( h ) Representative of - in - structures in which only host express Cathepsin - B - mCherry . Left , a live inner with no Cathepsin B in vacuole ( arrow ) ; middle , a live internalized with Cathepsin B from host inside of vacuole ( arrowhead ) ; right , a dead internalized with Cathepsin B from host throughout corpse ( asterisk ) . Scale bar , 15 μm .

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(h) Representative images of cell-in-cell structures in which only host cells express Cathepsin-B-mCherry. Left, a live inner cell with no Cathepsin B in the vacuole (arrow); middle, a live internalized cell with Cathepsin B from the host inside of the vacuole (arrowhead); right, a dead internalized cell with Cathepsin B from the host throughout the corpse (asterisk). Scale bar, 15 μm.


( i ) Five representative timings of GFP - LC3 recruitment to entotic vacuoles ( green bar ) of internalized ( cross ) from time - lapse microscopy . ( j ) Initiation duration of 2 × FYVE domain ( red bar , n = 11 ) , LC3 ( green bar , n = 20 ) Lamp1 ( orange bar , n = 7 ) recruitment to entotic vacuoles , determined using double - expressing shown in f , g . Error bars represent s . d .

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(i) Five representative timings of GFP-LC3 recruitment to entotic vacuoles (green bar) and death of internalized cells (cross) from time-lapse microscopy. (j) Initiation and duration of 2×FYVE domain (red bar, n=11), LC3 (green bar, n=20) and Lamp1 (orange bar, n=7) recruitment to entotic vacuoles, determined using double-expressing cells shown in f,g. Error bars represent s.d.


( a ) Left , model depicting multiple fates of internalized ; arrow thickness represents relative frequency of events . Right , of MCF10A - in - structures ( 1 , host ; 2 , internalized ) that show a ( top ) or release ( bottom ) event . Scale bar , 15 μm .

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(a) Left, model depicting the multiple cell fates of internalized cells; the arrow thickness represents the relative frequency of events. Right, images of MCF10A cell-in-cell structures (1, host cell; 2, internalized cell) that show a cell death (top images) or release (bottom images) event. Scale bar, 15 μm.


( b ) of MCF10A internalized fate over 20 h following autophagy inhibition . Data represent mean from at least three independent experiments ; n , total number of structures analysed ; * * * P0 . 007 , * * P0 . 004 , * P0 . 01 .

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(b) Quantification of MCF10A internalized cell fate over 20 h following autophagy inhibition. Data represent the mean from at least three independent experiments; n, total number of structures analysed; ***P0.007, **P0.004, *P0.01.


( c , d ) of LC3 recruitment ( c ) , fate ( d ) , of mixed - in - structures for which host or internalized were treated with ATG5 siRNA . Data represent mean ± s . e . m . from three separate experiments ; n , total number of structures analysed ; * P0 . 01 , * * P0 . 004 .

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(c,d) Quantification of LC3 recruitment (c), and cell fate (d), of mixed cell-in-cell structures for which host or internalized cells were treated with ATG5 siRNA. Data represent mean±s.e.m.from three separate experiments; n, total number of structures analysed; *P0.01, **P0.004.


( e ) Left , of type of internalized after autophagy inhibition . Data represent mean of three independent experiments ; n , total events analysed ; * * P0 . 03 . Right , representative of non - apoptotic apoptotic of an internalized ; arrow points to fragmented apoptotic nuclei . Scale bars , 15 μm .

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(e) Left, quantification of type of internalized cell death after autophagy inhibition. Data represent the mean of three independent experiments; n, total death events analysed; **P0.03. Right, representative images of non-apoptotic and apoptotic death of an internalized cell; the arrow points to fragmented apoptotic nuclei. Scale bars, 15 μm.


( f ) of mCherry / GFP of live MCF10A single or internalized expressing tandem GFP - mCherry - LC3 , with or without siRNA treatment as indicated . Data represent mean ± s . d . ; numbers of analysed for each data point ( from left to right ) 30 , 30 , 30 , 25 , 30 , 25 , 25 , 25 , 30 , 27 ; * P0 . 03 , * * P0 . 003 , * * * P0 . 0004 .

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(f) Quantification of mCherry/GFP ratio of live MCF10A single cells or internalized cells expressing tandem GFP-mCherry-LC3, with or without siRNA treatment as indicated. Data represent mean±s.d.; numbers of cells analysed for each data point (from left to right) 30, 30, 30, 25, 30, 25, 25, 25, 30, 27; *P0.03, **P0.003, ***P0.0004.


( g ) of autophagosome autolysosome in control MCF10A ( n = 10 ) internalized ( n = 10 ) cytoplasm imaged by electron microscopy ; * * P0 . 005 . ( h ) Representative electron of - in - structures ( left ) internalized cytoplasm ( middle right ) showing autophagosomes ( arrow ) autolysosomes ( arrowhead ) .

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(g) Quantification of autophagosome and autolysosome area in control MCF10A (n=10) and internalized cell (n=10) cytoplasm imaged by electron microscopy; **P0.005. (h) Representative electron micrographs of cell-in-cell structures (left) and internalized cell cytoplasm (middle and right) showing autophagosomes (arrow) and autolysosomes (arrowhead).


( a ) fate of internalized ( control MCF10A MCF10A expressing E7 Bcl2 ) was measured with without autophagy inhibition . Data show of internalized release with autophagy inhibition , compared with each control without autophagy inhibition ; mean ± s . e . m . from at least three independent experiments ( total numbers analysed : ATG5 siRNA , 261 ; Bcl2 ATG5 siRNA , 97 ; 3 - MA , 170 ; Bcl2 3 - MA , 112 ) ; * P0 . 05 , * * P0 . 02 .

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(a) The fate of internalized cells (control MCF10A and MCF10A expressing E7 and Bcl2) was measured with and without autophagy inhibition. Data show the percentage of internalized cell release with autophagy inhibition, compared with each control without autophagy inhibition; mean±s.e.m. from at least three independent experiments (total cell numbers analysed: ATG5 siRNA, 261 cells; Bcl2 ATG5 siRNA, 97 cells; 3-MA, 170 cells; Bcl2 3-MA, 112 cells); *P0.05, **P0.02.


( b ) Effects of Y27632 , 3 - MA VPS34 ATG5 siRNA on MCF10A - in - formation in suspension for 7 h ; > 300 per condition were scored for - in - formation ; ± s . e . m . from three independent experiments ; * * P0 . 004 .

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(b) Effects of Y27632, 3-MA and VPS34 and ATG5 siRNA on MCF10A cell-in-cell formation in suspension for 7 h; >300 cells per condition were scored for cell-in-cell formation; ±s.e.m.from three independent experiments; **P0.004.


( c ) Representative of an MCF10A E7 + Bcl2 - in - structure formed in soft agar . arrow marks LC3 recruitment around internalized . Scale bar , 5 μm .

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(c) Representative images of an MCF10A E7+Bcl2 cell-in-cell structure formed in soft agar. The arrow marks LC3 recruitment around internalized cells. Scale bar, 5 μm.


( d ) of MCF10A E7 + Bcl2 - in - structures 48 h after seeding into soft agar ( bars ) , colony formation after 2 ( grey bars ) . Data represent mean ± s . e . m . from three independent experiments ; * P0 . 03 , * * P0 . 004 , * * * P0 . 001 . ( e ) Representative wells for data in d . insets show higher magnifications .

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(d) Quantification of MCF10A E7+Bcl2 cell-in-cell structures 48 h after seeding into soft agar (white bars), and colony formation after 2 weeks (grey bars). Data represent mean±s.e.m.from three independent experiments; *P0.03, **P0.004, ***P0.001. (e) Representative wells for data in d. The insets show higher magnifications.


( f ) of MCF10A E7 + Bcl2 colony formation with Atg5 knockdown with two separate siRNAs . Data represent mean ± s . e . m . from three independent experiments ; * P0 . 001 . ( g ) Representative wells for data in f . insets show higher magnifications . See also Supplementary Movie S6 .

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(f) Quantification of MCF10A E7+Bcl2 colony formation with Atg5 knockdown with two separate siRNAs. Data represent mean±s.e.m.from three independent experiments; *P0.001. (g) Representative wells for data in f. The insets show higher magnifications. See also Supplementary Movie S6.


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

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

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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 - electron microscopy of an apoptotic phagosome with GFP - LC3 recruitment ( top left image , arrow ) . LC3 - loaded phagosome consists of a single ( black arrow in right image , which is a higher magnification of outlined in bottom left image ; EM , electron microscopy ; AP , apoptotic 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 of apoptotic phagocytosis degradation in control ( n = 21 ) Atg5 shRNA ( n = 21 ) J774 GFP - LC3 ; * * * P0 . 0001 , chi - squared . 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 of a J774 macrophage expressing GFP - LC3 incubated in media containing red dextran . arrow marks LC3 recruitment to a dextran - containing macropinosome . Scale bar , 2 μm . ( b ) of LC3 recruitment to macropinosomes in control Atg5 shRNA J774 macrophages ; n , number of macropinosomes analysed ; * * * P0 . 0001 , chi - squared . 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 expressing GFP - LC3 incubated with red dextran . Scale bar , 2 μm . ( d ) of LC3 recruitment to macropinosomes in MCF10A treated with siRNA against FIP200 or ATG5 ; n , number of macropinosomes analysed ; * * * P0 . 0001 , chi - squared .

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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 imaged by confocal microscopy for 1 h , followed by 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 expressing mCherry : : RAB - 5 GFP : : LGG - 1 . arrows point to two apoptotic phagosomes . See Supplementary Movie S11 . Scale bar , 10 μm . ( b ) Cropped time - lapse showing RAB - 5 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 of apoptotic phagosomes in from control - RNAi - or bec - 1 - RNAi - fed worms . insets show higher magnifications of areas outlined in main panels . ( d ) of GFP : : LGG - 1 - mCherry : : RAB - 5 - positive phagosomes in control - or bec - 1 - RNAi determined by time - lapse imaging . Control , 21 , 52 phagosomes ; bec - 1 , 9 , 25 phagosomes ; * * * P0 . 0001 , chi - squared .

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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 from control - bec - 1 - RNAi at different stages of development ( minutes after first division ) . yellow arrowheads mark apoptotic corpses . Scale bar , 10 μm . ( f ) of apoptotic corpses in control - bec - 1 - RNAi ; 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 of CED - 1 : : GFP with control or bec - 1 RNAi . 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 . mtHSF1 was examined by WB . 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 were exposed to 5 mM 3 - NP for 2 h . mtHSF1 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 mitochondria - enriched fractions of HD transgenic YAC128 mice ( 9 months old ) or - matched . ( n = 5 biological replicates ) .

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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 fibroblasts ( GM04208 , GM04222 , GM21756 ) fibroblasts from healthy individuals . ( n = 5 for Con1 / GM04208 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 in mitochondria isolated from - derived iPSCs 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 of striatal organoids . Left , immunostaining for MSN marker DARPP32 ( green ) radial glial marker SOX2 ( red ) in striatal organoids at D60 . Middle , immunostaining for LGE progenitor markers Ctip2 ( green ) GSH2 ( red ) at D30 . Right panel , immunostaining for neuronal marker NeuN ( green ) proliferation marker Ki67 ( red ) at D30 . are representative of 12 independent differentiation experiments of H9 IMR90 - 4 . HO = Hoechst ( blue ) . 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 scatterplot confirming increased HSF1 ( green ) translocation to mitochondria ( Tom20 , red ) . data were obtained from 3 independent biological experiments . Con : n = 15 organoids ; HD : n = 14 organoids . Two - unpaired t - ; Means ± SEMs . 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).


subcellular localization of HSF1 in HD striatal organoids was observed by immunoelectron microscopy . arrows ( red ) mark HSF1 in mitochondria . negative control lacked HSF1 antibody . 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 by lentiviral infection . 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 were transfected with Flag - mtHSF1 stained with anti - Tom20 ( green ) anti - Flag ( red ) antibodies . Mitochondrial was examined by confocal microscopy . Scatterplot with bar showed of with mitochondrial fragmentation . scale bar represents 40 µm . ( n = 3 biological replicates , at least 100 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).


total of p - Drp1 S616 , p - Drp1S637 , Drp1 , OPA1 , MFN2 , Flag - mtHSF1 actin ( loading control ) were determined by WB . ( 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 ) DAPI ( blue ) . Mitochondrial was analyzed in Flag + or Flag - regions by confocal microscopy . scale bar presents 2 mm , 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 was analyzed by electron microscopy ( EM ) in mice injected with AAV - Con or AAV - mtHSF1 . Scatterplot showed of mitochondria . scale bar presents 4 µm , 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 scatterplot of mitochondrial ( Tom20 , red ) in Flag + or Flag - . 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 scatterplot showing that number of p - Drp1 S616 in Flag + was greater than that in Flag - . data were obtained from 3 independent biological experiments . Flag - : n = 70 from 10 organoids ; Flag + : n = 55 from 10 organoids . 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).


mtDNA content was examined with primers of D - loop , mtCO1 , mtCO2 mtND2 by qPCR in control or Flag - mtHSF1 - expressing striatal . Scatterplot with bar showed relative 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 . ( n = 9 - 10 biological replicates )

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


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 . was determined by detection of LDH release into medium cleavage of PARP . Scatterplot with bar showed relative 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 of mtDNA content of AAV - Con - injected mice or AAV - mtHSF1 - injected mice . Scatterplot with bar showed relative 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).


- Caspase3 in AAV - Con - or AAV - mtHSF1 - injected mice was detected by WB . ( 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 scatterplot showing number of mtDNA ( purple ) colocalized with mitochondria ( Tom20 , green ) in striatal organoids injected with AAV - Con or AAV - mtHSF1 . data were obtained from 3 independent biological experiments . Con : n = 65 from 20 organoids ; Flag - mtHSF1 : n = 70 from 20 organoids . Two - unpaired t - was used . 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).


typical fragmentation of AAV - Con - ( GFP ) or AAV - mtHSF1 - injected striatal organoids was measured . Scatterplot showed quantitative result of fragmentation . arrows ( yellow ) mark fragmented nuclei . Two - unpaired t - was used . 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 lysates were subjected to immunoprecipitation with an anti - HSF1 antibody followed by WB . ( 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 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 fibroblasts ( GM04208 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 ) .

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


were transfected with an empty vector or Flag - mtHSF1 for 48 h . total lysates were harvested 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 striata of WT mice for three . 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 via lentiviral infection . expression of SSBP1 mtHSF1 was detected by WB . mtDNA content was examined by qPCR . ( 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).


neurons expressing Flag - mtHSF1 were stained with anti - DARPP32 ( green ) anti - Flag ( red ) antibodies . arrows ( ) mark Flag + or Flag - neurons . neurite of Flag + or Flag - neurons was measured by Image J showed by scatterplot . scale bar represents 50 µm . ( n = 155 for Flag - group 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 of NeuN + ( red ) among Flag + or GFP + ( green ) after injection for 2 . data were obtained from 3 independent biological experiments . Con : n = 11 organoids ; Flag - mtHSF1 : n = 13 organoids . 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 of immunostaining with DARPP32 ( red ) Flag ( green ) . DARPP32 density was analyzed in Flag + or Flag - sections by confocal microscopy showed by scatterplot . Thickness : 30 µm . scale bar is 2 mm , 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).


striatum of AAV - Con - or AAV - mtHSF1 - injected mice were harvested . DARPP32 were tested by WB . ( 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).


of NeuN + in Flag + or Flag - sections was determined by confocal microscopy 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 motor activity were recorded post AAV injection . ( n = 7 - 13 mice / group ) .

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


Total lysates of HdhQ7 or HdhQ111 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 HSF1 binding in vitro was analyzed by WB . ( 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 . of Drp1 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 of Drp1 HSF1 in 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 ( 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 - matched ( 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 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 ) .

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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 . 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 . fibroblasts 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).


Immunostaining for mitochondria ( Tom20 , green ) mtDNA ( anti - , red ) in striatal organoids treated with DH1 or TAT at D60 . Scatterplots with bar showed of mitochondria number of mtDNA colocalized with mitochondria . data were obtained from 3 independent biological experiments . Left , n = 222 - 311 mitochondria per group ; Right , n = 14 - 20 organoids per group . were taken using Structured Illumination Microscopy ( SIM ) . 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 ) p - Drp1 S616 ( green ) in striatal treated with DH1 or TAT at D60 . Scatterplot with bar showed number of p - Drp1 S616 per in organoids . data were obtained from 3 independent biological experiments . n = 29 - 34 from at least 10 organoids . 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 of MSNs ( DARPP32 , green ) in striatal organoids in presence or absence of DH1 . 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 of DARPP32 + neurons treated with DH1 or TAT at D45 . Scatterplot with bar showed results of longest neurites . 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 scatterplot with bar of fragmentation in striatal organoids treated with DH1 . arrows ( ) mark fragmented nuclei . data were obtained from 3 independent biological experiments . n = 7 - 10 organoids per group . 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 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 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).


) harboring CRISPR - generated deletion alleles in y48g1c . 1 ( gg682 or gg683 ) were treated with dpy - 11 RNAi progeny , grown in absence of dpy - 11 RNAi , were scored for Dpy . of Dpy 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 ) micrograph of GFP : : ZNFX - 1 PGL - 1 : : TagRFP in 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.


micrograph of gfp : : zsp - 1 at 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 , : 5μm , germline : 10μm . Dashed lines indicate outline of 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 expressing indicated were fixed stained with α - GFP α - HA or α - FLAG antibodies , followed by secondary antibodies conjugated with Alexa 488 CF430 , respectively . Pachytene germ - nuclei were imaged with a Leica TCS SP8 STED 3X microscope using 592nm to selectively deactivate fluorophores . Bottom panel . GFP : : ZNFX - 1 HA : : TagRFP : : ZNFX - 1 expressing were crossed germlines of trans - heterozygous progeny from this cross were fixed stained 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 from pachytene germ from of indicated genotypes were analysed as described in Fig . EV4B Materials Methods to determine ; of P granules ( panel B ) Granules from 9 total nuclei from 3 different ( 3 per ) were quantified for these analyses . Black lines indicate mean of data points in each group . P value , student ' s t , two .

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

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

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

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

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

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

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

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) 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 sensitive ( ts ) lethal ( embryonic arrest at temperatures > 20 ° C ) allele of oma - 1 ( R . Lin 2003 ) . oma - 1 RNAi suppresses oma - 1 ( zu405ts ) lethality . were exposed to oma - 1 RNAi number of that hatched was scored at 20 ° C . N = 3 . Error bars are + / - s . d . harboring mutations in genes specifically required for 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 , therefore , is generally required for RNAi ( see also Fig . EV5B - D ) .

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

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

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

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


FMN / FAD measured in eNOS immunoprecipitates from expressing WT , YF or YD eNOS ; n = 6 independent experiments ( 1 - way ANOVA 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 growth factor - induced formation of eNOS complexes with Hsp90 calmodulin ( CaM ) ; n = 6 - 19 independent batches ( 1 - way ANOVA Newman - Keuls ) .

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

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

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 expressing FLAG - tagged wild - type ( WT ) , Y657F ( YF ) or Y657D ( YD ) eNOS ; n = 6 independent batches ( 1 - way ANOVA 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 B-CONTROLLED_VAR 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 expressing WT , YF , YD or S633D ( SD ) eNOS ; n = 5 - 9 independent experiments ( 1 - way ANOVA Bonferroni ) .

B-MEASURED_VAR 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

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

O O O O B-MEASURED_VAR 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

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 expressing YF or YD eNOS 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 SNO signal .

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

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 co - expressing YF or YD eNOS either WT PKM2 or CS PKM2 mutant ; n = 6 independent experiments ( 2 - way ANOVA & Bonferroni ) .

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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 expressing YF or YD eNOS . data were fit with Michaelis - Menten equation to determine Vmax Km ; n = 4 independent experiments ( 2 - way ANOVA & Bonferroni ) .

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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).


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 ) erythrose - 4 - P ( E4P ) in human endothelial expressing YF or YD eNOS ; n = 6 - 8 independent batches ( Unpaired Student ' s t ) .

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

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

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

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

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

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 ) YF - eNOS ( YF ) mice ; n = 9 per group ( Unpaired Student ' s t ) .

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

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 YF mice . Experiments were performed in absence presence of L - NAME ( LN , 300 µmol / L ) ; n = 6 - 10 per group ( 2 - way ANOVA Bonferroni ) .

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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 YF mice . Experiments were performed in absence presence of L - NAME ( LN , 300 µmol / L ) ; n = 6 - 10 per group ( 2 - way ANOVA 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

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

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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 from WT YF mice ; n = 5 - 6 independent batches ( Unpaired Student ' s t ) . Dithiothreitol ( DTT ) treatment was included to demonstrate specificity of SNO signal . blots show non adjacent bands cropped from 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

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

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

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 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 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 ) 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 treated for 7 days , n = 6 - 7 mice per group ( Unpaired Student ' s t ) .

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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 ) 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 YF mice treated for 28 days , n = 10 mice per group ( Unpaired Student ' s t ) .

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

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 ) 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 same as in panel B , n = 8 mice per group ( 2 - way ANOVA Bonferroni ) .

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. 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 ) 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 YF mice administered AII for 28 days , n = 7 - 8 mice per group ( Unpaired Student ' s t ) . DTT treatment was included to demonstrate specificity of SNO signal . blots show non adjacent lanes from same membranes .

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

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 ) 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 + GSH / GSSG ratios in lungs from WT YF mice treated with AII for 28 days ; n = 8 - 9 mice per group ( Unpaired Student ' s t ) .

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

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 ) 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 ) in lungs from WT YF mice treated with vehicle ( Veh ) or AII ( AII ) for 28 days ; n = 7 - 9 mice per group ( 2 - way ANOVA Bonferroni ) .

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

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 ) 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 + GSH / GSSG ratios in pulmonary endothelial from WT YF mice treated with AII for 30 min ; n = 8 batches ( 2 - way ANOVA Tukey ) .

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. 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 ) YF - eNOS ( YF ) mice were treated with vehicle ( Veh ) or angiotensin II ( AII , 1 . 44 mg / kg / day ) for up to 28 days . H2O2 in pulmonary endothelial from WT YF mice after treatment with AII ( 1 µmol / L ) for 30 min ; n = 8 batches ( 2 - way ANOVA 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 - / - ( - / - ) ApoEYF ( YF ) mice were subjected to partial ligation of left carotid artery ( LCA ) given a cholesterol - rich diet . right , non - ligated artery ( RCA ) was used as a control . Proximity ligation assays showing S - nitrosated PKM2 ( yellow ) in RCA LCA 2 days after ligation . Phalloidin ( blue ) was included to highlight vessel wall ; n = 4 - 5 mice per group ( 2 - way ANOVA Bonferroni ) . Scale bars : 50 µm .

B-CONTROLLED_VAR 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 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

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

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

B-CONTROLLED_VAR 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 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

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

B-CONTROLLED_VAR 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

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

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 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

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

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 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

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

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

B-CONTROLLED_VAR 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

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 of ICAM1 ( green ) VCAM1 ( red ) in human endothelial pre - treated with PKM2 inhibitor shikonin ( SKN , 1 µmol / L ) or solvent ( Sol ) for 45 minutes 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 batches ( 2 - way ANOVA Bonferroni ) . Scale bars : 20 µm .

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

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.


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

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

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 - / - ( - / - ) ApoEYF ( YF ) mice fed a high cholesterol diet for 21 days ; scale bars : 2 mm . Intimal 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 ) .

O O O O O O O B-CONTROLLED_VAR 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

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 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 ) .

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

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

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

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 for Bu - 1 loss in wild - type DT40 two independent timeless clones generated by CRISPR - Cas9 targeting ( clones 1 2 ; , timeless ( clone 1 ) complemented by expression of human Timeless cDNA a timeless mutant on a background in which 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 for Bu - 1 loss in DT40 wild - type tipin .

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


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


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

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

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


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

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

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 for 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 removing CTD containing both DBD PARP binding domains . Then , complementation of timeless # 1 with human Timeless ( hTim ) , hTim ∆ 816 - 1208 ( lacking both DBD PBD ) , hTim ∆ 816 - 965 ( lacking DBD ) hTim [ 1 : 1000 ] , lacking PBD . At least two independent fluctuation analyses were performed with 24 - 36 clones each per repeat .

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

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 associated PCNA following exposure to 4 µM PDS for 24 hours . PCNA was precipitated from cross - linked 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 for Bu - 1a loss in wild - type DT40 , two independent ddx11 clones generated by CRISPR - Cas9 targeting ( clones 1 2 ) one ddx11 clone generated by conventional homologous recombination gene targeting ( clone 3 ) . Each symbol represents of in an clone expanded for 2 - 3 that have lost Bu - 1ahigh expression .

O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_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 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 B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O

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 for Bu - 1a loss variant generation in wild - type , ddx11 ( clone 1 ) , 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 , a ddx11 clone generated in in which endogenous + 3 . 5 G4 has been deleted ( ΔG4 ) .

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

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

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

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 ddx11 / timeless , with without 4 μM pyridostatin ( PDS ) . were seeded at 5 x104 / ml on day 0 viable 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 or exposed to 4 μM PDS for 3 days . Pale histogram , untreated ; histogram , treated ; black dotted , positive control treated with 0 . 1 µM cisplatin , also for 3 days . C . of γ - H2AX in DT40 wild type , ddx11 , timeless ddx11 / timeless treated with 4 μM PDS for 3 days . central band represents median , box 25th - 75th centile whiskers minimum to maximum range of three independent experiments performed in duplicate .

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

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 direction of change of genes dysregulated ( relative to wild type ) in timeless vs . ddx11 DT40 . 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.