( E ) Representative of wt , cest - 2 . 2 O / E cest - 2 . 2 ( lf ) stained with ORO . Arrows indicate mislocalized lipid droplets . ( F ) ( mean ± ) of ORO in hindgut ( left panel ) , foregut ( center panel ) whole ( right panel ) of 5 - day - old nematodes ( one - way ANOVA with Dunnett ' s post hoc correction , n = 11 - 17 ) . Data information : , bars represent mean ± Across experiments , p value summary is ns = not significant , * p < 0 . 05 , * * p < 0 . 01 ; * * * p < 0 . 001 , * * * * p < 0 . 0001 .

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

(E) Representative images of wt, cest-2.2 O/E and cest-2.2 (lf) animals stained with ORO. Arrows indicate mislocalized lipid droplets. (F) Quantification (mean intensity ± SEM) of ORO staining in hindgut (left panel), foregut (center panel) and whole body (right panel) of 5-day-old nematodes (one-way ANOVA with Dunnett's post hoc correction, n=11-17 animals). Data information: , bars represent mean ±SEM Across experiments, p value summary is ns= not significant, *p<0.05, **p < 0.01; ***p < 0.001, ****p<0.0001.


( G ) Representative of 5 - day - old wt , - 1 ( fc21 ) - 1 ( fc21 ) ; cest - 2 . 2 O / E mutant stained with LipidTOX ( scale bar = 100 μm ) . ( H ) Statistical of whole - LipidTOX of 5 - day - old nematodes ( ordinary one - way ANOVA with Tukey ' s multiple comparisons , n = 25 - 37 ) . Data information : bars represent mean ± ; Across experiments , p value summary is ns = not significant , * p < 0 . 05 , * * p < 0 . 01 ; * * * p < 0 . 001 , * * * * p < 0 . 0001 .

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

(G) Representative images of 5-day-old wt, gas-1(fc21) and gas-1(fc21); cest-2.2 O/E mutant animals stained with LipidTOX (scale bar= 100 μm). (H) Statistical analysis of whole-body LipidTOX intensity of 5-day-old nematodes (ordinary one-way ANOVA with Tukey's multiple comparisons test, n=25-37 animals). Data information: bars represent mean ±SEM ; Across experiments, p value summary is ns= not significant, *p<0.05, **p < 0.01; ***p < 0.001, ****p<0.0001.


( I - J ) Representative of nematodes stained with LipidTOX ( I ) ( scale bar = 100 μm ) , with ( J ) ( mean ± ) of LipidTOX . Data information : bars represent mean ± Across experiments , p value summary is ns = not significant , * p < 0 . 05 , * * p < 0 . 01 ; * * * p < 0 . 001 , * * * * p < 0 . 0001 .

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

(I-J) Representative images of nematodes stained with LipidTOX (I) (scale bar= 100 μm), with quantification (J) (mean intensity ± SEM) of LipidTOX staining. Data information: bars represent mean ±SEM Across experiments, p value summary is ns= not significant, *p<0.05, **p < 0.01; ***p < 0.001, ****p<0.0001.


( K ) Representative thin layer chromatography ( TLC ) plate of samples from 5 - day - old - 1 ( fc21 ) - 1 ( fc21 ) ; cest - 2 . 2 O / E mutant nematodes . ( L ) Densitometry of TLC - separated free fatty ( FFA ) across three biological replicates in 5 - day - old mutant . Data information : bars represent mean ± ; Across experiments , p value summary is ns = not significant , * p < 0 . 05 , * * p < 0 . 01 ; * * * p < 0 . 001 , * * * * p < 0 . 0001 .

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

(K) Representative thin layer chromatography (TLC) plate of samples from 5-day-old gas-1(fc21) and gas-1(fc21); cest-2.2 O/E mutant nematodes. (L) Densitometry of TLC-separated free fatty acids (FFA) across three biological replicates in 5-day-old mutant animals. Data information: bars represent mean ±SEM ; Across experiments, p value summary is ns= not significant, *p<0.05, **p < 0.01; ***p < 0.001, ****p<0.0001.


( N ) Sum of TLC - separated FFA of 5 - day old ( n = 5 biological replicates ) . ( O ) of FFA detected after TLC separation in 5 - day - old - 1 ( fc21 ) - 1 ( fc21 ) ; cest - 2 . 2 O / E mutant nematodes ( n = 5 biological replicates , n . d . = not detected ) . Data information : bars represent mean ± ; Across experiments , p value summary is ns = not significant , * p < 0 . 05 , * * p < 0 . 01 ; * * * p < 0 . 001 , * * * * p < 0 . 0001 .

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

(N) Sum of TLC-separated FFA of 5-day old animals (n=5 biological replicates). (O) Quantification of individual FFA detected after TLC separation in 5-day-old gas-1(fc21) and gas-1(fc21); cest-2.2 O/E mutant nematodes (n=5 biological replicates, n.d.= not detected). Data information: bars represent mean ±SEM ; Across experiments, p value summary is ns= not significant, *p<0.05, **p < 0.01; ***p < 0.001, ****p<0.0001.


Representative Western blots showing GAPDH association with mitochondria - enriched fractions in different HD models . of GAPDH were normalized to mitochondrial matrix , aconitase . n = 3 . * P = 0 . 0005 ; * * P = 0 . 019 ; # P = 0 . 042 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Western blots showing GAPDH association with mitochondria-enriched fractions in different HD models. The levels of GAPDH were normalized to mitochondrial matrix protein, aconitase. n = 3. *P = 0.0005; **P = 0.019; #P = 0.042.


Measurement of GAPDH activity in mitochondria - enriched fraction isolated from PC12 . n = 3 . * P = 0 . 0005 .

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

Measurement of GAPDH activity in the mitochondria-enriched fraction isolated from PC12 cells. n = 3. *P = 0.0005.


Western blots showing oxidized GAPDH in total lysates of PC12 isolated mitochondria . of oxidized GAPDH were normalized to beta - actin ( total lysate ) aconitase ( mitochondrial fraction ) , respectively . n = 2 . * P = 0 . 023 ; * * P = 0 . 039 .

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

Western blots showing oxidized GAPDH in the total lysates of PC12 cells and isolated mitochondria. The levels of oxidized GAPDH were normalized to beta-actin (total lysate) and aconitase (mitochondrial fraction), respectively. n = 2. *P = 0.023; **P = 0.039.


Western blot showing co - immunoprecipitation of GAPDH from total lysates of PC12 in RIPA buffer without detergent ( Triton X - 100 ) . Immunoprecipitated samples were immunoblotted with antibodies against mitochondrial ( VDAC Tom20 ) , endoplasmic reticulum ( calnexin ) , lysosomal markers ( LAMP1 ) . ctr : control . n = 2 .

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

Western blot showing co-immunoprecipitation of GAPDH from the total lysates of PC12 cells in RIPA buffer without detergent (Triton X-100). Immunoprecipitated samples were immunoblotted with antibodies against mitochondrial (VDAC and Tom20), endoplasmic reticulum (calnexin), and lysosomal markers (LAMP1). ctr: control. n = 2.


Representative electron microscopy showing mitochondrial in PC12 expressing Q23 ( top panel , left ) Q74 ( bottom panel , left ) in brain tissues from wild - type ( WT ) ( top panel , right ) HD transgenic mice ( bottom panel , right ) . An indicated portion of each image ( in yellow ) is expanded to illustrate mitochondrial . Scale bars : 1 μm .

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

Representative electron microscopy showing mitochondrial morphology in PC12 cells expressing Q23 (top panel, left) and Q74 (bottom panel, left) and in brain tissues from wild-type (WT) (top panel, right) and HD transgenic mice (bottom panel, right). An indicated portion of each image (in yellow) is expanded to illustrate the mitochondrial morphology. Scale bars: 1 μm.


Measurement of mitochondrial ROS production ( using Mitosox ) in different HD models . Results are presented as of control [ Q23 , normal fibroblast ( Nor ) , Q7 ] . n = 3 . * P = 0 . 001 ; * * P = 0 . 0001 ; * * * P = 0 . 0001 ; # P = 0 . 0045 .

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

Measurement of mitochondrial ROS production (using Mitosox) in different HD models. Results are presented as percent of control [Q23, normal fibroblast (Nor), Q7]. n = 3. *P = 0.001; **P = 0.0001; ***P = 0.0001; #P = 0.0045.


Mitochondrial potential measured by using JC - 1 dye in models of HD control ( MMP , ΔΨm ) . n = 3 . * P = 0 . 008 ; * * P = 0 . 044 ; * * * P = 0 . 025 ; # P = 0 . 005 .

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

Mitochondrial membrane potential measured by using JC-1 dye in models of HD and control cells (MMP, ΔΨm). n = 3. *P = 0.008; **P = 0.044; ***P = 0.025; #P = 0.005.


Mitochondrial respiratory function in PC12 , represented by respiratory control ( RCR ; [ state3 ] / [ state4 ] ) . n = 2 . * P = 0 . 028 .

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

Mitochondrial respiratory function in PC12 cells, represented by respiratory control ratio (RCR; [state3]/[state4]). n = 2. *P = 0.028.


ATP in HD - derived fibroblast . n = 2 . * P = 0 . 003 ; * * P = 0 . 004 .

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

ATP levels in HD patient-derived fibroblast cells. n = 2. *P = 0.003; **P = 0.004.


Western blots showing cytochrome c release from mitochondria . n = 2 . * P = 0 . 003 ; * * P = 0 . 003 ; * * * P = 0 . 01 ; # P = 0 . 008 .

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

Western blots showing cytochrome c release from mitochondria. n = 2. *P = 0.003; **P = 0.003; ***P = 0.01; #P = 0.008.


viability determined by colorimetric assays utilizing WST8 conversion to WST8 formazan in different HD models . n = 3 . * P , * * P , * * * P , # P < 0 . 005 .

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

Cell viability determined by colorimetric assays utilizing WST8 conversion to WST8 formazan in different HD models. n = 3. *P, **P, ***P, #P < 0.005.


Representative immunofluorescence of normal HD - derived fibroblasts stained for GAPDH mitochondrial ROS with Mitosox . Three independent per condition were analyzed to calculate Mitosox Pearson ' s correlation coefficient for of colocalization between GAPDH mitochondrial ROS . # P = 0 . 03 ; # # P = 0 . 02 ; * P = 0 . 006 ; * * P = 0 . 005 . were acquired at 63 × magnification , brightness contrast of were adjusted by 50 % .

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 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

Representative immunofluorescence of normal and HD patient-derived fibroblasts stained for GAPDH and mitochondrial ROS with Mitosox. Three independent images per condition were analyzed to calculate Mitosox intensity and the Pearson's correlation coefficient for quantification of colocalization between GAPDH and mitochondrial ROS. #P = 0.03; ##P = 0.02; *P = 0.006; **P = 0.005. Images were acquired at 63× magnification, and brightness and contrast of images were adjusted by 50%.


A Representative Western blots showing separation of lysosome - mitochondria - enriched fractions by a density gradient . Total extracts from different HD models were fractionated by density gradient . Eight fractions were collected from top ( # 1 ) to bottom ( # 8 ) . amount of mitochondria in lysosomal fractions was presented as a of VDAC present in entire gradient . n = 3 .

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

A Representative Western blots showing separation of lysosome- and mitochondria-enriched fractions by a density gradient. Total extracts from different HD models were fractionated by the density gradient. Eight fractions were collected from top (#1) to the bottom (#8). The amount of mitochondria in the lysosomal fractions was presented as a percentage of VDAC present in the entire gradient. n = 3.


B , C Representative electron microscopy of PC12 expressing Q23 or Q74 brain tissues from wild - type ( WT ) HD transgenic R6 / 2 mice showing mitochondrial ( as indicated by yellow arrows ) association with lysosomal vacuoles ( shown by red arrows ) . vacuoles containing mitochondrial remnants are presented as a of total vacuoles counted . ( of six independent ) was carried out by an observer blinded to experimental conditions . * P < 0 . 023 ; * * P < 0 . 035 . Scale bar : 1 μm .

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

B, C Representative electron microscopy of PC12 cells expressing Q23 or Q74 and brain tissues from wild-type (WT) and HD transgenic R6/2 mice showing mitochondrial (as indicated by yellow arrows) association with lysosomal vacuoles (shown by red arrows). The vacuoles containing mitochondrial remnants are presented as a percentage of the total vacuoles counted. Analysis (of six independent images) was carried out by an observer blinded to the experimental conditions. *P < 0.023; **P < 0.035. Scale bar: 1 μm.


D Western blot showing association of mitochondrial with lysosomal . presence of membranes of each organelle was assessed using anti - MFN1 , VDAC ( as mitochondria markers ) , LAMP1 ( as a lysosomal marker ) . n = 3 .

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

D Western blot showing association of mitochondrial membrane with lysosomal membrane. The presence of membranes of each organelle was assessed using anti-MFN1, VDAC (as mitochondria markers), and LAMP1 (as a lysosomal marker). n = 3.


E Representative Western blot showing mitochondrial contents ( VDAC , Tom20 , aconitase ) in total lysates of PC12 with Q23 or Q74 . Mitochondrial were normalized to beta - actin . n = 3 . * P = 0 . 024 ; * * P = 0 . 0008 ; * * * P = 0 . 03 .

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

E Representative Western blot showing mitochondrial contents (VDAC, Tom20, and aconitase) in the total lysates of PC12 cells with Q23 or Q74. Mitochondrial protein levels were normalized to beta-actin. n = 3. *P = 0.024; **P = 0.0008; ***P = 0.03.


F Representative Western blot showing mitochondrial contents ( VDAC , Tom20 , aconitase ) in total lysates of normal fibroblast HD - derived fibroblasts . Mitochondrial were normalized to , enolase . n = 3 . * P = 0 . 008 ; * * P = 0 . 002 ; # P = 0 . 0008 .

F Representative Western blot showing mitochondrial contents (VDAC, Tom20, and aconitase) in the total lysates of normal fibroblast cells and HD patient-derived fibroblasts. Mitochondrial protein levels were normalized to the cytosolic protein, enolase. n = 3. *P = 0.008; **P = 0.002; #P = 0.0008.


G Representative Western blot showing mitochondrial contents ( VDAC , Tom20 , aconitase ) in total lysates of mice striatal with Q7 Q111 . n = 3 . * P = 0 . 0008 ; * * P = 0 . 0007 ; # P = 0 . 001 .

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

G Representative Western blot showing mitochondrial contents (VDAC, Tom20, and aconitase) in the total lysates of mice striatal cells with Q7 and Q111. n = 3. *P = 0.0008; **P = 0.0007; #P = 0.001.


H Western blot showing mitochondrial contents ( Tom20 aconitase ) in total lysates of brain tissues from wild - type ( WT ) HD transgenic mice . n = 2 . * P = 0 . 02 ; * * P = 0 . 03 * § P = 0 . 01 ; * * § P < 0 . 04 .

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

H Western blot showing mitochondrial contents (Tom20 and aconitase) in the total lysates of brain tissues from wild-type (WT) and HD transgenic mice. n = 2. *P = 0.02; **P = 0.03 *§P = 0.01; **§P < 0.04.


Western blots showing co - immunoprecipitation of GAPDH from total lysates isolated mitochondria of PC12 with anti - GAPDH antibody . Anti - EGFP antibody detects presence of polyglutamine repeats . Bottom panel shows co - immunoprecipitation of GAPDH in total lysate of PC12 treated with formaldehyde to cross - link . Ctr * = control . n = 3 .

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

Western blots showing co-immunoprecipitation of GAPDH from the total lysates and isolated mitochondria of PC12 cells with anti-GAPDH antibody. Anti-EGFP antibody detects the presence of polyglutamine repeats. Bottom panel shows co-immunoprecipitation of GAPDH in the total lysate of PC12 cells treated with formaldehyde to cross-link proteins. Ctr* = control. n = 3.


Western blots showing co - immunoprecipitation of GAPDH from total lysate ( left ) isolated mitochondria ( right ) of brain tissues of wild - type ( WT ) transgenic R6 / 2 ( HD ) mice with anti - GAPDH antibody . presence of huntingtin was examined with anti - huntingtin antibody anti - polyglutamine antibody , respectively . Co - immunoprecipitated huntingtin or polyglutamine ( polyQ ) was normalized to GAPDH . Left : n = 2 . * P = 0 . 03 ; * * P = 0 . 02 . Right : One representative experiment from two repeats .

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

Western blots showing co-immunoprecipitation of GAPDH from the total lysate (left) and isolated mitochondria (right) of brain tissues of wild-type (WT) and transgenic R6/2 (HD) mice with anti-GAPDH antibody. The presence of huntingtin was examined with anti-huntingtin antibody and anti-polyglutamine antibody, respectively. Co-immunoprecipitated huntingtin or polyglutamine (polyQ) was normalized to GAPDH. Left: n = 2. *P = 0.03; **P = 0.02. Right: One representative experiment from two repeats.


Sub - mitochondrial localization of GAPDH expanded polyglutamine repeats . Isolated mitochondria were left untreated or subjected to proteinase K treatment in presence or absence of detergent ( Triton X - 100 ) . n = 3 .

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

Sub-mitochondrial localization of GAPDH and expanded polyglutamine repeats. Isolated mitochondria were left untreated or subjected to the proteinase K treatment in the presence or absence of detergent (Triton X-100). n = 3.


Representative Western blot showing of mitochondrial , as indicated by presence of aconitase , VDAC , Tom20 , in total lysates of PC12 with Q23 or Q74 before ( represented as NT : no treatment ) after overexpression of inactive GAPDH ( represented as G ) . n = 3 . * P < 0 . 035 ; * * P = 0 . 001 ; # P = 0 . 001 .

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

Representative Western blot showing levels of mitochondrial proteins, as indicated by the presence of aconitase, VDAC, and Tom20, in the total lysates of PC12 cells with Q23 or Q74 before (represented as NT: no treatment) and after overexpression of inactive GAPDH (represented as G). n = 3. *P < 0.035; **P = 0.001; #P = 0.001.


Representative Western blot showing of mitochondrial in total lysates of mice striatal with Q7 or Q111 before ( NT ) after overexpression of inactive GAPDH ( G ) . n = 2 . * P = 0 . 006 ; * * P = 0 . 007 ; # P = 0 . 001 .

Representative Western blot showing levels of mitochondrial proteins in the total lysates of mice striatal cells with Q7 or Q111 before (NT) and after overexpression of inactive GAPDH (G). n = 2. *P = 0.006; **P = 0.007; #P = 0.001.


Determination of mitochondrial ROS production in different HD models . Results are shown as of control . n = 3 . * P = 0 . 008 ; # P = 0 . 054 ; * * P = 0 . 03 .

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

Determination of mitochondrial ROS production in different HD models. Results are shown as percent of control. n = 3. *P = 0.008; #P = 0.054; **P = 0.03.


Determination of mitochondrial potential using JC - 1 in different HD models control ( MMP , ΔΨm ) . difference in potential between NT ( no inactive GAPDH overexpression ) G ( inactive GAPDH overexpression ) in with expanded polyglutamine repeats ( PC12 with Q74 HD - derived fibroblasts ) was not statistically significant . n = 3 .

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

Determination of mitochondrial membrane potential using JC-1 in different HD models and control cells (MMP, ΔΨm). The difference in membrane potential between NT (no inactive GAPDH overexpression) and G (inactive GAPDH overexpression) in cells with expanded polyglutamine repeats (PC12 cells with Q74 and HD patient-derived fibroblasts) was not statistically significant. n = 3.


ATP in normal HD - derived fibroblasts . difference in ATP between NT ( no inactive GAPDH overexpression ) G ( inactive GAPDH overexpression ) in HD - derived fibroblasts was not statistically significant . n = 2 .

B-MEASURED_VAR 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

ATP levels in normal and HD patient-derived fibroblasts. The difference in ATP levels between NT (no inactive GAPDH overexpression) and G (inactive GAPDH overexpression) in HD patient-derived fibroblasts was not statistically significant. n = 2.


viability determined by colorimetric assays in different HD models control . n = 3 . * P = 0 . 02 ; * * P = 0 . 02 ; # # P = 0 . 01 .

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

Cell viability determined by colorimetric assays in different HD models and control cells. n = 3. *P = 0.02; **P = 0.02; ##P = 0.01.


Immunofluorescence of normal HD - derived fibroblasts , with without overexpression of inactive GAPDH , stained for late endosome with anti - Rab7 antibody mitochondria with anti - Tom20 antibody . were acquired at 63 × magnification , brightness contrast of were adjusted by 30 % . Correlation coefficient ( juvenile ) : * * P = 0 . 007 ; * # P = 0 . 005 . Mitochondrial interconnectivity ( juvenile ) : # # P = 0 . 001 ; # # * P = 0 . 02 . Correlation coefficient ( adult ) : § § P = 0 . 001 ; * § P = 0 . 005 . Mitochondrial interconnectivity ( adult ) : ≠ ≠ P = 0 . 043 ; ≠ ≠ * P = 0 . 015 .

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

Immunofluorescence of normal and HD patient-derived fibroblasts, with and without overexpression of inactive GAPDH, stained for late endosome with anti-Rab7 antibody and mitochondria with anti-Tom20 antibody. Images were acquired at 63× magnification, and brightness and contrast of all images were adjusted by 30%. Correlation coefficient (juvenile): **P = 0.007; *#P = 0.005. Mitochondrial interconnectivity (juvenile): ##P = 0.001; ##*P = 0.02. Correlation coefficient (adult): §§P = 0.001; *§P = 0.005. Mitochondrial interconnectivity (adult): ≠≠P = 0.043; ≠≠*P = 0.015.


Representative Western blot showing of mitochondrial , as indicated by presence of aconitase , VDAC , Tom20 , in total lysates of PC12 with Q23 or Q74 in an in vitro reconstituted micro - mitophagic system . NT : no inactive GAPDH treatment ; G : inactive GAPDH treatment . n = 3 . * P = 0 . 0007 ; * * P = 0 . 001 ; # P = 0 . 008 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Western blot showing levels of mitochondrial proteins, as indicated by the presence of aconitase, VDAC, and Tom20, in the total lysates of PC12 cells with Q23 or Q74 in an in vitro reconstituted micro-mitophagic system. NT: no inactive GAPDH treatment; G: inactive GAPDH treatment. n = 3. *P = 0.0007; **P = 0.001; #P = 0.008.


Representative Western blots showing of mitochondrial ( aconitase Tom20 ) in total lysates of fibroblast ( juvenile adult ) mice striatal with Q7 or Q111 in an in vitro reconstituted mitophagic system . n = 2 . § P = 0 . 01 ; § § P = 0 . 02 ; * P = 0 . 04 ; * * P = 0 . 02 ; # P = 0 . 001 ; # # P = 0 . 024 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Western blots showing levels of mitochondrial proteins (aconitase and Tom20) in the total lysates of fibroblast cells (juvenile and adult) and mice striatal cells with Q7 or Q111 in an in vitro reconstituted mitophagic system. n = 2. §P = 0.01; §§P = 0.02; *P = 0.04; **P = 0.02; #P = 0.001; ##P = 0.024.


Representative Western blot showing of mitochondrial ( aconitase Tom20 ) in total lysates of PC12 . were treated with 1 mM of 3 - methyladenine ( 3MA ) for 5 h prior to micro - mitophagy reconstitution . n = 2 . * P = 0 . 001 ; * * P = 0 . 01 ; * # P = 0 . 003 ; * * # P = 0 . 01 ; # P = 0 . 02 ; § P = 0 . 049 ; # § P = 0 . 004 ; # # § P = 0 . 007 .

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 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

Representative Western blot showing levels of mitochondrial proteins (aconitase and Tom20) in the total lysates of PC12 cells. The cells were treated with 1 mM of 3-methyladenine (3MA) for 5 h prior to the micro-mitophagy reconstitution assay. n = 2. *P = 0.001; **P = 0.01; *#P = 0.003; **#P = 0.01; #P = 0.02; §P = 0.049; #§P = 0.004; ##§P = 0.007.


Western blot showing level of Atg5 expression in Atg5 + / + ( WT ) Atg5 − / − ( KO ) MEF .

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Western blot showing the level of Atg5 expression in the Atg5+/+ (WT) and Atg5−/− (KO) MEF cells.


Western blot showing of GAPDH association with mitochondria in Atg5 KO MEF . n = 2 . * P = 0 . 04 .

Western blot showing levels of GAPDH association with mitochondria in Atg5 KO MEF cells. n = 2. *P = 0.04.


Western blot showing relative mitochondrial mass in total lysates of Atg5 KO MEF . n = 2 . * * P = 0 . 03 ; * * * P = 0 . 02 . Expression of Q23 or Q73 were detected with anti - His antibody .

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

Western blot showing relative mitochondrial mass in the total lysates of Atg5 KO MEF cells. n = 2. **P = 0.03; ***P = 0.02. Expression levels of Q23 or Q73 were detected with anti-His antibody.


Western blot showing relative mitochondrial mass in total lysates of Atg5 KO MEF without with recombinant inactive GAPDH treatment ( represented as NT G , respectively ) . n = 2 . * P = 0 . 02 ; * * P = 0 . 03 ; * # P = 0 . 03 ; # P = 0 . 005 .

Western blot showing relative mitochondrial mass in the total lysates of Atg5 KO MEF cells without and with recombinant inactive GAPDH treatment (represented as NT and G, respectively). n = 2. *P = 0.02; **P = 0.03; *#P = 0.03; #P = 0.005.


A Left , kymograph from sequential epifluorescence ( 20 × ) showing Ca2 + oscillations in speed ‐ fluctuating immature DC after slow motility phases ( upper lower panel ) a change in direction ( lower panel ) . DCs were loaded with Ca2 + dye Oregon GreenBAPTA 1 ‐ AM , introduced in micro ‐ channels , imaged every 10 s . Right , sequential epifluorescence ( 20 × ) ( 1 image / 20 s is shown ) . Instantaneous velocities intracellular calcium oscillations were measured as described in Materials Methods . Scale bar : 10 μm .

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 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 O O O O 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

A Left, kymograph from sequential epifluorescence (20×) images showing cytosolic Ca2+ oscillations in speed‐fluctuating immature DC after slow motility phases (upper and lower panel) and a change in direction (lower panel). DCs were loaded with the Ca2+ dye Oregon GreenBAPTA 1‐AM, introduced in micro‐channels, and imaged every 10 s. Right, sequential epifluorescence (20×) images (1 image/20 s is shown). Instantaneous velocities and intracellular calcium oscillations were measured as described in Materials and Methods. Scale bar: 10 μm.


B , C Mean velocity of immature DCs ( n > 125 from two independent experiments ) ( B ) or CD8 + T lymphocytes ( n > 40 from one experiment ) ( C ) migrating in micro ‐ channels with or without BAPTA . Boxes illustrate 10 - 90 percentiles of values , whiskers represent range of values . P ‐ values were calculated using a Kruskal - Wallis .

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

B, C Mean cell velocity of immature DCs (n > 125 cells from two independent experiments) (B) or CD8+T lymphocytes (n > 40 cells from one experiment) (C) migrating in micro‐channels with or without BAPTA. Boxes illustrate 10-90 percentiles of values, and whiskers represent the range of values. P‐values were calculated using a Kruskal-Wallis test.


D Mean intracellular calcium in speed ‐ fluctuating ( ΔV / V0 > 0 . 3 ; ΔV / V0 relative speed variation divided by global median velocity ) non ‐ speed ‐ fluctuating ( ΔV / V0 0 . 3 ) immature DCs ( n = 187 from more than three independent experiments ) . P ‐ values were calculated using a Mann - Whitney .

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

D Mean intracellular calcium concentration in speed‐fluctuating (ΔV/V0 > 0.3; ΔV/V0 relative speed variation divided by the global median velocity) and non‐speed‐fluctuating (ΔV/V0 0.3) immature DCs (n = 187 cells from more than three independent experiments). P‐values were calculated using a Mann-Whitney test.


E , F Velocity fluctuations ( ΔV / V0 ) ( E ) or mean Ca2 + activities ( F ) displayed by DCs migrating in micro ‐ channels in presence of 100 ng / ml CCL3 . P ‐ values were calculated using a Mann - Whitney , with respect to non ‐ treated DCs ( NT ) .

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

E, F Velocity fluctuations (ΔV/V0) (E) or mean Ca2+ activities (F) displayed by DCs migrating in micro‐channels in the presence of 100 ng/ml CCL3. P‐values were calculated using a Mann-Whitney test, with respect to non‐treated DCs (NT).


G Mean Ca2 + activities during phases of fast ( > 50 % of max velocity ) slow ( 50 % of max velocity ) motility in speed ‐ fluctuating DCs ( Supplementary Fig S2F ) ( n = 64 , from three independent experiments ) . Activities were normalized to mean Ca2 + activity shown by each during entire movie . P ‐ value was calculated using a Mann - Whitney .

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

G Mean Ca2+ activities during phases of fast (> 50% of max velocity) and slow ( 50% of max velocity) motility in speed‐fluctuating DCs (Supplementary Fig S2F) (n = 64 cells, from three independent experiments). Activities were normalized to the mean Ca2+ activity shown by each cell during the entire movie. The P‐value was calculated using a Mann-Whitney test.


A Relative gene expression of IP3R1 ( blue ) , IP3R2 ( red ) , IP3R3 ( green ) in IP3R ‐ silenced DCs . DCs were infected with two lentiviruses encoding for different shRNAs for IP3R isoforms . ShScramble ‐ infected DCs were used as a control . Gene expression was determined by a quantitative PCR . It was calculated with respect to GAPDH expression normalized to observed in shScramble . median plus standard deviation of three independent experiments are shown .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Relative gene expression of IP3R1 (blue), IP3R2 (red), and IP3R3 (green) in IP3R‐silenced DCs. DCs were infected with two lentiviruses encoding for different shRNAs for IP3R isoforms. ShScramble‐infected DCs were used as a control. Gene expression was determined by a quantitative PCR. It was calculated with respect to GAPDH expression and normalized to the levels observed in the shScramble. The median plus standard deviation of three independent experiments are shown.


B Immunoblot for IP3R type 1 IP3R type 3 in IP3R ‐ silenced DCs . Actin was used as a control . IP3R3 appears as two bands as previously described ( 22 ) .

O O O O O O O O O O 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 Immunoblot for IP3R type 1 and IP3R type 3 in IP3R‐silenced DCs. Actin was used as a control. IP3R3 appears as two bands as previously described (22).


C Immunoblot for IP3R type 1 ( top ) IP3R type 3 ( bottom ) in DCs transduced with lentivirus encoding for different IP3R shRNAs . Actin was used as a control .

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C Immunoblot for IP3R type 1 (top) and IP3R type 3 (bottom) in DCs transduced with lentivirus encoding for different IP3R shRNAs. Actin was used as a control.


D Relative IP3R2 expression in ShScramble ‐ infected DCs ( gray ) , shIP3R1 ( blue ) , IP3R3 ( green ) ‐ silenced DCs . experiment was performed as described in ( A ) .

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D Relative IP3R2 expression in ShScramble‐infected DCs (gray), shIP3R1 (blue), and IP3R3 (green)‐silenced DCs. The experiment was performed as described in (A).


F Transmigration of shScramble ‐ , IP3R ( 1 , 3 ) A ( blue ) ‐ , IP3R ( 2 , 3 ) B ( red ) ‐ , IP3R ( 1 , 3 ) C ( green ) ‐ expressing DCs . were loaded in upper chamber of a 5 ‐ μm pore collagen ‐ coated transwell , recovered from upper lower chambers after 16 h , counted . median from three independent experiments is shown .

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F Transmigration assay of shScramble‐, IP3R(1,3)A (blue)‐, IP3R(2,3)B (red)‐, and IP3R(1,3)C (green)‐expressing DCs. Cells were loaded in the upper chamber of a 5‐μm pore collagen‐coated transwell assay, recovered from the upper and lower chambers after 16 h, and counted. The median from three independent experiments is shown.


G of mean velocity of shIP3R ( 1 , 3 ) A ( blue ) ‐ , shIP3R ( 2 , 3 ) B ( red ) ‐ , shIP3R ( 1 , 3 ) C ( green ) ‐ silenced immature DCs migrating in micro ‐ channels . shScramble ‐ infected DCs were used as a control ( gray ) ( n > 100 from three independent experiments for shIP3R ( 1 , 3 ) A shIP3R ( 1 , 3 ) C two independent experiments for shIP3R ( 2 , 3 ) B ) . Boxes illustrate 10 - 90 percentiles of values , whiskers represent range of values . P ‐ values were calculated using a Kruskal - Wallis .

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G Quantification of the mean cell velocity of shIP3R(1,3)A (blue)‐, shIP3R(2,3)B (red)‐, and shIP3R(1,3)C (green)‐silenced immature DCs migrating in micro‐channels. shScramble‐infected DCs were used as a control (gray) (n > 100 cells from three independent experiments for shIP3R(1,3)A and shIP3R(1,3)C and two independent experiments for shIP3R(2,3)B). Boxes illustrate 10-90 percentiles of values, and whiskers represent the range of values. P‐values were calculated using a Kruskal-Wallis test.


H of mean velocity of shIP3R ( 1 , 3 ) C ( green ) ‐ silenced immature DCs migrating in micro ‐ channels in presence of 2 mM BAPTA . shScramble ‐ infected DCs were used as a control ( gray ) ( n > 70 from two independent experiments ) . P ‐ values were calculated using a Kruskal - Wallis .

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

H Quantification of the mean cell velocity of shIP3R(1,3)C (green)‐silenced immature DCs migrating in micro‐channels in the presence of 2 mM BAPTA. shScramble‐infected DCs were used as a control (gray) (n > 70 cells from two independent experiments). P‐values were calculated using a Kruskal-Wallis test.


of shScramble ( gray ) ‐ , shIP3R ( 1 , 3 ) A ( blue ) ‐ , shIP3R ( 2 , 3 ) B ( red ) ‐ , shIP3R ( 1 , 3 ) C ( green ) ‐ expressing DCs migrating in micro ‐ channels ( n > 100 per condition from 3 independent experiments for shIP3R ( 1 , 3 ) A shIP3R ( 1 , 3 ) C two independent experiments for shIP3R ( 2 , 3 ) B ) . A Velocity fluctuations ( ΔV / V0 ) of immature DCs migrating in micro ‐ channels . Boxes illustrate 10 - 90 percentiles of values , whiskers represent range of values . P ‐ values were calculated using a Kruskal - Wallis .

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Analysis of shScramble (gray)‐, shIP3R(1,3)A (blue)‐, shIP3R(2,3)B (red)‐, and shIP3R(1,3)C (green)‐expressing DCs migrating in micro‐channels (n > 100 cells per condition from 3 independent experiments for shIP3R(1,3)A and shIP3R(1,3)C and two independent experiments for shIP3R(2,3)B).A Velocity fluctuations (ΔV/V0) of immature DCs migrating in micro‐channels. Boxes illustrate 10-90 percentiles of values, and whiskers represent the range of values. P‐values were calculated using a Kruskal-Wallis test.


of shScramble ( gray ) ‐ , shIP3R ( 1 , 3 ) A ( blue ) ‐ , shIP3R ( 2 , 3 ) B ( red ) ‐ , shIP3R ( 1 , 3 ) C ( green ) ‐ expressing DCs migrating in micro ‐ channels ( n > 100 per condition from 3 independent experiments for shIP3R ( 1 , 3 ) A shIP3R ( 1 , 3 ) C two independent experiments for shIP3R ( 2 , 3 ) B ) . B Dot plot showing velocity fluctuations migration speeds of shScramble ( gray ) ‐ shIP3R ( 1 , 3 ) C ( green ) ‐ expressing DCs .

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Analysis of shScramble (gray)‐, shIP3R(1,3)A (blue)‐, shIP3R(2,3)B (red)‐, and shIP3R(1,3)C (green)‐expressing DCs migrating in micro‐channels (n > 100 cells per condition from 3 independent experiments for shIP3R(1,3)A and shIP3R(1,3)C and two independent experiments for shIP3R(2,3)B).B Dot plot showing velocity fluctuations and migration speeds of shScramble (gray)‐ and shIP3R(1,3)C (green)‐expressing DCs.


of shScramble ( gray ) ‐ , shIP3R ( 1 , 3 ) A ( blue ) ‐ , shIP3R ( 2 , 3 ) B ( red ) ‐ , shIP3R ( 1 , 3 ) C ( green ) ‐ expressing DCs migrating in micro ‐ channels ( n > 100 per condition from 3 independent experiments for shIP3R ( 1 , 3 ) A shIP3R ( 1 , 3 ) C two independent experiments for shIP3R ( 2 , 3 ) B ) . C Inverted sequential epifluorescence ( 20 × ) of a representative control DC ( left ) an immature shIP3R ( 1 , 3 ) C ‐ silenced DC ( right ) migrating in micro ‐ channels . DCs were differentiated from LifeAct ‐ GFP transgenic mice imaged every 2 . 2 min .

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Analysis of shScramble (gray)‐, shIP3R(1,3)A (blue)‐, shIP3R(2,3)B (red)‐, and shIP3R(1,3)C (green)‐expressing DCs migrating in micro‐channels (n > 100 cells per condition from 3 independent experiments for shIP3R(1,3)A and shIP3R(1,3)C and two independent experiments for shIP3R(2,3)B).C Inverted sequential epifluorescence images (20×) of a representative control DC (left) and an immature shIP3R(1,3)C‐silenced DC (right) migrating in micro‐channels. DCs were differentiated from LifeAct‐GFP transgenic mice and imaged every 2.2 min.


of shScramble ( gray ) ‐ , shIP3R ( 1 , 3 ) A ( blue ) ‐ , shIP3R ( 2 , 3 ) B ( red ) ‐ , shIP3R ( 1 , 3 ) C ( green ) ‐ expressing DCs migrating in micro ‐ channels ( n > 100 per condition from 3 independent experiments for shIP3R ( 1 , 3 ) A shIP3R ( 1 , 3 ) C two independent experiments for shIP3R ( 2 , 3 ) B ) . D of DCs changing their direction of locomotion while migrating in micro ‐ channels . P ‐ values were calculated with a paired with respect to shScramble condition .

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

Analysis of shScramble (gray)‐, shIP3R(1,3)A (blue)‐, shIP3R(2,3)B (red)‐, and shIP3R(1,3)C (green)‐expressing DCs migrating in micro‐channels (n > 100 cells per condition from 3 independent experiments for shIP3R(1,3)A and shIP3R(1,3)C and two independent experiments for shIP3R(2,3)B).D Percentage of DCs changing their direction of locomotion while migrating in micro‐channels. P‐values were calculated with a paired test with respect to the shScramble condition.


of shScramble ( gray ) ‐ , shIP3R ( 1 , 3 ) A ( blue ) ‐ , shIP3R ( 2 , 3 ) B ( red ) ‐ , shIP3R ( 1 , 3 ) C ( green ) ‐ expressing DCs migrating in micro ‐ channels ( n > 100 per condition from 3 independent experiments for shIP3R ( 1 , 3 ) A shIP3R ( 1 , 3 ) C two independent experiments for shIP3R ( 2 , 3 ) B ) . E Dot plot showing frequency of changes in direction of immature DCs while migrating in micro ‐ channels . Bars show mean plus standard deviation . A Kruskal - Wallis was applied for statistical .

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Analysis of shScramble (gray)‐, shIP3R(1,3)A (blue)‐, shIP3R(2,3)B (red)‐, and shIP3R(1,3)C (green)‐expressing DCs migrating in micro‐channels (n > 100 cells per condition from 3 independent experiments for shIP3R(1,3)A and shIP3R(1,3)C and two independent experiments for shIP3R(2,3)B).E Dot plot showing the frequency of changes in direction of immature DCs while migrating in micro‐channels. Bars show the mean plus the standard deviation. A Kruskal-Wallis test was applied for statistical analysis.


of shScramble ( gray , n = 2 , 400 ) ‐ , shIP3R ( 1 , 3 ) A ( blue , n = 1 , 611 ) ‐ , shIP3R ( 1 , 3 ) C ( green , n = 1 , 454 ) ‐ expressing DCs migrating in device described in Supplementary Fig S3I , stained with Hoechst imaged every 2 min during 24 h at 10 × magnification . Values were obtained from three independent experiments . trajectories were obtained by tracking their nucleus . A Graph showing DC mean instantaneous speed . P ‐ values were calculated with a t ‐ . B Mean instantaneous speed distribution of migrating DCs .

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

Analysis of shScramble (gray, n = 2,400 cells)‐, shIP3R(1,3)A (blue, n = 1,611 cells)‐, and shIP3R(1,3)C (green, n = 1,454 cells)‐expressing DCs migrating in the device described in Supplementary Fig S3I, stained with Hoechst and imaged every 2 min during 24 h at 10× magnification. Values were obtained from three independent experiments. Cell trajectories were obtained by tracking their nucleus.A Graph showing the DC mean instantaneous speed. P‐values were calculated with a t‐test analysis.B Mean instantaneous speed distribution of migrating DCs.


of shScramble ( gray , n = 2 , 400 ) ‐ , shIP3R ( 1 , 3 ) A ( blue , n = 1 , 611 ) ‐ , shIP3R ( 1 , 3 ) C ( green , n = 1 , 454 ) ‐ expressing DCs migrating in device described in Supplementary Fig S3I , stained with Hoechst imaged every 2 min during 24 h at 10 × magnification . Values were obtained from three independent experiments . trajectories were obtained by tracking their nucleus . C Representation of DC trajectories .

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Analysis of shScramble (gray, n = 2,400 cells)‐, shIP3R(1,3)A (blue, n = 1,611 cells)‐, and shIP3R(1,3)C (green, n = 1,454 cells)‐expressing DCs migrating in the device described in Supplementary Fig S3I, stained with Hoechst and imaged every 2 min during 24 h at 10× magnification. Values were obtained from three independent experiments. Cell trajectories were obtained by tracking their nucleus.C Representation of individual DC trajectories.


of shScramble ( gray , n = 2 , 400 ) ‐ , shIP3R ( 1 , 3 ) A ( blue , n = 1 , 611 ) ‐ , shIP3R ( 1 , 3 ) C ( green , n = 1 , 454 ) ‐ expressing DCs migrating in device described in Supplementary Fig S3I , stained with Hoechst imaged every 2 min during 24 h at 10 × magnification . Values were obtained from three independent experiments . trajectories were obtained by tracking their nucleus . D Path persistence ( calculated as between effective total of trajectory ) of migrating DCs . P ‐ values were determined with a t ‐ .

Analysis of shScramble (gray, n = 2,400 cells)‐, shIP3R(1,3)A (blue, n = 1,611 cells)‐, and shIP3R(1,3)C (green, n = 1,454 cells)‐expressing DCs migrating in the device described in Supplementary Fig S3I, stained with Hoechst and imaged every 2 min during 24 h at 10× magnification. Values were obtained from three independent experiments. Cell trajectories were obtained by tracking their nucleus.D Path persistence (calculated as the ratio between the effective length and the total length of the cell trajectory) of migrating DCs. P‐values were determined with a t‐test analysis.


of shScramble ( gray , n = 2 , 400 ) ‐ , shIP3R ( 1 , 3 ) A ( blue , n = 1 , 611 ) ‐ , shIP3R ( 1 , 3 ) C ( green , n = 1 , 454 ) ‐ expressing DCs migrating in device described in Supplementary Fig S3I , stained with Hoechst imaged every 2 min during 24 h at 10 × magnification . Values were obtained from three independent experiments . trajectories were obtained by tracking their nucleus . E Bars showing proportion of effective displacement ( L ) of DCs . A Fisher ' s exact ( P ‐ value 2 . 2 × 10 − 16 ) was used for statistics .

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Analysis of shScramble (gray, n = 2,400 cells)‐, shIP3R(1,3)A (blue, n = 1,611 cells)‐, and shIP3R(1,3)C (green, n = 1,454 cells)‐expressing DCs migrating in the device described in Supplementary Fig S3I, stained with Hoechst and imaged every 2 min during 24 h at 10× magnification. Values were obtained from three independent experiments. Cell trajectories were obtained by tracking their nucleus.E Bars showing the proportion of the effective length displacement (L) of DCs. A Fisher's exact test (P‐value 2.2 × 10−16) was used for statistics.


A Immunoblot showing intracellular of phosphorylated MLC in DCs transduced with shScramble or shRNAs for IP3Rs ( shIP3R ( 1 , 3 ) A ′ , shIP3R ( 2 , 3 ) B ′ , shIP3R ( 1 , 3 ) C ′ ) . Actin was used as a control .

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A Immunoblot showing the intracellular levels of phosphorylated MLC in DCs transduced with the shScramble or shRNAs for IP3Rs (shIP3R(1,3)A′, shIP3R(2,3)B′, and shIP3R(1,3)C′). Actin was used as a control.


B Inverted sequential epifluorescence ( 20 × ) showing myosin IIA ‐ GFP localization of a representative shScramble ( left ) ‐ shIP3R ( 1 , 3 ) C ‐ expressing DC ( right ) migrating in micro ‐ channels . Myosin IIA ‐ GFP knock ‐ in DCs were imaged every 1 min ( 1 out of 2 is shown ) . Asterisks highlight changes of direction during locomotion .

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B Inverted sequential epifluorescence images (20×) showing myosin IIA‐GFP localization of a representative shScramble (left)‐ and shIP3R(1,3)C‐expressing DC (right) migrating in micro‐channels. Myosin IIA‐GFP knock‐in DCs were imaged every 1 min (1 out of 2 images is shown). Asterisks highlight changes of direction during locomotion.


C , D Myosin IIA ‐ GFP localization ( red ) versus velocity fluctuations ( blue , ΔV / V0 ) in a representative shScramble ‐ transduced DC ( C ) or shIP3R ( 1 , 3 ) C ‐ silenced ( D ) migrating in micro ‐ channels . DCs were recorded as in ( B ) . E of interval between curves ( highlighted in D ) obtained for myosin IIA positioning velocity from shScramble ‐ shIP3R ( 1 , 3 ) C ‐ expressing DCs . were recorded as in ( B ) ( n = 20 from two experiments ) . Mann - Whitney was applied for statistical .

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C, D Myosin IIA‐GFP localization (red) versus velocity fluctuations (blue, ΔV/V0) in a representative shScramble‐transduced DC (C) or shIP3R(1,3)C‐silenced (D) migrating in micro‐channels. DCs were recorded as in (B).E Quantification of the interval between curves (highlighted in D) obtained for myosin IIA positioning and velocity from shScramble‐ and shIP3R(1,3)C‐expressing DCs. Cells were recorded as in (B) (n = 20 cells from two experiments). The Mann-Whitney test was applied for statistical analysis.


A Immunoblot showing intracellular of phosphorylated MLC in DCs treated overnight with increasing amounts of MLCK inhibitor , ML7 .

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A Immunoblot showing the intracellular levels of phosphorylated MLC in DCs treated overnight with increasing amounts of the MLCK inhibitor, ML7.


B , C of immature DCs migrating in micro ‐ channels in presence of ML7 or DMSO as a control ( n > 170 from two independent experiments ) . Velocity fluctuations ( ΔV / V0 ) of immature DCs migrating in micro ‐ channels ( B ) . Whiskers show 10 - 90 percentiles plus median . P ‐ values were calculated using a Kruskal - Wallis . Frequency of changes ( C ) in direction of DCs migrating in micro ‐ channels . Mean plus SD are shown . A Kruskal - Wallis was applied for statistical .

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B, C Analysis of immature DCs migrating in micro‐channels in the presence of ML7 or DMSO as a control (n > 170 cells from two independent experiments). Velocity fluctuations (ΔV/V0) of immature DCs migrating in micro‐channels (B). Whiskers show 10-90 percentiles plus the median. P‐values were calculated using a Kruskal-Wallis test. Frequency of changes (C) in direction of DCs migrating in micro‐channels. Mean plus SD are shown. A Kruskal-Wallis test was applied for statistical analysis.


D , E of shScramble ( gray ) ‐ shIP3R ( 1 , 3 ) C ( green ) ‐ expressing DCs migrating in micro ‐ channels in presence of 20 μM ML7 ( n > 70 per condition from 3 independent experiments ) . Velocity fluctuations ( ΔV / V0 ) of immature DCs migrating in micro ‐ channels ( D ) . Boxes illustrate 10 - 90 percentiles of values , whiskers represent range of values . P ‐ values were calculated using a Kruskal - Wallis . Frequency of changes ( E ) in direction observed in DCs migrating in micro ‐ channels . Means plus SD are shown . A Kruskal - Wallis was applied for statistical .

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D, E Analysis of shScramble (gray)‐ and shIP3R(1,3)C (green)‐expressing DCs migrating in micro‐channels in the presence of 20 μM ML7 (n > 70 cells per condition from 3 independent experiments). Velocity fluctuations (ΔV/V0) of immature DCs migrating in micro‐channels (D). Boxes illustrate 10-90 percentiles of values, and whiskers represent the range of values. P‐values were calculated using a Kruskal-Wallis test. Frequency of changes (E) in direction observed in DCs migrating in micro‐channels. Means plus SD are shown. A Kruskal-Wallis test was applied for statistical analysis.


by western - blot of EZH2 H3K27me3 at each stage of de novo transformation . UT : untransformed , PN : pre - neoplastic , TR : transformed . Histone H3 is used as a loading control . graph on right displays H3 normalized densitometric values of western - blot band from untreated DMSO treated . Values represent mean ± from three biological replicates . One asterisk indicates p - value < 0 . 05 ( one - unpaired Student ' s t - ) . ns : non - significant .

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Quantification by western-blot analysis of EZH2 and H3K27me3 levels at each stage of de novo transformation. UT: untransformed, PN: pre-neoplastic, TR: transformed. Histone H3 is used as a loading control. The graph on the right displays the H3 normalized densitometric values of western-blot band from untreated and DMSO treated cells. Values represent mean ± SEM from three biological replicates. One asterisk indicates p-value < 0.05 (one-tailed unpaired Student's t-test). ns: non-significant.


Volcano plot showing relative enrichment of EZH2 binding at sites detected in either untransformed or transformed . Numbers within volcano plot indicate differential binding sites with a p - value ≤ 1e - 20 a fold - change in normalised tag ≥ 1 . 5 . Enrichment significance calculated on a Poisson distribution using ' getDifferentialPeaks ' function in homer ( see : methods ) . P - values of > 1e - 100 were set to 1e - 100 for display reasons . Tracks on right show representative high - low - magnitude EZH2 differential peaks . ChIP - seq signal normalised to sequencing depth is shown . Tracks are scaled to be of same height to make samples comparable . Blue bars represent regions called as an EZH2 binding site . UT : untransformed , TR : transformed .

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Volcano plot showing the relative enrichment of EZH2 binding at all sites detected in either untransformed or transformed cells. Numbers within the volcano plot indicate differential binding sites with a p-value ≤1e-20 and a fold-change in normalised tag count ≥1.5. Enrichment significance calculated based on a Poisson distribution using the 'getDifferentialPeaks' function in homer (see: methods). P-values of > 1e-100 were set to 1e-100 for display reasons. Tracks on the right show representative high- and low-magnitude EZH2 differential peaks. ChIP-seq signal normalised to sequencing depth is shown. Tracks are scaled to be of the same height to make samples comparable. Blue bars represent regions called as an EZH2 binding site. UT: untransformed, TR: transformed.


Western - blot of H3K27me3 after treatment of with an EZH2 inhibitor ( EZH2i ) or DMSO as control for 12 days . Histone H3 is used as a loading control . Values represent H3 - normalized densitometric values of H3K27me3 bands , are expressed relative to relevant DMSO control . UT : untransformed , PN : pre - neoplastic , TR : transformed .

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Western-blot analysis of H3K27me3 levels after treatment of cells with an EZH2 inhibitor (EZH2i) or DMSO as control for 12 days. Histone H3 is used as a loading control. Values represent H3-normalized densitometric values of the H3K27me3 bands, and are expressed relative to the relevant DMSO control. UT: untransformed, PN: pre-neoplastic, TR: transformed.


of differentially expressed genes ( false discovery rate ( FDR ) ≤ 0 . 01 , Log2FC ≥ 1 / ≤ - 1 maximal transcripts per million ( maxTPM ) ≥ 1 ) detected by - seq in each state upon EZH2i treatment . UT : untransformed , PN : pre - neoplastic , TR : transformed .

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Quantification of differentially expressed genes (false discovery rate (FDR) ≤ 0.01, Log2FC ≥ 1/≤-1 and maximal transcripts per million (maxTPM) ≥ 1) detected by RNA-seq in each cellular state upon EZH2i treatment. UT: untransformed, PN: pre-neoplastic, TR: transformed.


showing relative expression of genes identified by multi - step filtering strategy in indicated states . UT : untransformed , PN : pre - neoplastic , TR : transformed . Colours represent log2 - transformed , row - normalised TPM values .

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Heatmap showing the relative expression of genes identified by the multi-step filtering strategy in the indicated cellular states. UT: untransformed, PN: pre-neoplastic, TR: transformed. Colours represent log2-transformed, row-normalised TPM values.


ChIP - seq signal for EZH2 H3K27me3 ( left ) at EMX2 HOXB9 loci . ChIP - seq signal normalised to sequencing depth is shown . Tracks are scaled to be of same height to make samples comparable . mRNA expression of EMX2 HOXB9 ( right ) as detected by - seq . Expression values represent mean ± from three biological replicates . UT : untransformed , PN : pre - neoplastic , TR : transformed . TPM : transcripts per million .

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ChIP-seq signal for EZH2 and H3K27me3 (left) at EMX2 and HOXB9 loci. ChIP-seq signal normalised to sequencing depth is shown. Tracks are scaled to be of the same height to make samples comparable. mRNA expression of EMX2 and HOXB9 (right) as detected by RNA-seq. Expression values represent mean ± SEM from three biological replicates. UT: untransformed, PN: pre-neoplastic, TR: transformed. TPM: transcripts per million.


qRT - PCR showing expression of EMX2 HOXB9 in indicated treated with EZH2i or a DMSO control for 12 days . Values represent mean ± from three biological replicates . Two asterisks indicate p - value < 0 . 01 ( one - unpaired Student ' s t - ) . UT : untransformed , TR : transformed .

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qRT-PCR showing the expression levels of EMX2 and HOXB9 in the indicated cells treated with EZH2i or a DMSO control for 12 days. Values represent mean ± SEM from three biological replicates. Two asterisks indicate p-value < 0.01 (one-tailed unpaired Student's t-test). UT: untransformed, TR: transformed.


Expression of EMX2 HOXB9 in human neural , as detected by - seq . Data sourced from Brainseq2 . Expression values represent mean ± from four , twelve , one five biological replicates of astrocytes , mature astrocytes , neurons oligodendrocytes , respectively . FPKM : fragments per kilobase of transcript per million .

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Expression levels of EMX2 and HOXB9 in primary human neural cells, as detected by RNA-seq. Data sourced from Brainseq2. Expression values represent mean ± SEM from four, twelve, one and five biological replicates of fetal astrocytes, mature astrocytes, neurons and oligodendrocytes, respectively. FPKM: fragments per kilobase of transcript per million.


Expression of EMX2 HOXB9 in 62 glioma lines . Data sourced from CCLE . Every dot represents a . RPKM : reads per kilobase of transcript per million . In boxplot , top , middle bottom box delimiters represent 75th , 50th 25th percentiles of data , respectively . Top bottom whiskers show 75th percentile + 1 . 5 * interquartile range 25th percentile - 1 . 5 * interquartile range , respectively .

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Expression levels of EMX2 and HOXB9 in 62 glioma cell lines. Data sourced from CCLE. Every dot represents a cell line. RPKM: reads per kilobase of transcript per million. In the boxplot, the top, middle and bottom box delimiters represent the 75th, 50th and 25th percentiles of the data, respectively. Top and bottom whiskers show the 75th percentile + 1.5*interquartile range and 25th percentile - 1.5*interquartile range, respectively.


of EZH2 binding at EMX2 , HOXB9 GAPDH ( negative control ) promoters by ChIP - qPCR in M059K GBM . Values from two biological replicates are shown . Three asterisks indicate p - value < 0 . 001 of EZH2 samples relative to negative control GAPDH ( two - way ANOVA followed by pairwise comparisons using Holm - Sidak method ) . Ns : non - significant .

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Quantification of EZH2 binding at EMX2, HOXB9 and GAPDH (negative control) promoters by ChIP-qPCR in M059K GBM cells. Values from two biological replicates are shown. Three asterisks indicate p-value < 0.001 of EZH2 samples relative to the negative control GAPDH (two-way ANOVA followed by pairwise comparisons using Holm-Sidak method). Ns: non-significant.


of EMX2 by qRT - PCR in five different GBM lines upon treatment with 1μM , 3μM or 10μM of EZH2i for eight days , or a DMSO control . Values represent mean ± from three technical replicates . is indicated to show reliability of RT - PCR values , due to low endogenous of EMX2 . Two asterisks indicate p - value < 0 . 01 comparing EZH2i - DMSO - treated ( Two - way ANOVA ) .

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Quantification of EMX2 levels by qRT-PCR in five different GBM cell lines upon treatment with 1μM, 3μM or 10μM of EZH2i for eight days, or a DMSO control. Values represent mean ± SEM from three technical replicates. The SEM is indicated to show reliability of the RT-PCR values, due to the low endogenous levels of EMX2. Two asterisks indicate p-value <0.01 comparing EZH2i- and DMSO-treated cells (Two-way ANOVA).


Relationship between extent of EMX2 upregulation induced by EZH2i methylation level at EMX2 promoter in GBM lines . Values for EMX2 upregulation are fold - change in mRNA expression induced upon treatment with 10μM EZH2i ( see : panel G ) , are expressed relative to a DMSO control . Values represent mean ± from three technical replicates . is indicated to show reliability of RT - PCR values , due to low endogenous of EMX2 . Methylation values are averages of methylation at CpGs across promoter of EMX2 in GBM lines . data sourced from CCLE . significance of anti - correlation between mRNA methylation across lines is indicated ( Spearman rank correlation ) .

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Relationship between the extent of EMX2 upregulation induced by EZH2i and the DNA methylation level at the EMX2 promoter in GBM cell lines. Values for EMX2 upregulation are the fold-change in mRNA expression induced upon treatment with 10μM EZH2i (see: panel G), and are expressed relative to a DMSO control. Values represent mean ± SEM from three technical replicates. The SEM is indicated to show reliability of the RT-PCR values, due to the low endogenous levels of EMX2. Methylation values are averages of DNA methylation at CpGs across the promoter of EMX2 in GBM cell lines. All data sourced from the CCLE. The significance of the anti-correlation between mRNA levels and DNA methylation levels across cell lines is indicated (Spearman rank correlation).


visualizing fold change in EMX2 expression as detected by qRT - PCR after treatment of indicated PDX - derived ( blue ) established cancer lines ( black ) with EZH2i for eight days . Values are from three technical replicates .

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Heatmap visualizing the fold change in EMX2 expression as detected by qRT-PCR after treatment of the indicated PDX-derived (blue) and established cancer cell lines (black) with EZH2i for eight days. Values are from three technical replicates.


Expression of EMX2 in normal brain GBM samples . Data sourced from Repository of Molecular Brain Neoplasia Data ( REMBRANDT ) . Four asterisks indicate p - value < 0 . 0001 ( two - Mann Whitney U ) . Bars represent median ± interquartile range . Values are expressed relative to mean of normal brain samples . N : 21 for normal brain , 214 for GBM . Note that extent of EMX2 repression in GBM is likely underestimated due to possible presence of normal adjacent tissue in analysed samples , due to intrinsic background noise of microarrays , which limits detection of truly silenced genes .

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Expression of EMX2 in normal brain and GBM patient samples. Data sourced from the Repository of Molecular Brain Neoplasia Data (REMBRANDT). Four asterisks indicate p-value < 0.0001 (two-tailed Mann Whitney U test). Bars represent median ± interquartile range. Values are expressed relative to the mean of normal brain samples. N: 21 for normal brain, 214 for GBM. Note that the extent of EMX2 repression in GBM patients is likely underestimated due to the possible presence of normal adjacent tissue in the analysed samples, and due to the intrinsic background noise of microarrays, which limits detection of truly silenced genes.


Relative expression of HOX genes in normal brain GBM samples as detected by microarray . Data sourced from REMBRANDT . Values are expressed relative to median of normal brain samples . In boxplots , top , middle bottom box delimiters represent 75th , 50th 25th percentiles of data , respectively . Top bottom whiskers show 75th percentile + 1 . 5 * interquartile range 25th percentile - 1 . 5 * interquartile range , respectively . One asterisk indicates p - value < 0 . 05 ( two - Mann Whitney U corrected for multiple comparison using Holm ' s method ) . N : 21 for normal brain , 214 for GBM . Data was not available for HOXD8 .

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Relative expression of HOX genes in normal brain and GBM patient samples as detected by microarray analysis. Data sourced from REMBRANDT. Values are expressed relative to the median of normal brain samples. In the boxplots, the top, middle and bottom box delimiters represent the 75th, 50th and 25th percentiles of the data, respectively. Top and bottom whiskers show the 75th percentile + 1.5*interquartile range and 25th percentile - 1.5*interquartile range, respectively. One asterisk indicates p-value <0.05 (two-tailed Mann Whitney U test corrected for multiple comparison using Holm's method). N: 21 for normal brain, 214 for GBM. Data was not available for HOXD8.


Covariance between EMX2 EZH2 expression in GBM samples , as detected by microarray . Data sourced from REMBRANDT . P - value correlation coefficient ( r ) of covariance are shown ( Spearman rank correlation ) . Every dot is a . N : 214 .

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Covariance between EMX2 and EZH2 expression levels in GBM patient samples, as detected by microarray analysis. Data sourced from REMBRANDT. P-value and correlation coefficient (r) of the covariance are shown (Spearman rank correlation). Every dot is a patient. N: 214.


Expression of EMX2 ( top ) EZH2 ( bottom ) in tumour or adjacent normal regions microdissected from human GBM tumours as detected by - seq . Data sourced from Ivy Glioblastoma Atlas . FPKM : fragments per kilobase of transcript per million . significance of differential expression in normal tumour regions across is indicated ( two - way ANOVA ) . Bars represent mean ± . N : 3 regions sampled for each GBM tumour , 2 , 3 , 2 , 3 , 2 , 3 , 3 1 regions for normal tissue of 1 - 8 , respectively .

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Expression levels of EMX2 (top) and EZH2 (bottom) in tumour or adjacent normal regions laser microdissected from human GBM tumours as detected by RNA-seq. Data sourced from the Ivy Glioblastoma Atlas. FPKM: fragments per kilobase of transcript per million. The significance of the differential expression in normal and tumour regions across patients is indicated (two-way ANOVA). Bars represent mean ± SEM. N: 3 regions sampled for each GBM tumour, 2, 3, 2, 3, 2, 3, 3 and 1 regions for normal tissue of patients 1-8, respectively.


Hematoxylin eosin ( H & E ) of a human GBM tumour . black square represents approximate location of field shown in G ( left image ) . Serial sections were used for H & E - FISH . Scale bar : 300 µm .

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Hematoxylin and eosin (H&E) staining of a human GBM tumour. The black square represents the approximate location of the field shown in G (left image). Serial sections were used for H&E and RNA-FISH. Scale bar: 300 µm.


Visualisation of EZH2 ( green ) EMX2 mRNA ( red ) in a human GBM tumour by FISH . Nuclei were counterstained with DAPI . Scale bar : 20 µm .

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Visualisation of EZH2 (green) and EMX2 mRNA (red) in a human GBM tumour by RNA FISH. Nuclei were counterstained with DAPI. Scale bar: 20 µm.


Expression of EMX2 ( left ) EZH2 ( right ) in glioma samples , grouped according to tumour grade , as detected by - seq . Data sourced from Chinese Glioma Genome Atlas . Four asterisks indicate p - value < 0 . 0001 ( Kruskal - Wallis ) . Bars represent median ± interquartile range . N : 109 for grade II - , 72 for grade III , 144 for grade IV . RPKM : Reads per kilobase of transcript per million .

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Expression of EMX2 (left) and EZH2 (right) in glioma patient samples, grouped according to tumour grade, as detected by RNA-seq. Data sourced from the Chinese Glioma Genome Atlas. Four asterisks indicate p-value < 0.0001 (Kruskal-Wallis test). Bars represent median ± interquartile range. N: 109 for grade II -, 72 for grade III, 144 for grade IV. RPKM: Reads per kilobase of transcript per million.


Proliferation examining effect of EMX2 ectopic expression in U - 87 MG DBTRG - 05MG GBM , RFP is used as a control . Values represent mean ± from three biological replicates . fold change in number after eight days of proliferation whilst expressing exogenous is shown relative to an uninduced control . Two asterisks indicate p - value < 0 . 01 ( one - unpaired Student ' s t - ) . Similar results were reported in [ 103 ] using other GBM lines .

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Proliferation assay examining the effect of EMX2 ectopic expression in U-87 MG and DBTRG-05MG GBM cells, RFP is used as a control. Values represent mean ± SEM from three biological replicates. The fold change in cell number after eight days of proliferation whilst expressing the exogenous protein is shown relative to an uninduced control. Two asterisks indicate p-value < 0.01 (one-tailed unpaired Student's t-test). Similar results were reported in [103] using other GBM cell lines.


Transplantation comparing growth kinetics of subcutaneous DBTRG - 05MG - induced tumours expressing EMX2 at comparable to those expressed in normal , or expressing RFP as a control . Values represent mean ± from six tumours . Three asterisks indicate p - value < 0 . 001 ( two - single sample Student ' s t - ) . Similar results were obtained in an independent experiment using a distinct batch of transduced .

Transplantation assay comparing the growth kinetics of subcutaneous DBTRG-05MG-induced tumours expressing EMX2 at levels comparable to those expressed in normal cells, or expressing RFP as a control. Values represent mean ± SEM from six tumours. Three asterisks indicate p-value < 0.001 (two-tailed single sample Student's t-test). Similar results were obtained in an independent experiment using a distinct batch of transduced cells.


MRI scans showing representative brain tumours six post - injection of DBTRG - 05MG expressing EMX2 or RFP . Axial anatomical scan coronal signal map indicate location of tumour in RFP control ( yellow arrow ) . Contrast enhancement in post - contrast indicates tumour blood brain barrier breakdown . Signal was not present in pre - contrast . show same MRI slice position between mice . Scale bar : 2mm . of brain tumour in mice injected with EMX2 - or RFP - expressing DBTRG - 05MG . Values represent mean ± from four control three EMX2 tumours . Five mice per conditions were injected but three ( one for RFP , two for EMX2 ) had to be excluded from study due to complications from procedure .

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MRI scans showing representative brain tumours six weeks post-injection of DBTRG-05MG cells expressing EMX2 or RFP. Axial anatomical scan and coronal signal intensity map indicate location of tumour in RFP control (yellow arrow). Contrast enhancement in post-contrast images indicates tumour blood brain barrier breakdown. Signal was not present in pre-contrast images. Images show the same MRI slice position between mice. Scale bar: 2mm. Quantification of brain tumour size in mice injected with EMX2- or RFP- expressing DBTRG-05MG cells. Values represent mean ± SEM from four control and three EMX2 tumours. Five mice per conditions were injected but three (one for RFP, and two for EMX2) had to be excluded from the study due to complications from the procedure.


Representative of euploid control or ArCK interacting with NK . NK - mediated killing was measured at a 2 . 5 : 1 ( NK : target ) was recorded by live imaging for 36 hours at a 30 - min interval . TO - PRO - 3 ( 1 μM ) was added to medium at same time of NK addition to measure integrity . Phase contrast ( top ) TO - PRO - 3 signal ( bottom ) from same field were presented . Arrowheads indicate ArCK . were acquired at same exposure time . Scale bar 20 μM .

Representative images of euploid control or ArCK cells interacting with NK cells. The NK cell-mediated killing was measured at a 2.5:1 (NK cells: target cells) ratio and was recorded by live cell imaging for 36 hours at a 30-min interval. TO-PRO-3 (1 μM) was added to the medium at the same time of NK cell addition to measure cell membrane integrity. Phase contrast (top) and TO-PRO-3 signal (bottom) from the same field were presented. Arrowheads indicate ArCK cell death. All images were acquired at the same exposure time and light intensity. Scale bar 20 μM.


NK - mediated cytotoxicity across various NK to target ratios . Either euploid control ( Ctrl ) or ArCK were co - cultured with NK at indicated NK : target ratios . cumulative killing of target was measured . n = 3 biological replicates ; mean ± . p < 0 . 0001 for four NK : target ratios , KS .

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NK cell-mediated cytotoxicity across various NK cell to target cell ratios. Either euploid control (Ctrl) or ArCK cells were co-cultured with NK cells at the indicated NK cell: target cell ratios. The cumulative killing of target cells was measured. n=3 biological replicates; mean± SEM. p< 0.0001 for all four NK cell: target cell ratios, KS test.


NK - mediated killing for doxorubicin ( Doxo ) , palbociclib ( Palbo ) , nutlin3 ( Nutlin ) treated samples ( NK : target = 2 . 5 : 1 ) . n = 3 biological replicates ; mean ± . Ctrl vs . Doxo , p < 0 . 0001 ; Ctrl vs . Palbo , p < 0 . 0001 ; Ctrl vs . Nutlin , p < 0 . 0001 ; KS .

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NK cell-mediated killing for doxorubicin (Doxo), palbociclib (Palbo), and nutlin3 (Nutlin) treated samples (NK cell: target cell =2.5:1). n=3 biological replicates; mean± SEM. Ctrl vs. Doxo, p< 0.0001; Ctrl vs. Palbo, p< 0.0001; Ctrl vs. Nutlin, p< 0.0001; KS test.


NK - mediated cytotoxicity towards torin1 - treated . Torin1 treated ( Torin ) were generated as described in ( F ) NK killing was performed as described in Figure 1 . n = 3 biological replicates ; mean ± . Ctrl vs . Torin , p = 0 . 79 , not significant ( n . s . ) ; KS .

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NK cell-mediated cytotoxicity towards torin1-treated cells. Torin1 treated (Torin) cells were generated as described in (F) and the NK cell killing assay was performed as described in Figure 1. n=3 biological replicates; mean± SEM. Ctrl vs. Torin, p= 0.79, not significant (n.s.); KS test.


γ - H2AX foci were analyzed in ArCK G1 arrested ( generated as described in Figure 2 ) . At least 50 were analyzed per condition per replicate . n = 2 biological replicates ; values mean are shown . distribution of foci number in each treated condition was compared to that of control using Kolmogorov - Smirnov . Ctrl vs . Doxo , p < 0 . 0001 ; Ctrl vs . Palbo , p = 0 . 07 , n . s . ; Ctrl vs . Nutlin , p = 0 . 11 , n . s . ; Ctrl vs . ArCK , p = 0 . 0007 .

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γ-H2AX foci were analyzed in ArCK and G1 arrested cells (generated as described in Figure 2). At least 50 cells were analyzed per condition per replicate. n=2 biological replicates; individual values and mean are shown. The distribution of the foci number in each treated condition was compared to that of control using Kolmogorov-Smirnov test. Ctrl vs. Doxo, p< 0.0001; Ctrl vs. Palbo, p= 0.07, n.s.; Ctrl vs. Nutlin, p= 0.11, n.s.; Ctrl vs. ArCK, p= 0.0007.


Secreted cytokine interferon were determined in supernatants . Media were collected after 36 hours of incubation with grown as described in Figure 1 2 . Cytokine interferon were shown as fold change normalized to euploid control . values mean are shown ; n = 2 biological replicates .

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Secreted cytokine and interferon levels were determined in cell supernatants. Media were collected after 36 hours of incubation with cells grown as described in Figure 1 and 2. Cytokine and interferon levels were shown as fold change normalized to euploid control cells. Individual values and mean are shown; n=2 biological replicates.


NK medium was incubated with either euploid control or ArCK for 12 hours . At time of NK addition , media were switched between ArCK euploid control ( Ctrl ) . NK killing was measured as described in Figure 1C . For reference , NK killing of ArCK euploid control ( Ctrl ) without medium switch were performed side by side plotted on graph . Black , euploid control without medium switch ; red , ArCK without medium switch ; blue , euploid control in ArCK condition medium ; green , ArCK in euploid control condition medium . n = 3 biological replicates ; mean ± . Ctrl vs . ArCK , p < 0 . 0001 ; Ctrl vs . Ctrl in ArCK med , p < 0 . 0001 ; Ctrl vs . ArCK in Ctrl med , p < 0 . 0001 ; ArCK vs . ArCK in Ctrl med , p = 0 . 0002 ; KS .

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NK cell medium was incubated with either euploid control or ArCK cells for 12 hours. At the time of NK cell addition, media were switched between ArCK and euploid control cells (Ctrl). NK cell killing was measured as described in Figure 1C. For reference, NK cell killing of ArCK and euploid control cells (Ctrl) without medium switch were performed side by side and plotted on the graph. Black, euploid control cells without medium switch; red, ArCK cells without medium switch; blue, euploid control cells in ArCK cell condition medium; green, ArCK cells in euploid control cell condition medium. n=3 biological replicates; mean± SEM. Ctrl vs. ArCK, p< 0.0001; Ctrl vs. Ctrl in ArCK med, p< 0.0001; Ctrl vs. ArCK in Ctrl med, p< 0.0001; ArCK vs. ArCK in Ctrl med, p= 0.0002; KS test.


RT - qPCR quantifying NF - κB downstream target gene expression in ArCK euploid control ( Ctrl ) . n = 6 biological replicates ; mean ± . IL - 1b , * p = 0 . 029 ; IL - 6 , * p = 0 . 032 ; IL - 8 , * * p = 0 . 008 ; unpaired t - .

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RT-qPCR quantifying NF-κB downstream target gene expression in ArCK and euploid control (Ctrl) cells. n=6 biological replicates; mean± SEM. IL-1b, *p= 0.029; IL-6, *p= 0.032; IL-8, **p= 0.008; unpaired t-test.


ArCK or euploid proliferating control were treated with either DMSO or NF - κB inhibitor BMS - 345541 ( 5 μM ) for 48 hours before assessing NK - mediated cytotoxicity . drug was washed out during NK co - culture . n = 3 biological replicates ; mean ± . ArCK vs ArCK NF - κB inhibitor , p < 0 . 0001 ; KS . ( H , I ) effect of inactivating both RELA RELB on NK - mediated cytotoxicity in 7 - day doxorubicin ( H ) nutlin3 - treated ( I ) . n2 biological replicates ; Doxo - c1 vs . Doxo RELA RELB - c1 , p = 0 . 02 . Nutlin - c1 vs . Nutlin RELA RELB - c1 , p = 0 . 44 , n . s . ; KS .

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 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 B-CONTROLLED_VAR 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 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

ArCK or euploid proliferating control cells were treated with either DMSO or the NF-κB inhibitor BMS-345541 (5 μM) for 48 hours before assessing NK cell-mediated cytotoxicity. The drug was washed out during the NK cell co-culture assay. n=3 biological replicates; mean± SEM. ArCK vs ArCK NF-κB inhibitor, p< 0.0001; KS test. (H, I) The effect of inactivating both RELA and RELB on NK cell-mediated cytotoxicity in 7-day doxorubicin (H) and nutlin3-treated (I) cells. n2 biological replicates; Doxo-c1 vs. Doxo RELA RELB-c1, p = 0.02. Nutlin-c1 vs. Nutlin RELA RELB-c1, p = 0.44, n.s.; KS test.


HCT116 ( left ) or DLD1 ( right ) were treated with either DMSO or Mps1 inhibitor reversine ( 500 nM ) for 48 hours before assessing NK - mediated cytotoxicity . drug was washed out during NK co - culture . n = 3 biological replicates ; mean ± . HCT116 vs . HCT116 Mps1 inhibitor , p = 0 . 44 , n . s . ; DLD1 vs . DLD1 Mps1 inhibitor , p = 0 . 44 , n . s . ; KS .

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HCT116 (left) or DLD1 cells (right) were treated with either DMSO or the Mps1 inhibitor reversine (500 nM) for 48 hours before assessing NK cell-mediated cytotoxicity. The drug was washed out during the NK cell co-culture assay. n=3 biological replicates; mean±SEM. HCT116 vs. HCT116 Mps1 inhibitor, p= 0.44, n.s.; DLD1 vs. DLD1 Mps1 inhibitor, p= 0.44, n.s.; KS test.


A Representative siliques from wild - type ( WT ; Col - 0 ) , herk1 , anj herk1 anj prior to dehiscence . Siliques were placed on double - sided sticky tape carpel walls separated from replum to expose developing seeds . Scale bar = 5 mm .

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A Representative siliques from wild-type (WT; Col-0), herk1, anj and herk1 anj plants prior to dehiscence. Siliques were placed on double-sided sticky tape and carpel walls separated from the replum to expose the developing seeds. Scale bar = 5 mm.


B Developing seeds per silique in wild - type , herk1 , anj herk1 anj . Fully expanded siliques were dissected photographed under a stereomicroscope . n = 15 ( four independent experiments with at least three per five siliques per ) . Data presented are means ± . * * * p < 0 . 001 ( Student ' s t - ) .

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B Developing seeds per silique in wild-type, herk1, anj and herk1 anj plants. Fully expanded siliques were dissected and photographed under a stereomicroscope. n = 15 (four independent experiments with at least three plants per line and five siliques per plant). Data presented are means ± SEM. *** p<0.001 (Student's t-test).


C of pollen tubes with normal reception at gametophyte ( black bars ; representative image middle centre of figure ) with overgrowth ( grey bars ; representative image lower centre ) as assessed by aniline blue . 15 self - pollinated stage 16 flowers from wild - type , herk1 , anj herk1 anj were analysed . Legend scale bars = 50 µm . * * * p < 0 . 001 ( χ - square ) .

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C Percentage of pollen tubes with normal reception at the female gametophyte (black bars; representative image middle centre of figure) and with overgrowth (grey bars; representative image lower centre) as assessed by aniline blue staining. 15 self-pollinated stage 16 flowers from wild-type, herk1, anj and herk1 anj were analysed. Legend scale bars = 50 µm. *** p<0.001 (χ-square tests).


D Aniline blue of pollen tube reception in reciprocal crosses between wild - type herk1 anj with at least two siliques per cross . Legend as per ( C ) . * * * p < 0 . 001 ( χ - square ) .

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D Aniline blue staining of pollen tube reception in reciprocal crosses between wild-type and herk1 anj plants with at least two siliques per cross. Legend as per (C). *** p<0.001 (χ-square tests).


A , B Expression of pHERK1 : : H2B - TdTomato in mature ovules . dotted lines delineate egg synergid . Data information : Scale bars = 50 µm . M , micropyle . CC , central . EC , egg . SC , synergid .

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A,B Expression of pHERK1::H2B-TdTomato in mature ovules. White dotted lines delineate the egg cell and synergid cells. Data information : Scale bars = 50 µm. M, micropyle. CC, central cell. EC, egg cell. SC, synergid cell.


C , D Expression of pANJ : : H2B - TdTomato in mature ovules . dotted lines delineate egg synergid . Data information : Scale bars = 50 µm . M , micropyle . CC , central . EC , egg . SC , synergid .

C,D Expression of pANJ::H2B-TdTomato in mature ovules. White dotted lines delineate the egg cell and synergid cells. Data information : Scale bars = 50 µm. M, micropyle. CC, central cell. EC, egg cell. SC, synergid cell.


E , F Localisation of HERK1 - GFP in synergid from pFER : : HERK1 - GFP construct in ( F ) corresponding differential interference contrast ( DIC ) image in ( E ) . red dotted lines delineate egg synergid , respectively . Data information : Scale bars = 50 µm . M , micropyle . Arrows , filiform apparatus .

E,F Localisation of HERK1-GFP in the synergid cell from the pFER::HERK1-GFP construct in (F) and corresponding differential interference contrast (DIC) image in (E). White and red dotted lines delineate the egg cell and synergid cells, respectively. Data information : Scale bars = 50 µm. M, micropyle. Arrows, filiform apparatus.


G , H Localisation of ANJ - GFP in synergid from pANJ : : ANJ - GFP construct in ( H ) corresponding DIC image in ( G ) . red dotted lines delineate egg synergid , respectively . Data information : Scale bars = 50 µm . M , micropyle . Arrows , filiform apparatus .

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G,H Localisation of ANJ-GFP in the synergid cell from the pANJ::ANJ-GFP construct in (H) and corresponding DIC image in (G). White and red dotted lines delineate the egg cell and synergid cells, respectively. Data information : Scale bars = 50 µm. M, micropyle. Arrows, filiform apparatus.


A Localisation of HERK1 , ANJ , LRE , FER NTA in synergid of wild - type ( Col - 0 ; WT ) , herk1 anj lre - 5 in unfertilised ovules , as shown by pFER : : HERK1 - GFP , pANJ : : ANJ - GFP , pLRE : : LRE - Citrine , pFER : : FER - GFP pMYB98 : : NTA - GFP . DIC are shown , left to right , respectively . red dotted lines delineate egg synergid , respectively . Scale bars = 25 µm .

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A Localisation of HERK1, ANJ, LRE, FER and NTA in the synergid cell of wild-type (Col-0; WT), herk1 anj and lre-5 in unfertilised ovules, as shown by pFER::HERK1-GFP, pANJ::ANJ-GFP, pLRE::LRE-Citrine, pFER::FER-GFP and pMYB98::NTA-GFP. DIC and fluorescence images are shown, left to right, respectively. White and red dotted lines delineate the egg cell and synergid cells, respectively. Scale bars = 25 µm.


B Localisation of NTA in synergid of wild - type herk1 anj before ( upper panels ) after ( lower panels ) pollen tube arrival . In green , NTA localisation as shown by pMYB98 : : NTA - GFP . In magenta , callose of filiform apparatus pollen tube stained with SR2200 . From left to right , shown are DIC , merged , merged of DIC . red dotted lines delineate pollen tube synergid , respectively . Scale bars = 25 µm . M , micropyle .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Localisation of NTA in the synergid cell of wild-type and herk1 anj plants before (upper panels) and after (lower panels) pollen tube arrival. In green, NTA localisation as shown by pMYB98::NTA-GFP fluorescence. In magenta, callose of the filiform apparatus and pollen tube stained with SR2200. From left to right, images shown are DIC, merged fluorescence images, and merged images of DIC and fluorescence. White and red dotted lines delineate the pollen tube and synergid cells, respectively. Scale bars = 25 µm. M, micropyle.


Profile of relative of NTA - GFP along synergid of wild - type herk1 anj ovules ( C ) before ( virgin ) after ( pollinated ) pollen arrival . Data shown are means ± , n = 25 . * * * p < 0 . 001 ( Student ' s t - ) . FA , filiform apparatus .

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Profile of relative fluorescence intensity of NTA-GFP along the synergid cells of wild-type and herk1 anj ovules (C) before (virgin) and after (pollinated) pollen arrival. Data shown are means ± SEM, n = 25. *** p<0.001 (Student's t-test). FA, filiform apparatus.


D Profile of relative of NTA - GFP along synergid of wild - type lre - 5 ovules ( D ) before ( virgin ) after ( pollinated ) pollen arrival . Data shown are means ± , n = 25 . * * * p < 0 . 001 ( Student ' s t - ) . FA , filiform apparatus .

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D Profile of relative fluorescence intensity of NTA-GFP along the synergid cells of wild-type and lre-5 ovules (D) before (virgin) and after (pollinated) pollen arrival. Data shown are means ± SEM, n = 25. *** p<0.001 (Student's t-test). FA, filiform apparatus.


A Representative of ovules from wild - type ( Col - 0 ) , herk1 anj , lre - 5 fer - 4 20 hours after emasculation ( HAE ) displaying mature gametophyte structure . presented here are maximum projections from confocal microscopy across several z - planes of ovules stained as per [ 70 ] . Scale bars = 50 µm .

A Representative images of ovules from wild-type (Col-0), herk1 anj, lre-5 and fer-4 20 hours after emasculation (HAE) displaying the mature female gametophyte structure. Images presented here are maximum intensity projections from confocal microscopy images across several z-planes of ovules stained as per [70]. Scale bars = 50 µm.


B of H2DCF - DA of ROS in ovules from wild - type , herk1 anj , lre - 5 fer - 4 at 20 HAE . Categories are listed in legend Ovules dissected from at least five siliques per . * * * p < 0 . 001 ( χ - square ) .

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B Quantification of H2DCF-DA staining of ROS in ovules from wild-type, herk1 anj, lre-5 and fer-4 plants at 20 HAE. Categories are listed in the legend Ovules dissected from at least five siliques per line. *** p<0.001 (χ-square tests).


C of pollen tubes with normal reception at gametophyte ( black bars ) displaying overgrowth ( grey bars ) in wild - type , herk1 anj , lre - 5 fer - 4 , manually selfed at 20 HAE . Fertilisation events counted from at least three siliques per . * * * p < 0 . 001 ( Student ' s t - ) .

C Percentage of pollen tubes with normal reception at the female gametophyte (black bars) and displaying overgrowth (grey bars) in wild-type, herk1 anj, lre-5 and fer-4 plants, manually selfed at 20 HAE. Fertilisation events counted from at least three siliques per line. *** p<0.001 (Student's t-test).


A Yeast two hybrid ( Y2H ) assays of extracellular juxtamembrane domains of HERK1 ANJ ( HERK1exJM ANJexJM , respectively ) with LRE ( residues 23 - 138 ; signal peptide C - terminal domains excluded ) .

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A Yeast two hybrid (Y2H) assays of the extracellular juxtamembrane domains of HERK1 and ANJ (HERK1exJM and ANJexJM, respectively) with LRE (residues 23-138; signal peptide and C-terminal domains excluded).


B Y2H assays with extracellular domains of HERK1 , ANJ FER ( HERK1 - ECD , ANJ - ECD FER - ECD , respectively ) . Ø represents negative controls where no was cloned into activating domain ( AD ) or - binding domain ( BD ) constructs . - L - W - H , growth medium depleted of leucine ( - L ) , tryptophan ( - W ) histidine ( - H ) .

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B Y2H assays with the extracellular domains of HERK1, ANJ and FER (HERK1-ECD, ANJ-ECD and FER-ECD, respectively). Ø represents negative controls where no sequence was cloned into the activating domain (AD) or DNA-binding domain (BD) constructs. -L-W-H, growth medium depleted of leucine (-L), tryptophan (-W) and histidine (-H).