouse pulmonary endothelial cells ; the Golgi apparatus and endosomes were stained with Golph4 ( green ) , the plasma membrane was stained with CD144 and nuclei were highlighted with DAPI ( gray ) . Only rare PLA foci were found in samples incubated with control mouse and rabbit IgGs , demonstrating the specificity of the reaction ; n = 3 independent cell batches . Scale bars : 20 µm .

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

Representative images of eNOS-PKM2 interaction (PLA foci, red) in mouse pulmonary endothelial cells; the Golgi apparatus and endosomes were stained with Golph4 (green), the plasma membrane was stained with CD144 and nuclei were highlighted with DAPI (gray). Only rare PLA foci were found in samples incubated with control mouse and rabbit IgGs, demonstrating the specificity of the reaction; n=3 independent cell batches. Scale bars: 20 µm.


Wild - type ( WT ) and YF - eNOS ( YF ) mice were treated with vehicle ( Veh ) or angiotensin II ( AII , 1 . 44 mg / kg / day ) for up to 28 days . Phosphorylation of eNOS on Tyr656 in lung lysates from animals treated for 7 days , n = 6 - 7 mice per group ( Unpaired Student ' s t test ) .

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

Wild-type (WT) and YF-eNOS (YF) mice were treated with vehicle (Veh) or angiotensin II (AII, 1.44 mg/kg/day) for up to 28 days. Phosphorylation of eNOS on Tyr656 in lung lysates from animals treated for 7 days, n=6-7 mice per group (Unpaired Student's t test).


Wild - type ( WT ) and YF - eNOS ( YF ) mice were treated with vehicle ( Veh ) or angiotensin II ( AII , 1 . 44 mg / kg / day ) for up to 28 days . Systolic blood pressure ( SBP ) in WT and YF mice treated for 28 days , n = 10 mice per group ( Unpaired Student ' s t test ) .

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

Wild-type (WT) and YF-eNOS (YF) mice were treated with vehicle (Veh) or angiotensin II (AII, 1.44 mg/kg/day) for up to 28 days. Systolic blood pressure (SBP) in WT and YF mice treated for 28 days, n=10 mice per group (Unpaired Student's t test).


Wild - type ( WT ) and YF - eNOS ( YF ) mice were treated with vehicle ( Veh ) or angiotensin II ( AII , 1 . 44 mg / kg / day ) for up to 28 days . Acetylcholine ( ACh ) - induced relaxation of aortic rings from the same animals as in panel B , n = 8 mice per group ( 2 - way ANOVA and Bonferroni ) .

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

Wild-type (WT) and YF-eNOS (YF) mice were treated with vehicle (Veh) or angiotensin II (AII, 1.44 mg/kg/day) for up to 28 days. Acetylcholine (ACh)-induced relaxation of aortic rings from the same animals as in panel B, n=8 mice per group (2-way ANOVA and Bonferroni).


Wild - type ( WT ) and YF - eNOS ( YF ) mice were treated with vehicle ( Veh ) or angiotensin II ( AII , 1 . 44 mg / kg / day ) for up to 28 days . PKM2 S - nitrosation in lungs from WT and YF mice administered AII for 28 days , n = 7 - 8 mice per group ( Unpaired Student ' s t test ) . DTT treatment was included to demonstrate specificity of the SNO signal . The blots show non adjacent lanes from the same membranes .

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

Wild-type (WT) and YF-eNOS (YF) mice were treated with vehicle (Veh) or angiotensin II (AII, 1.44 mg/kg/day) for up to 28 days. PKM2 S-nitrosation in lungs from WT and YF mice administered AII for 28 days, n=7-8 mice per group (Unpaired Student's t test). DTT treatment was included to demonstrate specificity of the SNO signal. The blots show non adjacent lanes from the same membranes.


Wild - type ( WT ) and YF - eNOS ( YF ) mice were treated with vehicle ( Veh ) or angiotensin II ( AII , 1 . 44 mg / kg / day ) for up to 28 days . NADPH / NADP + and GSH / GSSG ratios in lungs from WT and YF mice treated with AII for 28 days ; n = 8 - 9 mice per group ( Unpaired Student ' s t test ) .

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

Wild-type (WT) and YF-eNOS (YF) mice were treated with vehicle (Veh) or angiotensin II (AII, 1.44 mg/kg/day) for up to 28 days. NADPH/NADP+ and GSH/GSSG ratios in lungs from WT and YF mice treated with AII for 28 days; n=8-9 mice per group (Unpaired Student's t test).


Wild - type ( WT ) and YF - eNOS ( YF ) mice were treated with vehicle ( Veh ) or angiotensin II ( AII , 1 . 44 mg / kg / day ) for up to 28 days . 3 - Nitrotyrosine ( 3 - NT ) levels in lungs from WT and YF mice treated with vehicle ( Veh ) or AII ( AII ) for 28 days ; n = 7 - 9 mice per group ( 2 - way ANOVA and Bonferroni ) .

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

Wild-type (WT) and YF-eNOS (YF) mice were treated with vehicle (Veh) or angiotensin II (AII, 1.44 mg/kg/day) for up to 28 days. 3-Nitrotyrosine (3-NT) levels in lungs from WT and YF mice treated with vehicle (Veh) or AII (AII) for 28 days; n=7-9 mice per group (2-way ANOVA and Bonferroni).


Wild - type ( WT ) and YF - eNOS ( YF ) mice were treated with vehicle ( Veh ) or angiotensin II ( AII , 1 . 44 mg / kg / day ) for up to 28 days . NADPH / NADP + and GSH / GSSG ratios in pulmonary endothelial cells from WT and YF mice treated with AII for 30 min ; n = 8 cell batches ( 2 - way ANOVA and Tukey ) .

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

Wild-type (WT) and YF-eNOS (YF) mice were treated with vehicle (Veh) or angiotensin II (AII, 1.44 mg/kg/day) for up to 28 days. NADPH/NADP+ and GSH/GSSG ratios in pulmonary endothelial cells from WT and YF mice treated with AII for 30 min; n=8 cell batches (2-way ANOVA and Tukey).


Wild - type ( WT ) and YF - eNOS ( YF ) mice were treated with vehicle ( Veh ) or angiotensin II ( AII , 1 . 44 mg / kg / day ) for up to 28 days . H2O2 levels in pulmonary endothelial cells from WT and YF mice after treatment with AII ( 1 µmol / L ) for 30 min ; n = 8 cell batches ( 2 - way ANOVA and Tukey ) .

Wild-type (WT) and YF-eNOS (YF) mice were treated with vehicle (Veh) or angiotensin II (AII, 1.44 mg/kg/day) for up to 28 days. H2O2 levels in pulmonary endothelial cells from WT and YF mice after treatment with AII (1 µmol/L) for 30 min; n=8 cell batches (2-way ANOVA and Tukey).


ApoE - / - ( - / - ) and ApoEYF ( YF ) mice were subjected to partial ligation of the left carotid artery ( LCA ) and given a cholesterol - rich diet . The right , non - ligated artery ( RCA ) was used as a control . Proximity ligation assays showing S - nitrosated PKM2 ( yellow ) in the RCA and LCA 2 days after ligation . Phalloidin staining ( blue ) was included to highlight the vessel wall ; n = 4 - 5 mice per group ( 2 - way ANOVA and Bonferroni ) . Scale bars : 50 µm .

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

ApoE-/- (-/-) and ApoEYF (YF) mice were subjected to partial ligation of the left carotid artery (LCA) and given a cholesterol-rich diet. The right, non-ligated artery (RCA) was used as a control. Proximity ligation assays showing S-nitrosated PKM2 (yellow) in the RCA and LCA 2 days after ligation. Phalloidin staining (blue) was included to highlight the vessel wall; n=4-5 mice per group (2-way ANOVA and Bonferroni). Scale bars: 50 µm.


ApoE - / - ( - / - ) and ApoEYF ( YF ) mice were subjected to partial ligation of the left carotid artery ( LCA ) and given a cholesterol - rich diet . The right , non - ligated artery ( RCA ) was used as a control . GSH ( green ) levels in the RCA and LCA 2 days after ligation . CD31 ( red ) was included to label endothelial cells . n = 4 - 5 mice per group ( 2 - way ANOVA and Bonferroni ) . Scale bars : 50 µm .

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

ApoE-/- (-/-) and ApoEYF (YF) mice were subjected to partial ligation of the left carotid artery (LCA) and given a cholesterol-rich diet. The right, non-ligated artery (RCA) was used as a control. GSH (green) levels in the RCA and LCA 2 days after ligation. CD31 (red) was included to label endothelial cells. n=4-5 mice per group (2-way ANOVA and Bonferroni). Scale bars: 50 µm.


ApoE - / - ( - / - ) and ApoEYF ( YF ) mice were subjected to partial ligation of the left carotid artery ( LCA ) and given a cholesterol - rich diet . The right , non - ligated artery ( RCA ) was used as a control . 3 - nitrotyrosine ( 3NT , green ) levels in the RCA and LCA 2 days after ligation . CD31 ( red ) was included to label endothelial cells . n = 5 mice per group ( 2 - way ANOVA and Bonferroni ) . Scale bars : 50 µm .

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

ApoE-/- (-/-) and ApoEYF (YF) mice were subjected to partial ligation of the left carotid artery (LCA) and given a cholesterol-rich diet. The right, non-ligated artery (RCA) was used as a control. 3-nitrotyrosine (3NT, green) levels in the RCA and LCA 2 days after ligation. CD31 (red) was included to label endothelial cells. n=5 mice per group (2-way ANOVA and Bonferroni). Scale bars: 50 µm.


ApoE - / - ( - / - ) and ApoEYF ( YF ) mice were subjected to partial ligation of the left carotid artery ( LCA ) and given a cholesterol - rich diet . The right , non - ligated artery ( RCA ) was used as a control . Acetylcholine ( ACh ) - induced relaxation of the phenylephrine contracted LCA 7 days after ligation , n = 7 - 8 mice per group ( 2 - way ANOVA and Bonferroni ) . Acetylcholine ( ACh ) - induced relaxation of the phenylephrine contracted aorta 7 days after ligation , n = 7 - 8 mice per group ( 2 - way ANOVA and Bonferroni ) .

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

ApoE-/- (-/-) and ApoEYF (YF) mice were subjected to partial ligation of the left carotid artery (LCA) and given a cholesterol-rich diet. The right, non-ligated artery (RCA) was used as a control. Acetylcholine (ACh)-induced relaxation of the phenylephrine contracted LCA 7 days after ligation, n=7-8 mice per group (2-way ANOVA and Bonferroni). Acetylcholine (ACh)-induced relaxation of the phenylephrine contracted aorta 7 days after ligation, n=7-8 mice per group (2-way ANOVA and Bonferroni).


ApoE - / - mice were treated with vehicle ( Veh ) or the PKM2 inhibitor shikonin ( SKN , 1 . 2 mg / kg ) , subjected to partial ligation of the left carotid artery ( LCA ) and given a cholesterol - rich diet . The right , non - ligated artery ( RCA ) was used as a control . GSH ( green ) levels in the RCA and LCA 3 days after ligation . CD31 ( red ) was included to label endothelial cells . n = 5 mice per group ( 2 - way ANOVA and Bonferroni ) . Scale bars : 50 µm .

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

ApoE-/- mice were treated with vehicle (Veh) or the PKM2 inhibitor shikonin (SKN, 1.2 mg/kg), subjected to partial ligation of the left carotid artery (LCA) and given a cholesterol-rich diet. The right, non-ligated artery (RCA) was used as a control. GSH (green) levels in the RCA and LCA 3 days after ligation. CD31 (red) was included to label endothelial cells. n=5 mice per group (2-way ANOVA and Bonferroni). Scale bars: 50 µm.


ApoE - / - mice were treated with vehicle ( Veh ) or the PKM2 inhibitor shikonin ( SKN , 1 . 2 mg / kg ) , subjected to partial ligation of the left carotid artery ( LCA ) and given a cholesterol - rich diet . The right , non - ligated artery ( RCA ) was used as a control . 3 - Nitrotyrosine ( 3NT , green ) levels in the RCA and LCA 3 days after ligation . CD31 ( red ) was included to label endothelial cells . n = 5 mice per group ( 2 - way ANOVA and Bonferroni ) . Scale bars : 50 µm .

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ApoE-/- mice were treated with vehicle (Veh) or the PKM2 inhibitor shikonin (SKN, 1.2 mg/kg), subjected to partial ligation of the left carotid artery (LCA) and given a cholesterol-rich diet. The right, non-ligated artery (RCA) was used as a control. 3-Nitrotyrosine (3NT, green) levels in the RCA and LCA 3 days after ligation. CD31 (red) was included to label endothelial cells. n=5 mice per group (2-way ANOVA and Bonferroni). Scale bars: 50 µm.


ApoE - / - ( - / - ) and ApoEYF ( YF ) mice were subjected to partial ligation of the left carotid artery ( LCA ) and given a cholesterol - rich diet . ICAM1 and VCAM1 ( green ) expression in the LCA 2 days after ligation . CD31 ( red ) was included to label endothelial cells and nuclei are highlighted with DAPI ( gray ) . Comparable images were obtained in 4 additional animals in each group . Scale bars : 50 µm .

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ApoE-/- (-/-) and ApoEYF (YF) mice were subjected to partial ligation of the left carotid artery (LCA) and given a cholesterol-rich diet. ICAM1 and VCAM1 (green) expression in the LCA 2 days after ligation. CD31 (red) was included to label endothelial cells and nuclei are highlighted with DAPI (gray). Comparable images were obtained in 4 additional animals in each group. Scale bars: 50 µm.


ApoE - / - ( - / - ) and ApoEYF ( YF ) mice were subjected to partial ligation of the left carotid artery ( LCA ) and given a cholesterol - rich diet . Percentage of neutrophils and monocytes infiltrated into the LCA 7 days after ligation and high cholesterol diet feeding ; n = 9 - 10 mice per group ( Unpaired Student ' s t test ) .

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ApoE-/- (-/-) and ApoEYF (YF) mice were subjected to partial ligation of the left carotid artery (LCA) and given a cholesterol-rich diet. Percentage of neutrophils and monocytes infiltrated into the LCA 7 days after ligation and high cholesterol diet feeding; n=9-10 mice per group (Unpaired Student's t test).


Representative images and quantification of ICAM1 ( green ) and VCAM1 ( red ) staining in human endothelial cells pre - treated with the PKM2 inhibitor shikonin ( SKN , 1 µmol / L ) or solvent ( Sol ) for 45 minutes and then stimulated with interleukin - 1β ( IL - 1 , 20 ng / ml ) or solvent ( CTL ) for 90 min . Nuclei are highlighted with DAPI ( gray ) . n = 6 independent cell batches ( 2 - way ANOVA and Bonferroni ) . Scale bars : 20 µm .

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Representative images and quantification of ICAM1 (green) and VCAM1 (red) staining in human endothelial cells pre-treated with the PKM2 inhibitor shikonin (SKN, 1 µmol/L) or solvent (Sol) for 45 minutes and then stimulated with interleukin-1β (IL-1, 20 ng/ml) or solvent (CTL) for 90 min. Nuclei are highlighted with DAPI (gray). n=6 independent cell batches (2-way ANOVA and Bonferroni). Scale bars: 20 µm.


Percentage of neutrophils and macrophages infiltrated into the LCA from ApoE - / - mice treated with Veh or SKN ( 1 . 2 mg / kg ) 7 days after ligation and high cholesterol diet feeding ; n = 4 - 6 mice per group ( Unpaired Student ' s t test ) .

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Percentage of neutrophils and macrophages infiltrated into the LCA from ApoE-/- mice treated with Veh or SKN (1.2 mg/kg) 7 days after ligation and high cholesterol diet feeding; n=4-6 mice per group (Unpaired Student's t test).


Plaque burden in ligated carotid arteries from ApoE - / - ( - / - ) and ApoEYF ( YF ) mice fed a high cholesterol diet for 21 days ; scale bars : 2 mm . Intimal and medial thickness was determined at four specific locations ( proximal , middle 1 , middle 2 , distal ) in cryo - sectioned arteries stained with Oil Red O ; scale bars : 200 µm . n = 7 mice per group ( Unpaired Student ' s t test ) .

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Plaque burden in ligated carotid arteries from ApoE-/- (-/-) and ApoEYF (YF) mice fed a high cholesterol diet for 21 days; scale bars: 2 mm. Intimal and medial thickness was determined at four specific locations (proximal, middle 1, middle 2, distal) in cryo-sectioned arteries stained with Oil Red O; scale bars: 200 µm. n=7 mice per group (Unpaired Student's t test).


Oil Red O staining of plaques in aortae from mice fed a Western diet for four ( B ) or six months ( C ) ; scale bars : 3 mm . n = 6 ( B ) or n = 12 ( C ) mice per group ( Unpaired Student ' s t test ) .

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Oil Red O staining of plaques in aortae from mice fed a Western diet for four (B) or six months (C); scale bars: 3 mm. n=6 (B) or n=12 (C) mice per group (Unpaired Student's t test).


B . Instability of BU - 1 expression in timeless cells . FACS plots of wild type and timeless ( clone 1 ) DT40 cells stained with anti - Bu - 1 conjugated with phycoerythrin . Each line represents the Bu - 1 expression profile of an individual clonal population . Unstained controls are shown in blue .

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B. Instability of BU-1 expression in timeless cells. FACS plots of wild type and timeless (clone 1) DT40 cells stained with anti-Bu-1 conjugated with phycoerythrin. Each line represents the Bu-1 expression profile of an individual clonal population. Unstained controls are shown in blue.


C . Fluctuation analysis for Bu - 1 loss in wild - type DT40 cells and two independent timeless clones generated by CRISPR - Cas9 targeting ( clones 1 and 2 ; , timeless ( clone 1 ) complemented by expression of human Timeless cDNA and a timeless mutant on a background in which the endogenous + 3 . 5 G4 has been deleted ( ΔG4 )

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C. Fluctuation analysis for Bu-1 loss in wild-type DT40 cells and two independent timeless clones generated by CRISPR-Cas9 targeting (clones 1 and 2; , timeless (clone 1) complemented by expression of human Timeless cDNA and a timeless mutant on a background in which the endogenous +3.5 G4 has been deleted (ΔG4)


D . Fluctuation analysis for Bu - 1 loss in DT40 wild - type and tipin cells .

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D. Fluctuation analysis for Bu-1 loss in DT40 wild-type and tipin cells.


D . The DNA - binding affinity of DBD was measured by fluorescence anisotropy , titrating the DBD protein against Cy3 3 ' - labelled ssDNA , dsDNA and G4 DNA The top panel shows binding curves for ss - and dsDNA , the bottom panel shows the binding curve for the G4 DNA substrate .

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D. The DNA-binding affinity of DBD was measured by fluorescence anisotropy, titrating the DBD protein against Cy3 3'-labelled ssDNA, dsDNA and G4 DNA The top panel shows binding curves for ss- and dsDNA, the bottom panel shows the binding curve for the G4 DNA substrate.


Fluorescence anisotropy was used to measure the binding affinity of Timeless - Tipin for the indicated DNA sequences . A . ssG4 : G4 flanked by single - stranded DNA ; ssHP : hairpin flanked by single - stranded DNA ; ss : single - stranded DNA

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Fluorescence anisotropy was used to measure the binding affinity of Timeless-Tipin for the indicated DNA sequences. A. ssG4: G4 flanked by single-stranded DNA; ssHP: hairpin flanked by single-stranded DNA; ss: single-stranded DNA


Fluorescence anisotropy was used to measure the binding affinity of Timeless - Tipin for the indicated DNA sequences . B . Binding affinity of Timeless - Tipin for a range of G4 DNA sequences Single - stranded ( ss20 : 5 ′ - 6FAM - ATAAGAGTGGTTAGAGTGTA ) and double - stranded ( ds20 : ss20 annealed to complementary sequence ) DNA were also tested as controls .

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Fluorescence anisotropy was used to measure the binding affinity of Timeless-Tipin for the indicated DNA sequences. B. Binding affinity of Timeless-Tipin for a range of G4 DNA sequences Single-stranded (ss20: 5′-6FAM-ATAAGAGTGGTTAGAGTGTA) and double-stranded (ds20: ss20 annealed to complementary sequence) DNA were also tested as controls.


Fluctuation analysis for the generation of Bu - 1 loss variants . Top to bottom : wild type , timeless ( clone 1 ) , a timeless mutant ( timeless ∆ C ) generated by CRISPR - Cas9 targeting exon 16 which truncates the protein removing the CTD containing both the DBD and the PARP binding domains . Then , complementation of timeless # 1 with human Timeless ( hTim ) , hTim ∆ 816 - 1208 ( lacking both the DBD and PBD ) , hTim ∆ 816 - 965 ( lacking the DBD ) and hTim [ 1 : 1000 ] , lacking the PBD . At least two independent fluctuation analyses were performed with 24 - 36 individual clones each cell line per repeat .

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Fluctuation analysis for the generation of Bu-1 loss variants. Top to bottom: wild type, timeless (clone 1), a timeless mutant (timeless ∆C) generated by CRISPR-Cas9 targeting exon 16 which truncates the protein removing the CTD containing both the DBD and the PARP binding domains. Then, complementation of timeless#1 with human Timeless (hTim), hTim∆816-1208 (lacking both the DBD and PBD), hTim∆816-965 (lacking the DBD) and hTim[1:1000], lacking the PBD. At least two independent fluctuation analyses were performed with 24-36 individual clones each cell line per repeat.


A . Enhanced recruitment of DDX11 to chromatin associated PCNA following exposure to 4 µM PDS for 24 hours . PCNA was precipitated from cross - linked chromatin and the immunoprecipitate blotted for FLAG - DDX11 .

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A. Enhanced recruitment of DDX11 to chromatin associated PCNA following exposure to 4 µM PDS for 24 hours. PCNA was precipitated from cross-linked chromatin and the immunoprecipitate blotted for FLAG-DDX11.


B . Fluctuation analysis for Bu - 1a loss in wild - type DT40 cells , two independent ddx11 clones generated by CRISPR - Cas9 targeting ( clones 1 and 2 ) and one ddx11 clone generated by conventional homologous recombination gene targeting ( clone 3 ) . Each symbol represents the percentage of cells in an individual clone expanded for 2 - 3 weeks that have lost Bu - 1ahigh expression .

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B. Fluctuation analysis for Bu-1a loss in wild-type DT40 cells, two independent ddx11 clones generated by CRISPR-Cas9 targeting (clones 1 and 2) and one ddx11 clone generated by conventional homologous recombination gene targeting (clone 3). Each symbol represents the percentage of cells in an individual clone expanded for 2-3 weeks that have lost Bu-1ahigh expression.


C . Fluctuation analysis for Bu - 1a loss variant generation in wild - type cells , ddx11 ( clone 1 ) cells , ddx11 ( clone 1 ) complemented by expression of chicken DDX11 WT cDNA , ddx11 ( clone 1 ) complemented by expression of helicase - dead form of chicken DDX11 ( K87A ) cDNA , and a ddx11 clone generated in cells in which the endogenous + 3 . 5 G4 has been deleted ( ΔG4 ) .

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C. Fluctuation analysis for Bu-1a loss variant generation in wild-type cells, ddx11 (clone 1) cells, ddx11 (clone 1) complemented by expression of chicken DDX11 WT cDNA, ddx11 (clone 1) complemented by expression of helicase-dead form of chicken DDX11 (K87A) cDNA, and a ddx11 clone generated in cells in which the endogenous +3.5 G4 has been deleted (ΔG4).


D . Fluctuation analysis for Bu - 1 loss in two independent timeless / ddx11 double - mutant clones ( # 1 and # 2 ) , ddx11 expressing DDX11KAK and fancj and fancj / ddx11 double - mutants . Fluctuation analyses for wild type , timeless # 1 and ddx11 # 1 are shown for comparison .

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D. Fluctuation analysis for Bu-1 loss in two independent timeless/ddx11 double-mutant clones (#1 and #2), ddx11 expressing DDX11KAK and fancj and fancj/ddx11 double-mutants. Fluctuation analyses for wild type, timeless #1 and ddx11 #1 are shown for comparison.


A . Growth curves for DT40 wild type , ddx11 , timeless and ddx11 / timeless cells , with and without 4 μM pyridostatin ( PDS ) . Cells were seeded at 5 x104 cells / ml on day 0 and the viable cells were counted each 24 h for 4 days . Bars represent SD of two independent experiments performed in duplicate . Doubling times ( DMSO ) : WT 13 hours , timeless 18 hours , ddx11 16 hours , ddx11 / timeless 24 hours . Doubling times ( PDS ) : WT 13 . 6 hours , timeless 27 hours , ddx11 25 . 7 hours , ddx11 / timeless 47 . 5 hours .

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A. Growth curves for DT40 wild type, ddx11, timeless and ddx11/timeless cells, with and without 4 μM pyridostatin (PDS). Cells were seeded at 5 x104 cells/ml on day 0 and the viable cells were counted each 24 h for 4 days. Bars represent SD of two independent experiments performed in duplicate. Doubling times (DMSO): WT 13 hours, timeless 18 hours, ddx11 16 hours, ddx11/timeless 24 hours. Doubling times (PDS): WT 13.6 hours, timeless 27 hours, ddx11 25.7 hours, ddx11/timeless 47.5 hours.


B . DDR signalling detected by phosphorylation of histone H2AX ( γ - H2AX ) by flow cytometry in untreated cells or cells exposed to 4 μM PDS for 3 days . Pale histogram , untreated ; dark histogram , treated ; black dotted line , positive control cells treated with 0 . 1 µM cisplatin , also for 3 days . C . Quantification of γ - H2AX in DT40 wild type , ddx11 , timeless and ddx11 / timeless cells treated with 4 μM PDS for 3 days . The central band represents the median , the box the 25th - 75th centile and whiskers the minimum to maximum range of three independent experiments performed in duplicate .

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B. DDR signalling detected by phosphorylation of histone H2AX (γ-H2AX) by flow cytometry in untreated cells or cells exposed to 4 μM PDS for 3 days. Pale histogram, untreated; dark histogram, treated; black dotted line, positive control cells treated with 0.1 µM cisplatin, also for 3 days. C. Quantification of γ-H2AX in DT40 wild type, ddx11, timeless and ddx11/timeless cells treated with 4 μM PDS for 3 days. The central band represents the median, the box the 25th - 75th centile and whiskers the minimum to maximum range of three independent experiments performed in duplicate.


A . Correlation of magnitude and direction of change of genes dysregulated ( relative to wild type ) in timeless vs . ddx11 DT40 cells . rs ( Spearman rho ) is shown for each correlation .

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A. Correlation of magnitude and direction of change of genes dysregulated (relative to wild type) in timeless vs. ddx11 DT40 cells. rs (Spearman rho) is shown for each correlation.


B . Genes dysregulated in timeless , ddx11 and fancj mutants have a higher density of G4s around their TSS . Cumulative fraction of the genes dysregulated in timeless ( red ) , ddx11 ( blue ) and fancj ( green ) containing n ( x - axis ) G4 motifs within 1 . 5kb of the TSS compared with all genes ( black ) .

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B. Genes dysregulated in timeless, ddx11 and fancj mutants have a higher density of G4s around their TSS. Cumulative fraction of the genes dysregulated in timeless (red), ddx11 (blue) and fancj (green) containing n (x-axis) G4 motifs within 1.5kb of the TSS compared with all genes (black).


C . Metagene analysis showing G4 frequency around the TSS of genes dysregulated ( up or down ) in timeless ( left panel ) , ddx11 ( centre panel ) and fancj ( right panel ) compared with all genes ( black line ) . G4 frequency is calculated separately for coding ( above the x - axis ) and template strands ( below the x - axis ) .

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C. Metagene analysis showing G4 frequency around the TSS of genes dysregulated (up or down) in timeless (left panel), ddx11 (centre panel) and fancj (right panel) compared with all genes (black line). G4 frequency is calculated separately for coding (above the x-axis) and template strands (below the x-axis).


B Confocal images of HeLa cells transiently expressing GFP - CBPs or GFP . The dotted lines show each cell shape . Scale bars , 10 µm .

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B Confocal images of HeLa cells transiently expressing GFP-CBPs or GFP. The dotted lines show each cell shape. Scale bars, 10 µm.


C Confocal images of HeLa cells transiently expressing GFP - CbpCT4 and mCherry - LC3 ( upper ) . The fluorescence intensities of GFP - CbpCT4 ( green ) and mCherry - LC3 ( red ) along the arrow are shown in the graph at the bottom .

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C Confocal images of HeLa cells transiently expressing GFP-CbpCT4 and mCherry-LC3 (upper). The fluorescence intensities of GFP-CbpCT4 (green) and mCherry-LC3 (red) along the arrow are shown in the graph at the bottom.


D Lysates from 293T cells transiently expressing GFP - Cbps or GFP were subjected to SDS - PAGE and analyzed by immunoblotting using antibodies against LC3 , GFP , or actin .

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D Lysates from 293T cells transiently expressing GFP-Cbps or GFP were subjected to SDS-PAGE and analyzed by immunoblotting using antibodies against LC3, GFP, or actin.


E Lysates from 293T cells transiently expressing GFP - CbpCT4 , GFP - LytR , or GFP in the presence or absence of chloroquine were subjected to SDS - PAGE and analyzed by immunoblotting using antibodies against LC3 , GFP , or actin .

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E Lysates from 293T cells transiently expressing GFP-CbpCT4, GFP-LytR, or GFP in the presence or absence of chloroquine were subjected to SDS-PAGE and analyzed by immunoblotting using antibodies against LC3, GFP, or actin.


F MEFs were infected with S . pneumoniae R6 WT or ΔcbpF for the indicated periods , and the intracellular survival of bacteria expressed as the number of CFUs .

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F MEFs were infected with S. pneumoniae R6 WT or ΔcbpF for the indicated periods, and the intracellular survival of bacteria expressed as the number of CFUs.


G p62 - KO MEF cells infected with S . pneumoniae R6 ΔcbpF / pCbpFR6 - FLAG for 2 h were fixed and stained with DAPI and an anti - FLAG antibody , and representative epifluorescence images are shown . Scale bars , 10 µm .

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G p62-KO MEF cells infected with S. pneumoniae R6 ΔcbpF/pCbpFR6-FLAG for 2 h were fixed and stained with DAPI and an anti-FLAG antibody, and representative epifluorescence images are shown. Scale bars, 10 µm.


A Quantification of the band intensities following streptavidin - based pulldown assays using the in vitro - translated biotinylated CbpCT4 and FLAG - tagged autophagy - related proteins shown in Fig EV2A .

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A Quantification of the band intensities following streptavidin-based pulldown assays using the in vitro-translated biotinylated CbpCT4 and FLAG-tagged autophagy-related proteins shown in Fig EV2A.