( e ) Representative confocal images of muscle 4synapses stained for both DVGLUT ( green ) and FasII ( red ) in wandering larvae of wild - type ( C155 - Gal4 / + ) , Atg1Gap - GFP ( C155 - Gal4 / + ; UAS - Gap - GFP / + ; UAS - Atg16B / + ) , and Atg1NTAP - Rae1 ( C155 - Gal4 / + ; UAS - NTAP - Rae1 / + ; UAS - Atg1 / + ) . Scale bar represents 10 μm . ( f ) Quantification of the number of boutons at muscle 4synapses in wild - type , Rae1 expression ( C155 - Gal4 / + ; UAS - NTAP - Rae1 / + ) , Atg1Gap - GFP and Atg1NTAP - Rae1 wandering larvae ( segment A2 - 4 , n = 28 , 27 , 33 and 34 , respectively ) . Error bars denote s . e . m . * P 0 . 001 , * * P 0 . 05 .

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

(e) Representative confocal images of muscle 4synapses stained for both DVGLUT (green) and FasII (red) in wandering larvae of wild-type (C155-Gal4/+), Atg1Gap-GFP (C155-Gal4/+; UAS-Gap-GFP/+; UAS-Atg16B/+), and Atg1NTAP-Rae1 (C155-Gal4/+; UAS-NTAP-Rae1/+; UAS-Atg1/+). Scale bar represents 10 μm. (f) Quantification of the number of boutons at muscle 4synapses in wild-type, Rae1 expression (C155-Gal4/+; UAS-NTAP-Rae1/+), Atg1Gap-GFP and Atg1NTAP-Rae1 wandering larvae (segment A2-4, n = 28, 27, 33 and 34, respectively). Error bars denote s.e.m. *P 0.001, **P 0.05.


a , TG levels in hepatocytes untreated ( None ) or treated with 3 - methyladenine ( 3MA ) and cultured in regular medium ( RM ) or oleate ( OL ) ( * P 0 . 02 , * * P 0 . 002 , 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

a, TG levels in hepatocytes untreated (None) or treated with 3-methyladenine (3MA) and cultured in regular medium (RM) or oleate (OL) (*P 0.02, **P 0.002, n = 3).


b , TG levels in vector - infected ( VEC ) and siAtg5 cells in RM , OL or in MCDM ( * P 0 . 001 , n = 5 ) . OL values are in mM .

O O O O O O O O O O O O O O O O O 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, TG levels in vector-infected (VEC) and siAtg5 cells in RM, OL or in MCDM (*P  0.001, n = 5). OL values are in mM.


c , TG levels in wild - type ( WT ) or Atg5 knockout mice embryonic fibroblasts ( Atg5 - / - ) ( * P 0 . 01 or * * P 0 . 0001 , n = 5 ) .

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c, TG levels in wild-type (WT) or Atg5 knockout mice embryonic fibroblasts (Atg5-/-) (*P 0.01 or **P 0.0001, n = 5).


d f , Cells from a and b stained with BODIPY 493 / 503 ( d ) . Nuclei are highlighted with 4 , 6 - diamidino - 2 - phenylindole ( DAPI ; d ) . Error bars , s . e . m .

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

df, Cells from a and b stained with BODIPY 493/503 (d). Nuclei are highlighted with 4,6-diamidino-2-phenylindole (DAPI; d). Error bars, s.e.m.


d f , Cells from a and b stained with oil red O ( e ) or visualized by electron microscopy ( f ) . Right : quantifications of LD number and size ( * P 0 . 01 , * * P 0 . 001 with untreated wild - type cells ; # P 0 . 01 , # # P 0 . 001 with cells in RM ; n = 4 6 ) .

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

df, Cells from a and b stained with oil red O (e) or visualized by electron microscopy (f). Right: quantifications of LD number and size (*P  0.01, **P  0.001 with untreated wild-type cells; #P  0.01, # #P  0.001 with cells in RM; n = 46).


a , b , VEC and siAtg5 cells cultured with oleate ( OL ) or in MCDM were examined for their rates of TG synthesis ( a ) and β - oxidation ( b ) as compared to cells in regular medium alone ( * P 0 . 03 , * * P 0 . 004 with VEC cells in the same medium , n = 3 4 ) .

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

a, b, VEC and siAtg5 cells cultured with oleate (OL) or in MCDM were examined for their rates of TG synthesis (a) and β-oxidation (b) as compared to cells in regular medium alone (*P  0.03, **P  0.004 with VEC cells in the same medium, n = 34).


c , d , Rates of TG decay in OL ( c ) and MCDM ( d ) ( * P 0 . 05 , * * P 0 . 01 , * * * P 0 . 001 as compared to VEC cells , n = 3 7 ) .

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

c, d, Rates of TG decay in OL (c) and MCDM (d) (*P  0.05, **P  0.01, ***P  0.001 as compared to VEC cells, n = 37).


e , TG levels in wild - type cells treated with dimethyl sulphoxide vehicle ( DMSO ) , 3 - methyladenine ( 3MA ) or diethylumbelliferyl phosphate ( DEUP ) ( * P 0 . 00001 with DMSO - treated cells , # P 0 . 003 with 3MA - treated cells , n = 6 ) . Error bars , s . e . m .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, TG levels in wild-type cells treated with dimethyl sulphoxide vehicle (DMSO), 3-methyladenine (3MA) or diethylumbelliferyl phosphate (DEUP) (*P  0.00001 with DMSO-treated cells, #P  0.003 with 3MA-treated cells, n = 6). Error bars, s.e.m.


f , Co - localization of BODIPY 493 / 503 ( green ) with LAMP1 ( red ) or LC3 ( red ) in hepatocytes in MCDM .

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

f, Co-localization of BODIPY 493/503 (green) with LAMP1 (red) or LC3 (red) in hepatocytes in MCDM.


g , High - magnification regions of hepatocytes in MCDM alone ( none ) or treated with vinblastine and stained as labelled . Arrows indicate co - localization .

O O O O O 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, High-magnification regions of hepatocytes in MCDM alone (none) or treated with vinblastine and stained as labelled. Arrows indicate co-localization.


a , Electron micrographs of cultured hepatocytes . dm , double - membrane cytosolic vesicles . Arrows indicate membranes in LDs . Insets show the double membrane in nucleus ( N ) , lipid - containing vesicles ( L ) and mitochondria ( mit ) , but not in LDs .

a, Electron micrographs of cultured hepatocytes. dm, double-membrane cytosolic vesicles. Arrows indicate membranes in LDs. Insets show the double membrane in nucleus (N), lipid-containing vesicles (L) and mitochondria (mit), but not in LDs.


b , Mouse liver LC3 immunogold . Insets show higher magnification . Arrowheads indicate gold particles ( black ) and LC3 - labelled bilayer membranes ( white ) . c , Percentages of autophagic vacuoles ( AVs ) containing only lipid ( Lipids , * ) , other cargo ( Other ) or mixed cargo ( Mixed ) in cells treated as in b ( * P 0 . 01 , * * P 0 . 001 with cells in RM , n = 4 6 ) . Left : representative examples . Error bars , s . e . m .

O O O O B-MEASURED_VAR 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

b, Mouse liver LC3 immunogold. Insets show higher magnification. Arrowheads indicate gold particles (black) and LC3-labelled bilayer membranes (white). c, Percentages of autophagic vacuoles (AVs) containing only lipid (Lipids, *), other cargo (Other) or mixed cargo (Mixed) in cells treated as in b (*P  0.01, **P  0.001 with cells in RM, n = 46). Left: representative examples. Error bars, s.e.m.


a , Immunoblots of cellular homogenates ( Hom ) and LDs from fed ( F ) or 24 - h starved ( S ) mice . IκB , inhibitor of the nuclear factor of kappa light polypeptide gene enhancer ; GPDH , glyceraldehyde - 3 - phosphate dehydrogenase .

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

a, Immunoblots of cellular homogenates (Hom) and LDs from fed (F) or 24-h starved (S) mice. IκB, inhibitor of the nuclear factor of kappa light polypeptide gene enhancer; GPDH, glyceraldehyde-3-phosphate dehydrogenase.


b , Percentages of autophagic vacuoles ( AVs ) containing only lipid , other cargo or mixed cargo calculated from samples processed as in Supplementary Fig . 16b ( * P 0 . 001 , * * P 0 . 0001 , n = 4 6 ) . ( Supplementary Figure 16 Effects of starvation on hepatic lipid droplets . a , Mouseliverelectron micrographs . Arrows : Lipid - containing double membrane vesicles . b , Electron micrographs of autophagic vacuoles ( AVs ) isolated from mice fed or starved for the indicated time periods . The percentages of double membrane vesicles containing lipid in isolated AVs from 3 - 4 mice per condition are shown ( * P < 0 . 01 as compared to fractions from fed animals ) . ( * ) indicate lipid )

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, Percentages of autophagic vacuoles (AVs) containing only lipid, other cargo or mixed cargo calculated from samples processed as in Supplementary Fig. 16b (*P  0.001, **P  0.0001, n = 46). (Supplementary Figure 16 Effects of starvation on hepatic lipid droplets. a, Mouseliverelectron micrographs. Arrows: Lipid-containing double membrane vesicles. b, Electron micrographs of autophagic vacuoles (AVs) isolated from mice fed or starved for the indicated time periods. The percentages of double membrane vesicles containing lipid in isolated AVs from 3-4 mice per condition are shown (*P<0.01 as compared to fractions from fed animals). (*) indicate lipid)


c , Immunoblots of Hom , AV and lysosomes ( Lys ) . L - 1 , LAMP1 .

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

c, Immunoblots of Hom, AV and lysosomes (Lys). L-1, LAMP1.


d , Immunoblots of liver Hom and LDs from HFD - fed mice .

d, Immunoblots of liver Hom and LDs from HFD-fed mice.


e , Liver homogenate immunoblots . PDI , protein disulphide isomerase .

e, Liver homogenate immunoblots. PDI, protein disulphide isomerase.


f , Total hepatic TG and cholesterol content ( * P 0 . 01 , * * P 0 . 00001 , n = 817 ) .

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

f, Total hepatic TG and cholesterol content (*P 0.01, **P 0.00001, n = 817).


g , Hepatic TG concentration ( * P 0 . 05 , n = 3 ) .

g, Hepatic TG concentration (*P  0.05, n = 3).


h , Percentage of cellular cholesterol in lysosomes ( * P 0 . 02 , n = 4 ) .

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

h, Percentage of cellular cholesterol in lysosomes (*P  0.02, n = 4).


i , Immunoblots of homogenates and isolated LDs . Error bars , s . e . m .

i, Immunoblots of homogenates and isolated LDs. Error bars, s.e.m.


The rate of glucose uptake ( a , e ) , lactate released after 4 h of incubation ( b , f ) , glycolytic flux ( c , g ) and pentose - phosphate flux ( d , h ) were assessed in MEF and mouse cortical neurons in primary culture , as indicated , obtained from either WT or Pink1 KO ( Pink1 − / − ) mice .

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

The rate of glucose uptake (a,e), lactate released after 4 h of incubation (b,f), glycolytic flux (c,g) and pentose-phosphate flux (d,h) were assessed in MEF and mouse cortical neurons in primary culture, as indicated, obtained from either WT or Pink1 KO (Pink1−/−) mice.


Blood concentrations of glucose ( i ) and lactate ( j ) , and the rate of 14C - lactate appearance in the blood from acutely injected [ U - 14C ] glucose in the tail vein ( k ) of WT and Pink1 KO mice were analysed . Data are expressed as mean ± s . e . m . * P0 . 05 ( Student ' s t - test ; n = 3 - 4 independent experiments ) .

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

Blood concentrations of glucose (i) and lactate (j), and the rate of 14C-lactate appearance in the blood from acutely injected [U-14C]glucose in the tail vein (k) of WT and Pink1 KO mice were analysed. Data are expressed as mean±s.e.m. *P0.05 (Student's t-test; n=3-4 independent experiments).


Quantitative real - time PCR ( RT - qPCR analysis of the relative mRNA abundances of Glut1 , Glut3 , Hk2 and Gapdh in WT and Pink1 KO miceMEF ( a ) ß - Actin mRNA was used for normalization , and the Pink1 KO data are expressed as the change relative to WT .

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

Quantitative real-time PCR (RT-qPCR analysis of the relative mRNA abundances of Glut1, Glut3, Hk2 and Gapdh in WT and Pink1 KO miceMEF (a) ß-Actin mRNA was used for normalization, and the Pink1 KO data are expressed as the change relative to WT.


Western blot analysis of the protein abundances of PINK1 , GLUT1 , GLUT3 ( not detectable in MEF ) , HK2 , GAPDH and HIF1α in WT and Pink1 - null mice MEF ( b ) ß - Actin was used as loading control .

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

Western blot analysis of the protein abundances of PINK1, GLUT1, GLUT3 (not detectable in MEF), HK2, GAPDH and HIF1α in WT and Pink1-null mice MEF (b) ß-Actin was used as loading control.


Quantitative real - time PCR ( RT - qPCR analysis of the relative mRNA abundances of Glut1 , Glut3 , Hk2 and Gapdh in WT and Pink1 KO neurons ( c ) . ß - Actin mRNA was used for normalization , and the Pink1 KO data are expressed as the change relative to WT .

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

Quantitative real-time PCR (RT-qPCR analysis of the relative mRNA abundances of Glut1, Glut3, Hk2 and Gapdh in WT and Pink1 KO neurons (c). ß-Actin mRNA was used for normalization, and the Pink1 KO data are expressed as the change relative to WT.


Western blot analysis of the protein abundances of PINK1 , GLUT1 , GLUT3 ( not detectable in MEF ) , HK2 , GAPDH and HIF1α in WT and Pink1 - null neurons ( d ) ; ß - Actin was used as loading control .

Western blot analysis of the protein abundances of PINK1, GLUT1, GLUT3 (not detectable in MEF), HK2, GAPDH and HIF1α in WT and Pink1-null neurons (d); ß-Actin was used as loading control.


( e ) Western blot against PINK1 , GLUT1 , GLUT3 and HIF1α in WT and Pink1 KO mice skeletal muscle samples ; α - Tubulin was used as loading control . Only representative western blots are shown per condition ; the replicates and the semi - quantitative estimation of the band intensities are shown in the Supplementary Information . The mean ± s . e . m . values of the mRNA data were calculated from the fold change of each ß - Actin - normalized transcript abundance in the Pink1 KO samples versus that in the WT . Thus , in all cases , the WT values were considered = 1 . 00 . * P0 . 05 versus the corresponding WT ( Student ' s t - test ; n = 3 - 4 independent experiments ) .

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

(e) Western blot against PINK1, GLUT1, GLUT3 and HIF1α in WT and Pink1 KO mice skeletal muscle samples; α-Tubulin was used as loading control. Only representative western blots are shown per condition; the replicates and the semi-quantitative estimation of the band intensities are shown in the Supplementary Information. The mean±s.e.m. values of the mRNA data were calculated from the fold change of each ß-Actin-normalized transcript abundance in the Pink1 KO samples versus that in the WT. Thus, in all cases, the WT values were considered=1.00. *P0.05 versus the corresponding WT (Student's t-test; n=3-4 independent experiments).


( a ) RT - qPCR analysis of Hif1α mRNA abundance in MEF transfected with siHif1α , or siControl ( 100 nM ) , after 72 h .

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

(a) RT-qPCR analysis of Hif1α mRNA abundance in MEF transfected with siHif1α, or siControl (100 nM), after 72 h.


( b ) siHif1α effectively knockdowns expressed Hif1α cDNA WT , but not a Hif1α cDNA silent mutant form refractory to siHif1α ( Hif1α cDNA REFR . ) in mice MEF .

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

(b) siHif1α effectively knockdowns expressed Hif1α cDNA WT, but not a Hif1α cDNA silent mutant form refractory to siHif1α (Hif1α cDNA REFR.) in mice MEF.


( c ) RT - qPCR analysis of the relative mRNA abundances of Glut1 , Glut3 , Hk2 and Gapdh in WT and Pink1 KO mice MEF transfected with siControl or siHif1α . ß - Actin mRNA was used for normalization , and all data are expressed as the change relative to WT - siControl .

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

(c) RT-qPCR analysis of the relative mRNA abundances of Glut1, Glut3, Hk2 and Gapdh in WT and Pink1 KO mice MEF transfected with siControl or siHif1α. ß-Actin mRNA was used for normalization, and all data are expressed as the change relative to WT-siControl.


( d ) Western blot against HIF1α in WT and in Pink1 KO mice MEF transfected with siHif1α or siControl shows the efficacy of the siHif1α at knocking down endogenous HIF1α ; ß - actin was used as loading control . As shown , siHif1α partially prevented the increase in GLUT1 , HK2 and GAPDH protein abundances .

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

(d) Western blot against HIF1α in WT and in Pink1 KO mice MEF transfected with siHif1α or siControl shows the efficacy of the siHif1α at knocking down endogenous HIF1α; ß-actin was used as loading control. As shown, siHif1α partially prevented the increase in GLUT1, HK2 and GAPDH protein abundances.


The increased rate of lactate released after 4 h of incubation ( e ) and glycolytic flux ( f ) observed in the Pink1 KO MEF was prevented by siHif1α .

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

The increased rate of lactate released after 4 h of incubation (e) and glycolytic flux (f) observed in the Pink1 KO MEF was prevented by siHif1α.


siHif1α was also effective at preventing the endogenous increased HIF1α protein ( g ) , as well as the increased glycolytic flux ( h ) observed in Pink1 KO primary neurons . Only representative western blots are shown ; the replicates and the semi - quantitative estimation of the band intensities are shown in the Supplementary Information .

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 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

siHif1α was also effective at preventing the endogenous increased HIF1α protein (g), as well as the increased glycolytic flux (h) observed in Pink1 KO primary neurons. Only representative western blots are shown; the replicates and the semi-quantitative estimation of the band intensities are shown in the Supplementary Information.


( a ) Mitochondrial respiratory chain complexes activity values normalized to citrate synthase ( CS ) , and ( b ) mitochondrial inner membrane potential ( Δψm ) in WT and Pink1 KO mice MEF .

O O O O O O O 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

(a) Mitochondrial respiratory chain complexes activity values normalized to citrate synthase (CS), and (b) mitochondrial inner membrane potential (Δψm) in WT and Pink1 KO mice MEF.


( c ) Western blot against PDK1 and HIF1α in WT and Pink1 KO MEF transfected with siHif1α ( or its siControl ) ; ß - actin was used as loading control .

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

(c) Western blot against PDK1 and HIF1α in WT and Pink1 KO MEF transfected with siHif1α (or its siControl); ß-actin was used as loading control.


d ) PDH complex activity in WT and Pink1 KO MEF transfected with siHif1α or siControl .

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

d) PDH complex activity in WT and Pink1 KO MEF transfected with siHif1α or siControl.


Rate of whole - cell H2O2 ( e ) and mitochondrial O2 · − ( f ) detection in WT and Pink1 KO

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

Rate of whole-cell H2O2 (e) and mitochondrial O2·− (f) detection in WT and Pink1 KO MEF


( g ) Detection of mitochondrial O2 · − using the mitochondrial - specific probe MitoSox in MEF incubated in the absence ( − ) or in the presence ( + ) of the plasma membrane permeable GSH - EE , or by expressing mitochondrial - tagged forms of glutamate - cysteine ligase , ( + mitoGCL ; empty vector , pIRES2 - EGFP , denoted as − ) or catalase ( + mitoCAT ; empty vector denoted as − ) , shows effective mitochondrial ROS removal .

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

(g) Detection of mitochondrial O2·− using the mitochondrial-specific probe MitoSox in MEF incubated in the absence (−) or in the presence (+) of the plasma membrane permeable GSH-EE, or by expressing mitochondrial-tagged forms of glutamate-cysteine ligase, (+mitoGCL; empty vector, pIRES2-EGFP, denoted as −) or catalase (+ mitoCAT; empty vector denoted as −), shows effective mitochondrial ROS removal.


( h ) Western blot against HIF1α in Pink1 KO MEF incubated with ( or without ) GSH - EE , or transfected with empty vector , mitoGCL or mitoCAT , showing reduction in HIF1α protein abundance by the treatments that remove mitochondrial ROS ; α - TUBULIN was used as loading control . Only representative western blots are shown ; the replicates and the semi - quantitative estimation of the band intensities are shown in the Supplementary Information . Data are expressed as mean ± s . e . m . * P0 . 05 ( Student ' s t - test ; n = 3 - 4 independent experiments ) .

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 B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 against HIF1α in Pink1 KO MEF incubated with (or without) GSH-EE, or transfected with empty vector, mitoGCL or mitoCAT, showing reduction in HIF1α protein abundance by the treatments that remove mitochondrial ROS; α-TUBULIN was used as loading control. Only representative western blots are shown; the replicates and the semi-quantitative estimation of the band intensities are shown in the Supplementary Information. Data are expressed as mean±s.e.m. *P0.05 (Student's t-test; n=3-4 independent experiments).


( a ) Proportion of BrdU incorporation into DNA and cell cycle phases , and ( b ) cell growth in WT and Pink1 KO MEF after 72 h in the absence ( denoted as - 2DG ) or in the presence ( + 2DG ) of the glucose - metabolizing inhibitor , 2 - deoxyglucose ( 2DG , 1 mM ) .

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 O O O O O O O

(a) Proportion of BrdU incorporation into DNA and cell cycle phases, and (b) cell growth in WT and Pink1 KO MEF after 72 h in the absence (denoted as -2DG) or in the presence (+2DG) of the glucose-metabolizing inhibitor, 2-deoxyglucose (2DG, 1 mM).


( c ) Proportions of BrdU incorporation into DNA and cell cycle phases , and ( d ) cell growth in WT and Pink1 KO MEF 72 h after transfection with siHif1α ( + siHif1α ) or siControl ( denoted as - siHif1α ) .

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

(c) Proportions of BrdU incorporation into DNA and cell cycle phases, and (d) cell growth in WT and Pink1 KO MEF 72 h after transfection with siHif1α (+siHif1α) or siControl (denoted as -siHif1α).


( e ) Ki67 , Sox2 and DAPI immunostaining in the subventricular zone of WT and Pink1 KO mouse brain , and the corresponding quantification . * P0 . 05 ; # P0 . 05 versus all other conditions ( analysis of variance followed by Bonferroni test ; n = 3 - 4 independent experiments ) . All data are expressed as mean ± s . e . m . Scale bar , 100 μm .

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

(e) Ki67, Sox2 and DAPI immunostaining in the subventricular zone of WT and Pink1 KO mouse brain, and the corresponding quantification. *P0.05; #P0.05 versus all other conditions (analysis of variance followed by Bonferroni test; n=3-4 independent experiments). All data are expressed as mean±s.e.m. Scale bar, 100 μm.


a ) Rate of mitochondrial O2 · - detection in WT and Pink1 KO mice primary neurons .

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

a) Rate of mitochondrial O2·- detection in WT and Pink1 KO mice primary neurons.


( b ) Proportion of BrdU incorporation into DNA of WT and Pink1 KO neurons .

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

(b) Proportion of BrdU incorporation into DNA of WT and Pink1 KO neurons.


( c ) Proportion of apoptosis in WT and Pink1 KO neurons , as assessed by the proportion of annexin V - positive / 7 - AAD - negative cells , determined by flow cytometry . Data are expressed as mean ± s . e . m . * P0 . 05 ( Student ' s t - test ; n = 3 - 4 independent experiments ) .

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

(c) Proportion of apoptosis in WT and Pink1 KO neurons, as assessed by the proportion of annexin V-positive/7-AAD-negative cells, determined by flow cytometry. Data are expressed as mean±s.e.m. *P0.05 (Student's t-test; n=3-4 independent experiments).


A - E Double - immunolabeling of Sororin ( green ) and SYCP3 ( red ) on spread spermatocytes at ( A ) zygotene ( Zyg . ) , ( B ) pachytene ( Pac . ) , ( C ) diplotene ( Dip . ) , ( D ) late diplotene ( L . dip . ) , and ( E ) prometaphase I ( ProM I ) .

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A-E Double-immunolabeling of Sororin (green) and SYCP3 (red) on spread spermatocytes at (A) zygotene (Zyg.), (B) pachytene (Pac.), (C) diplotene (Dip.), (D) late diplotene (L. dip.), and (E) prometaphase I (ProM I).


F - H Double - immunolabeling of Sororin ( green ) and SYCP1 ( pseudocoloured in pink ) on spread spermatocytes at ( F ) zygotene , ( G ) pachytene , and ( H ) diplotene .

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F-H Double-immunolabeling of Sororin (green) and SYCP1 (pseudocoloured in pink) on spread spermatocytes at (F) zygotene, (G) pachytene, and (H) diplotene.


I - K Double - immunolabeling of Sororin ( green ) and HORMAD1 ( pseudocoloured in blue ) on spread spermatocytes at ( I ) zygotene , ( J ) pachytene , and ( K ) diplotene . The sex bivalent ( XY ) and their pseudoautosomal regions ( arrows ) , as well as the round body ( arrowheads ) are indicated . Scale bar : 10 µm .

I-K Double-immunolabeling of Sororin (green) and HORMAD1 (pseudocoloured in blue) on spread spermatocytes at (I) zygotene, (J) pachytene, and (K) diplotene. The sex bivalent (XY) and their pseudoautosomal regions (arrows), as well as the round body (arrowheads) are indicated. Scale bar: 10 µm.


A , C Double - immunolabeling of Sororin ( green ) and SYCP3 ( red ) on spread spermatocytes at ( A ) early zygotene ( E . zyg . ) and ( C ) late zygotene ( L . zyg . ) . B , D Double - immunolabeling of SMC3 ( pseudocoloured in cyan ) and SYCP3 ( red ) on spread spermatocytes at ( B ) early zygotene and ( D ) late zygotene .

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

A,C Double-immunolabeling of Sororin (green) and SYCP3 (red) on spread spermatocytes at (A) early zygotene (E. zyg.) and (C) late zygotene (L. zyg.).B,D Double-immunolabeling of SMC3 (pseudocoloured in cyan) and SYCP3 (red) on spread spermatocytes at (B) early zygotene and (D) late zygotene.


E Double - immunolabeling of Sororin ( green ) and REC8 - myc ( pseudocoloured in purple ) on a spread mid zygotene spermatocyte . The sex univalents ( X , Y ) are indicated . Scale bar : 10 µm .

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

E Double-immunolabeling of Sororin (green) and REC8-myc (pseudocoloured in purple) on a spread mid zygotene spermatocyte. The sex univalents (X,Y) are indicated. Scale bar: 10 µm.


Double - immunolabeling of Sororin ( green ) and SYCP3 ( red ) on spread pachytene - like ( Pac . - like ) and zygotene - like ( Zyg . - like ) spermatocytes of different genotypes . Unsynapsed axial elements of sex chromosomes ( X , Y ) are indicated . Arrowheads indicate nucleolar - associated round bodies . Scale bar : 10 µm .

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

Double-immunolabeling of Sororin (green) and SYCP3 (red) on spread pachytene-like (Pac.-like) and zygotene-like (Zyg.-like) spermatocytes of different genotypes. Unsynapsed axial elements of sex chromosomes (X,Y) are indicated. Arrowheads indicate nucleolar-associated round bodies. Scale bar: 10 µm.


Double - immunolabeling of Sororin ( green ) and SYCP3 ( red ) , and counterstaining of the chromatin with DAPI ( blue ) on spread spermatocytes at ( A ) metaphase I ( M I ) , ( G ) anaphase I ( A I ) , ( H ) interkinesis ( Int . ) , ( I ) prophase II ( Pro . II ) , and ( J ) metaphase II ( M II ) . The sex bivalent ( XY ) is indicated . Arrows indicate the centriolar area at cell poles . Arrowheads indicate the Sororin labeling at the centromere of Y chromosome . B - F Selected metaphase I autosomal ( B and C ) and sex ( E and F ) bivalents . The sex chromosomes ( X , Y ) are indicated . K , L Enlarged metaphase II centromeres . Scale bar : 10 µm .

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

Double-immunolabeling of Sororin (green) and SYCP3 (red), and counterstaining of the chromatin with DAPI (blue) on spread spermatocytes at (A) metaphase I (M I), (G) anaphase I (A I), (H) interkinesis (Int.), (I) prophase II (Pro. II), and (J) metaphase II (M II). The sex bivalent (XY) is indicated. Arrows indicate the centriolar area at cell poles. Arrowheads indicate the Sororin labeling at the centromere of Y chromosome.B-F Selected metaphase I autosomal (B and C) and sex (E and F) bivalents. The sex chromosomes (X,Y) are indicated.K,L Enlarged metaphase II centromeres. Scale bar: 10 µm.


Double - immunolabeling of Sororin ( green ) and kinetochores ( ACA serum , red ) , and counterstaining of the chromatin with DAPI ( blue ) on spread spermatocytes at ( A ) metaphase I ( M I ) , ( B ) metaphase II ( M II ) , and ( C ) anaphase II ( A II ) . Arrowheads indicate the cell poles . The centromere of the Y chromosome and chromatids ( Y ) is indicated . Scale bar : 10 µm .

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

Double-immunolabeling of Sororin (green) and kinetochores (ACA serum, red), and counterstaining of the chromatin with DAPI (blue) on spread spermatocytes at (A) metaphase I (M I), (B) metaphase II (M II), and (C) anaphase II (A II). Arrowheads indicate the cell poles. The centromere of the Y chromosome and chromatids (Y) is indicated. Scale bar: 10 µm.


Double - immunolabeling of Sororin ( green ) and SYCP3 ( red ) , and counterstaining of the chromatin with DAPI ( blue ) on spread Sgo2 - / - spermatocytes at ( A ) pachytene ( Pac . ) , ( B ) diplotene ( Dip . ) , ( C ) metaphase I ( M I ) , and ( D ) metaphase II - like ( M II - like ) . The sex bivalent ( XY ) is indicated . Arrowheads indicate round bodies . Arrows in ( A ) indicate the pseudoautomal region in the sex bivalent , and the centriolar area at cell poles in ( C , D ) . Scale bar : 10 µm .

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

Double-immunolabeling of Sororin (green) and SYCP3 (red), and counterstaining of the chromatin with DAPI (blue) on spread Sgo2-/- spermatocytes at (A) pachytene (Pac.), (B) diplotene (Dip.), (C) metaphase I (M I), and (D) metaphase II-like (M II-like). The sex bivalent (XY) is indicated. Arrowheads indicate round bodies. Arrows in (A) indicate the pseudoautomal region in the sex bivalent, and the centriolar area at cell poles in (C,D). Scale bar: 10 µm.


Double - immunolabeling of Sororin ( green ) and SYCP3 ( red ) on spread cultured wild - type control ( WT Control ) , and okadaic acid treated wild - type ( WT + OA ) and Smc1β - / - ( Smc1β - / - + OA ) spermatocytes . A - B WT control cultured spermatocytes in ( A ) late zygotene ( L . zyg . ) and ( B ) metaphase I ( M I ) . Arrows indicate the cell poles . C - D WT cultured spermatocytes treated with okadaic acid in ( C ) late zygotene ( L . zyg . ) and ( D ) metaphase I - like ( M I - like ) . E - F Smc1β - / - cultured spermatocytes treated with okadaic acid in ( E ) pachytene - like ( Pac . - like ) and ( F ) metaphase I - like ( M I - like ) . Arrowheads indicate nucleolar - associated round bodies . The unsynapsed axial elements of the sex chromosomes ( X , Y ) in prophase I , as well as the sex bivalent ( XY ) in metaphases I are indicated . Scale bar : 10 µm .

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

Double-immunolabeling of Sororin (green) and SYCP3 (red) on spread cultured wild-type control (WT Control), and okadaic acid treated wild-type (WT + OA) and Smc1β-/- (Smc1β-/- + OA) spermatocytes.A-B WT control cultured spermatocytes in (A) late zygotene (L. zyg.) and (B) metaphase I (M I). Arrows indicate the cell poles.C-D WT cultured spermatocytes treated with okadaic acid in (C) late zygotene (L. zyg.) and (D) metaphase I-like (M I-like).E-F Smc1β-/- cultured spermatocytes treated with okadaic acid in (E) pachytene-like (Pac.-like) and (F) metaphase I-like (M I-like). Arrowheads indicate nucleolar-associated round bodies. The unsynapsed axial elements of the sex chromosomes (X,Y) in prophase I, as well as the sex bivalent (XY) in metaphases I are indicated. Scale bar: 10 µm.


C - E . Logistic statistical model based on the ( C ) single cell sequencing , ( D ) ImageStream ( E ) analysis or automated FISH of RPE - 1 cells in untreated condition ( blue circles ) or treated with Auxin for 48 h ( red squares ) . Error bars represent the SEM based on the number of cells analyzed Dashed lines indicate the means of aneuploidy rates in untreated ( blue line ) or Auxin - treated ( red line ) condition . Red asterisks ( IAA ) and Blue ( Untreated ) indicate significance over the respective mean using a binomial test . * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 ; * * * * p < 0 . 0001 .

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C-E. Logistic statistical model based on the (C) single cell sequencing, (D) ImageStream (E) analysis or automated FISH of RPE-1 cells in untreated condition (blue circles) or treated with Auxin for 48 h (red squares). Error bars represent the SEM based on the number of cells analyzed Dashed lines indicate the means of aneuploidy rates in untreated (blue line) or Auxin-treated (red line) condition. Red asterisks (IAA) and Blue (Untreated) indicate significance over the respective mean using a binomial test. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.


F . Table summarizing whole - chromosome aneuploidy ( fold over the mean ) using the indicated methods to measure chromosome mis - segregation rate . Bold numbers represent a statistically significant difference from the mean for each method . Orange to dark red gradient highlights chromosomes that mis - segregate ( with at least one method ) at a significantly higher rate compared to the mean level ( weak chromosomes ) . Light green to dark green gradient highlights chromosomes that mis - segregate ( with at least one method ) at a significantly lower rate compared to the mean ( stronger chromosomes ) . Data obtained with only one method were excluded . * = centromere 4 probe with image stream was reported to lead to non - specific signal ( Worrall et al , 2018 ) . Im . St . = image stream ; Seq = single cell sequencing .

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F. Table summarizing whole-chromosome aneuploidy (fold over the mean) using the indicated methods to measure chromosome mis-segregation rate. Bold numbers represent a statistically significant difference from the mean for each method. Orange to dark red gradient highlights chromosomes that mis-segregate (with at least one method) at a significantly higher rate compared to the mean level (weak chromosomes). Light green to dark green gradient highlights chromosomes that mis-segregate (with at least one method) at a significantly lower rate compared to the mean (stronger chromosomes). Data obtained with only one method were excluded. * = centromere 4 probe with image stream was reported to lead to non-specific signal (Worrall et al, 2018). Im. St. = image stream; Seq = single cell sequencing.


B - C . ( B ) Micronuclei frequency formation by live cell imaging in untreated ( NT ) and Auxin - treated ( IAA ) condition . ( C ) Bar graph represents the type of chromosome mis - segregation ( independently of the dCas9 signal ) observed in the indicated conditions , with ( IAA ) or without ( NT ) auxin , in the cell lines expressing dCas9 mScarlet - I and sgRNA targeting chromosome 1 or 3 . Error bars represent the SEM of four independent experiments in which cells labeled for chromosome 1 and 3 were analyzed together ( n = 45 - 149 cells ) .

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B-C. (B) Micronuclei frequency formation by live cell imaging in untreated (NT) and Auxin-treated (IAA) condition. (C) Bar graph represents the type of chromosome mis-segregation (independently of the dCas9 signal) observed in the indicated conditions, with (IAA) or without (NT) auxin, in the cell lines expressing dCas9 mScarlet-I and sgRNA targeting chromosome 1 or 3. Error bars represent the SEM of four independent experiments in which cells labeled for chromosome 1 and 3 were analyzed together (n = 45 - 149 cells).


D . Representative live cell imaging of RPE - 1 cells dCas9 mScarlet - I with sgRNA targeting chromosome 1 ( red dots ) starting from metaphase and showing example of correct ( untreated ) or mis - aligned chromosome ( auxin ) leading to the formation of a micronucleus . Yellow arrows mark mis - aligned - and micronucleus - containing chromosome 1 . Cells were imaged every five minutes . The numbers indicate the time point at which the presented images were taken . Cells were fixed at the end of the movie to detect Cas9 with an antibody . Scale bar represents 10 µm .

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

D. Representative live cell imaging of RPE-1 cells dCas9 mScarlet-I with sgRNA targeting chromosome 1 (red dots) starting from metaphase and showing example of correct (untreated) or mis-aligned chromosome (auxin) leading to the formation of a micronucleus. Yellow arrows mark mis-aligned- and micronucleus-containing chromosome 1. Cells were imaged every five minutes. The numbers indicate the time point at which the presented images were taken. Cells were fixed at the end of the movie to detect Cas9 with an antibody. Scale bar represents 10 µm.


E . Bar graph representing the proportion of chromosome mis - segregation observed in both cell lines after auxin addition . Only movies in which the dCas9 mScarlet - I dots were clearly visible during the whole division were taken into consideration for the analysis ( N = 6 - 10 cells ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Bar graph representing the proportion of chromosome mis-segregation observed in both cell lines after auxin addition. Only movies in which the dCas9 mScarlet-I dots were clearly visible during the whole division were taken into consideration for the analysis (N = 6 - 10 cells).


F . Representative images of mitotic errors leading to aneuploidy in fixed cells after CENP - A depletion ( 28 h auxin ) . Chromosomes are stained using whole ( X ) or centromeric ( 6 ) FISH probes . Yellow arrows mark lagging chromosomes . Scale bar represents 5 µm . G - H . Bars represent the ( G ) proportion of chromosome mis - segregation and ( H ) the type of the different chromosome mis - segregations observed . Error bars represent the SEM of two replicates . n > 66 mis - segregation events . Paired t test lagging chromosomes vs mis - alignment chromosomes , * p = 0 . 0275 .

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F. Representative images of mitotic errors leading to aneuploidy in fixed cells after CENP-A depletion (28 h auxin). Chromosomes are stained using whole (X) or centromeric (6) FISH probes. Yellow arrows mark lagging chromosomes. Scale bar represents 5 µm. G-H. Bars represent the (G) proportion of chromosome mis-segregation and (H) the type of the different chromosome mis-segregations observed. Error bars represent the SEM of two replicates. n > 66 mis-segregation events. Paired t test lagging chromosomes vs mis-alignment chromosomes, *p = 0.0275.


Bar plot showing the mean of ( B ) centromere length ( n = 4 ) as determined by whole genome sequencing . Error bars represent the SEM of four independent experiments . Acrocentric chromosomes 13 , 14 , 21 and 22 were marked by a line as we could not assign the respective reads . Dashed lines indicate the mean . Bars were labeled with asterisks according to the significance of their difference from the mean ( t - test ) . * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 ; * * * * p < 0 . 0001 . Bars represent the sum of the

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

Bar plot showing the mean of (B) centromere length (n = 4) as determined by whole genome sequencing. Error bars represent the SEM of four independent experiments. Acrocentric chromosomes 13, 14, 21 and 22 were marked by a line as we could not assign the respective reads. Dashed lines indicate the mean. Bars were labeled with asterisks according to the significance of their difference from the mean (t-test). *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001. Bars represent the sum of the length


Bar plot showing the mean of ( C ) CENP - B boxes counts ( n = 4 ) as determined by whole genome sequencing . Error bars represent the SEM of four independent experiments . Acrocentric chromosomes 13 , 14 , 21 and 22 were marked by a line as we could not assign the respective reads . Dashed lines indicate the mean . Bars were labeled with asterisks according to the significance of their difference from the mean ( t - test ) . * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 ; * * * * p < 0 . 0001 . Bars represent the sum of counts of different HOR arrays CENP - B boxes counts are normalized to the average number of mapped reads in each replicate .

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

Bar plot showing the mean of (C) CENP-B boxes counts (n = 4) as determined by whole genome sequencing. Error bars represent the SEM of four independent experiments. Acrocentric chromosomes 13, 14, 21 and 22 were marked by a line as we could not assign the respective reads. Dashed lines indicate the mean. Bars were labeled with asterisks according to the significance of their difference from the mean (t-test). *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001. Bars represent the sum of counts of different HOR arrays CENP-B boxes counts are normalized to the average number of mapped reads in each replicate.


D . Schematic using representative images of the mFISH labeling followed by CENP - B box FISH method used to identify and quantify centromere specific CENP - B boxes signal in ( E ) . Scale bar represents 10 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

D. Schematic using representative images of the mFISH labeling followed by CENP-B box FISH method used to identify and quantify centromere specific CENP-B boxes signal in (E). Scale bar represents 10 µm.


E . Box and whisker plots of normalized CENP - B boxes intensity over the mean on metaphase spread from three independent experiments ( n > 50 cells ) using the Tukey plot . One - way ANOVA with post - hoc Tukey ' s multiple comparison test shows high diversity between chromosomes . t - test against the mean was used to estimate the statistical significance for each chromosome . * * p < 0 . 01 ; * * * p < 0 . 001 ; * * * * p < 0 . 0001 . .

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E. Box and whisker plots of normalized CENP-B boxes intensity over the mean on metaphase spread from three independent experiments (n>50 cells) using the Tukey plot. One-way ANOVA with post-hoc Tukey's multiple comparison test shows high diversity between chromosomes. t-test against the mean was used to estimate the statistical significance for each chromosome. **p < 0.01; ***p < 0.001; ****p < 0.0001..


Scatter plot showing a non - significant or significant negative correlation between the mean of ( F ) centromere length ( n = 4 ) and mis - segregation rate ( from Fig . 1F ) ( r = Spearman rank coefficient ) . Lines represent the linear regression with a 95 % confidence interval . Data from chr 13 , 14 , 21 and 22 were excluded from the analysis .

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

Scatter plot showing a non-significant or significant negative correlation between the mean of (F) centromere length (n = 4) and mis-segregation rate (from Fig. 1F) (r = Spearman rank coefficient). Lines represent the linear regression with a 95% confidence interval. Data from chr 13, 14, 21 and 22 were excluded from the analysis.


Scatter plot showing a non - significant or significant negative correlation between the mean of ( G ) centromere CENP - B boxes FISH signal ( n > 50 cells ) and mis - segregation rate , respectively ( r = Spearman rank coefficient ) . Lines represent the linear regression with a 95 % confidence interval . Data from chr 13 , 14 , 21 and 22 were excluded from the analysis .

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

Scatter plot showing a non-significant or significant negative correlation between the mean of (G) centromere CENP-B boxes FISH signal (n>50 cells) and mis-segregation rate, respectively (r = Spearman rank coefficient). Lines represent the linear regression with a 95% confidence interval. Data from chr 13, 14, 21 and 22 were excluded from the analysis.


B . Representative immunofluorescence FISH ( IF - FISH ) images to measure CENP - B intensity at specific chromosome in untreated cells . Scale bar represents 5 µm .

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B. Representative immunofluorescence FISH (IF-FISH) images to measure CENP-B intensity at specific chromosome in untreated cells. Scale bar represents 5 µm.


C . Bar plot showing the normalized CENP - B fluorescence intensity at every chromosome over the mean on each metaphase spread ( n > 37 per chromosome ) ± SEM . Acrocentric chromosomes 13 , 14 , 21 and 22 were not analyzed and are marked by a line . One - way ANOVA with post - hoc Tukey ' s multiple comparison test shows high diversity between chromosomes . t - test against the mean was used to estimate the statistical significance for each chromosome . * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 ; * * * * p < 0 . 0001 .

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

C. Bar plot showing the normalized CENP-B fluorescence intensity at every chromosome over the mean on each metaphase spread (n > 37 per chromosome) ± SEM. Acrocentric chromosomes 13, 14, 21 and 22 were not analyzed and are marked by a line. One-way ANOVA with post-hoc Tukey's multiple comparison test shows high diversity between chromosomes. t-test against the mean was used to estimate the statistical significance for each chromosome. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.


D . Scatter plot showing a significant negative correlation between the mean of CENP - B IF - FISH signal ( n > 37 ) and the mis - segregation rate ( r = Spearman rank coefficient ) . The lines represent linear regression with 95 % confidence band .

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D. Scatter plot showing a significant negative correlation between the mean of CENP-B IF-FISH signal (n > 37) and the mis-segregation rate (r = Spearman rank coefficient). The lines represent linear regression with 95% confidence band.


E - F . Bar graphs report the sum of the normalized reads counts of different HOR arrays representing CENP - C binding following CUT & RUN , sequencing and centromere mapping . Cells were treated for 6 h with IAA to deplete CENP - A . Error bars represent the SEM of three independent experiments and the dashed line represents the mean . Acrocentric chromosomes 13 - 14 , 21 and 22 were marked by a line . t - test against the mean was used to estimate the statistical significance for each chromosome . * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 .

O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Bar graphs report the sum of the normalized reads counts of different HOR arrays representing CENP-C binding following CUT&RUN, sequencing and centromere mapping. Cells were treated for 6 h with IAA to deplete CENP-A. Error bars represent the SEM of three independent experiments and the dashed line represents the mean. Acrocentric chromosomes 13-14, 21 and 22 were marked by a line. t-test against the mean was used to estimate the statistical significance for each chromosome. *p < 0.05; **p < 0.01; ***p < 0.001.


G . Scatter plot showing a significant positive correlation between the mean of CENP - C reads ( n = 3 ) and CENP - B reads ( n = 3 ) ( r = Spearman rank coefficient ) . Data from chr 1 , 5 and 19 were excluded from the analysis in addition to chr 13 , 14 , 21 and 22 to better assess correlation without the FISH correction The lines represent linear regression with a 95 % confidence interval .

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G. Scatter plot showing a significant positive correlation between the mean of CENP-C reads (n = 3) and CENP-B reads (n = 3) (r = Spearman rank coefficient). Data from chr 1, 5 and 19 were excluded from the analysis in addition to chr 13, 14, 21 and 22 to better assess correlation without the FISH correction The lines represent linear regression with a 95% confidence interval.


H . Scatter plot showing a significant negative correlation between the mean of CENP - C reads ( n = 3 ) and the mis - segregation rate ( r = Spearman rank coefficient ) . The lines represent linear regression with 95 % confidence interval . Data from chr 13 , 14 , 21 and 22 were excluded from the analysis . The lines represent linear regression with a 95 % confidence interval .

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H. Scatter plot showing a significant negative correlation between the mean of CENP-C reads (n = 3) and the mis-segregation rate (r = Spearman rank coefficient). The lines represent linear regression with 95% confidence interval. Data from chr 13, 14, 21 and 22 were excluded from the analysis. The lines represent linear regression with a 95% confidence interval.


B . Representative image of centromere 11 ( red ) and centromere X ( green ) FISH on RPE - 1 CENP - AAID CENP - B KO mutant in control ( top ) or IAA treated condition ( bottom ) . Scale bar represents 5 µm . Bar graphs show automatic FISH quantification of chromosome mis - segregation in the indicated cell lines with and without IAA addition for 24 h . Error bars represent the SEM of three independent experiments ( n > 200 cells per experiment ) . Dunnett ' s multiple comparisons test was used to compare conditions . Grey lines separate independent experiments . Fold changes between grey and red bars are also indicated .

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B. Representative image of centromere 11 (red) and centromere X (green) FISH on RPE-1 CENP-AAID CENP-B KO mutant in control (top) or IAA treated condition (bottom). Scale bar represents 5 µm. Bar graphs show automatic FISH quantification of chromosome mis-segregation in the indicated cell lines with and without IAA addition for 24 h. Error bars represent the SEM of three independent experiments (n> 200 cells per experiment). Dunnett's multiple comparisons test was used to compare conditions. Grey lines separate independent experiments. Fold changes between grey and red bars are also indicated.


C . Schematic of the centromere strength model with the microtubule destabilizer BAL27862 compound . D . Representative image of centromere 17 ( red ) and centromere X ( green ) FISH on RPE - 1 cells treated with the BAL27862 compound . Scale bar represents 5 µm .

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C. Schematic of the centromere strength model with the microtubule destabilizer BAL27862 compound. D. Representative image of centromere 17 (red) and centromere X (green) FISH on RPE-1 cells treated with the BAL27862 compound. Scale bar represents 5 µm.


E . Bars represent the frequency of chromosome mis - segregation into micronuclei after 28 h treatment with the BAL27862 drug at indicated concentration . Error bars represent the SEM of three independent experiments , dashed line represents the mean . n > 40 micronuclei were analyzed . Dunnett ' s multiple comparisons test , * : p < 0 . 05 . DMSO was used at the same concentration of BAL27862 .

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E. Bars represent the frequency of chromosome mis-segregation into micronuclei after 28 h treatment with the BAL27862 drug at indicated concentration. Error bars represent the SEM of three independent experiments, dashed line represents the mean. n > 40 micronuclei were analyzed. Dunnett's multiple comparisons test, *: p<0.05. DMSO was used at the same concentration of BAL27862.


A . Representative image of CENP - B box FISH and mFISH stained RPE - 1 metaphase spread cells . Chromosome 3 homologs are indicated by white arrows with single CENP - B box chromosomes signal magnified in insets . Scale bar represents 5 µm .

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A. Representative image of CENP-B box FISH and mFISH stained RPE-1 metaphase spread cells. Chromosome 3 homologs are indicated by white arrows with single CENP-B box chromosomes signal magnified in insets. Scale bar represents 5 µm.


B . Representative image of CENP - B box ( red ) and centromere 3 ( green ) FISH on RPE - 1 interphase nucleus after 48 h IAA treatment . CENP - B box signal at individual homologs are magnified in insets . Scale bar represents 5 µm .

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B. Representative image of CENP-B box (red) and centromere 3 (green) FISH on RPE-1 interphase nucleus after 48 h IAA treatment. CENP-B box signal at individual homologs are magnified in insets. Scale bar represents 5 µm.


C . Bar plots represent the quantification of the CENP - B box FISH intensity at the chromosome 3 homolog contained in the main nucleus versus the homolog present in the micronucleus Error bars represent the SEM of three independent experiments . n = 69 cells . Unpaired t test , * * * * p < 0 . 0001 .

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C. Bar plots represent the quantification of the CENP-B box FISH intensity at the chromosome 3 homolog contained in the main nucleus versus the homolog present in the micronucleus Error bars represent the SEM of three independent experiments. n = 69 cells. Unpaired t test, ****p < 0.0001.


E . Immunoblot shows expression of the ( ΔN ) CENP - B - dCas9 after doxycycline induction .

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E. Immunoblot shows expression of the (ΔN)CENP-B-dCas9 after doxycycline induction.


F . Representative images show a micronucleus or the main nucleus containing the Y chromosome ( red ) detected by FISH in no - doxycycline or doxycycline treated cells respectively . Chromosome X is shown in green . Scale bar represents 5 µm .

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F. Representative images show a micronucleus or the main nucleus containing the Y chromosome (red) detected by FISH in no-doxycycline or doxycycline treated cells respectively. Chromosome X is shown in green. Scale bar represents 5 µm.


G . Bar plot shows the mean ( n ≥ 3 ) frequency of micronuclei containing the chromosome Y or chromosome X ± doxycycline treatment for 72 h and IAA for 48 h ± SEM . n > 200 cells with a micronucleus . Mann - Whitney test . * p = 0 . 0173 .

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G. Bar plot shows the mean (n ≥ 3) frequency of micronuclei containing the chromosome Y or chromosome X ± doxycycline treatment for 72 h and IAA for 48 h ± SEM. n > 200 cells with a micronucleus. Mann-Whitney test. *p=0.0173.


B . Table summarizing whole - chromosome aneuploidy ( fold over the mean ) using the indicated methods to measure chromosome mis - segregation rate . Bold numbers represent significant statistical difference from the mean for each method . Orange to dark red gradient highlights chromosomes that mis - segregate ( with at least one method ) at a significantly higher rate compared to the mean level ( weak chromosomes ) . Light green to dark green gradient highlights chromosomes that mis - segregate ( with at least one method ) at a significantly lower rate compared to the mean ( stronger chromosomes ) . Data obtained with only one method were excluded . Im . St . = image stream .

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B. Table summarizing whole-chromosome aneuploidy (fold over the mean) using the indicated methods to measure chromosome mis-segregation rate. Bold numbers represent significant statistical difference from the mean for each method. Orange to dark red gradient highlights chromosomes that mis-segregate (with at least one method) at a significantly higher rate compared to the mean level (weak chromosomes). Light green to dark green gradient highlights chromosomes that mis-segregate (with at least one method) at a significantly lower rate compared to the mean (stronger chromosomes). Data obtained with only one method were excluded. Im. St. = image stream.


C . Plot shows the karyotype distribution of DLD - 1 and RPE - 1 cells in untreated or Auxin - treated

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C. Plot shows the karyotype distribution of DLD-1 and RPE-1 cells in untreated or Auxin-treated condition


E . Representative image of a chromosome spreads in DLD - 1 cells following mFISH and subsequently CENP - B boxes FISH . Inset shows the supernumerary ( SN ) chromosome . Scale bar represents 10 µm . F . Bar plot shows the mean variation in CENP - B boxes in DLD - 1 cells from data from E ( n = 10 cells ) and whole - genome sequencing and centromere mapping ± SD .

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E. Representative image of a chromosome spreads in DLD-1 cells following mFISH and subsequently CENP-B boxes FISH. Inset shows the supernumerary (SN) chromosome. Scale bar represents 10 µm. F. Bar plot shows the mean variation in CENP-B boxes in DLD-1 cells from data from E (n = 10 cells) and whole-genome sequencing and centromere mapping ± SD.


G - H . Scatter plot showing a significant positive correlation between the indicated features in DLD - 1 cells ( n = 1 ) . The lines represent linear regression with 95 % confidence band . Data from chr 13 , 14 , 21 , 22 , Y and SN were excluded from the analysis .

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G-H. Scatter plot showing a significant positive correlation between the indicated features in DLD-1 cells (n = 1). The lines represent linear regression with 95% confidence band. Data from chr 13, 14, 21, 22, Y and SN were excluded from the analysis.


J . Bars show frequency of micronuclei with or without siRNA treatment . Error bars represent the SEM of three independent experiments ( n > 170 cells per replicate ) .

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J. Bars show frequency of micronuclei with or without siRNA treatment. Error bars represent the SEM of three independent experiments (n > 170 cells per replicate).


K . ( Left ) Representative image of CENP - B box ( red ) staining after siRNA against CENP - A / HJURP for 48 h . CENP - B box signals at individual micronuclei are magnified in insets . Scale bar represent 5 µm . ( Right ) Bar graph represents the mean of the CENP - B box FISH intensity contained in the main nucleus versus the one present in the micronucleus ± SEM . Color dots represent independent experiments . ( n > 43 micronuclei and 840 main nucleus ) Mann - Whitney U test : * * * p = 0 . 0001 .

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K. (Left) Representative image of CENP-B box (red) staining after siRNA against CENP-A/HJURP for 48 h. CENP-B box signals at individual micronuclei are magnified in insets. Scale bar represent 5 µm. (Right) Bar graph represents the mean of the CENP-B box FISH intensity contained in the main nucleus versus the one present in the micronucleus ± SEM. Color dots represent independent experiments. (n > 43 micronuclei and 840 main nucleus) Mann-Whitney U test: ***p = 0.0001.


A - B . Scatter plot showing ( A ) a significant positive or ( B ) a negative correlation between chromosome length and the mis - segregation rate or CENP - C ( n = 3 ) reads over chromosome length and mis - segregation rate ( r = Spearman rank coefficient ) . The lines represent linear regression with 95 % confidence interval . Data from chr 13 , 14 , 21 and 22 were excluded from the analysis . Numbers indicate chromosomes with low or high mis - segregation frequency .

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A-B. Scatter plot showing (A) a significant positive or (B) a negative correlation between chromosome length and the mis-segregation rate or CENP-C (n = 3) reads over chromosome length and mis-segregation rate (r = Spearman rank coefficient). The lines represent linear regression with 95% confidence interval. Data from chr 13, 14, 21 and 22 were excluded from the analysis. Numbers indicate chromosomes with low or high mis-segregation frequency.


NE activates mTORC2 in vitro via cAMP , PI3K and Epac1 . ( A ) Immunoblot analysis of BAT cells stimulated with norepinephrine ( NE ) for the indicated proteins .

NE activates mTORC2 in vitro via cAMP, PI3K and Epac1. (A) Immunoblot analysis of BAT cells stimulated with norepinephrine (NE) for the indicated proteins.


( B ) Immunoblot analysis of BAT cells stimulated with NE for 5 minutes in the presence of Rapamycin ( Rapa ) , Torin or Wortmannin ( Wrtm ) for the indicated proteins .

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(B) Immunoblot analysis of BAT cells stimulated with NE for 5 minutes in the presence of Rapamycin (Rapa), Torin or Wortmannin (Wrtm) for the indicated proteins.


( C ) Immunoblot analysis of BAT cells stimulated with 8 - Br - cAMP for 5 minutes in the presence of Rapa , Torin or Wrtm for the indicated proteins .

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(C) Immunoblot analysis of BAT cells stimulated with 8-Br-cAMP for 5 minutes in the presence of Rapa, Torin or Wrtm for the indicated proteins.


( D ) Immunoblot analysis of BAT cells stimulated with NE for 5 minutes in the presence of Wrtm , H89 or ESI - 09 for the indicated proteins .

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(D) Immunoblot analysis of BAT cells stimulated with NE for 5 minutes in the presence of Wrtm, H89 or ESI-09 for the indicated proteins.


( E ) Immunoblot analysis of BAT cells stimulated with 8 - Br - cAMP for 5 minutes in the presence of Wrtm , H89 or ESI - 09 for the indicated proteins . All experiments were performed in triplicates , a representative replicate is presented .

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(E) Immunoblot analysis of BAT cells stimulated with 8-Br-cAMP for 5 minutes in the presence of Wrtm, H89 or ESI-09 for the indicated proteins. All experiments were performed in triplicates, a representative replicate is presented.


NE and cold activate mTORC2 in vivo . ( A ) Immunoblot analysis of BAT from control mice treated with either norepinephrine ( NE ) or vehicle for 30 minutes for the indicated proteins ( n = 3 / group ) .

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NE and cold activate mTORC2 in vivo. (A) Immunoblot analysis of BAT from control mice treated with either norepinephrine (NE) or vehicle for 30 minutes for the indicated proteins (n=3/group).


( B ) Immunoblot analysis of BAT from AdRiKO and control mice treated with either norepinephrine ( NE ) or vehicle for 30 minutes for the indicated proteins ( n = 3 / group ) .

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(B) Immunoblot analysis of BAT from AdRiKO and control mice treated with either norepinephrine (NE) or vehicle for 30 minutes for the indicated proteins (n=3/group).


( C ) Immunoblot analysis of BAT from AdRiKO and control mice housed at either 22 ° C or 4 ° C for 2h for the indicated proteins ( n = 6 / group , each lane represents a mix of 3 mice ) .

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(C) Immunoblot analysis of BAT from AdRiKO and control mice housed at either 22°C or 4°C for 2h for the indicated proteins (n=6/group, each lane represents a mix of 3 mice).


( D ) Body temperature of AdRiKO and control mice housed at 22 ° C ( n = 11 ( control ) , n = 9 ( AdRiKO ) ) .

(D) Body temperature of AdRiKO and control mice housed at 22°C (n=11 (control), n=9 (AdRiKO)).


( E ) Body temperature of AdRiKO and control mice housed at 30 ° C for 2 weeks ( n = 8 / group ) .

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(E) Body temperature of AdRiKO and control mice housed at 30°C for 2 weeks (n=8/group).


( F ) Body temperature loss upon cold exposure of AdRiKO and control mice ( n = 20 ( control ) , n = 17 ( AdRiKO ) ) . Data represent mean ± SEM . Statistically significant differences between AdRiKO and control mice were determined with unpaired Student ' s t - test and are indicated with asterisks ( * = p < 0 . 05 ; * * = p < 0 . 01 , * * * = p < 0 . 001 ) . Statistically significant differences between temperatures are indicated with a number sign ( # = p < 0 . 05 ; # # = p < 0 . 01 ; # # # = p < 0 . 001 ) . The exact p - value for each significant difference can be found in Appendix Table S2 .

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(F) Body temperature loss upon cold exposure of AdRiKO and control mice (n=20 (control), n=17 (AdRiKO)). Data represent mean ± SEM. Statistically significant differences between AdRiKO and control mice were determined with unpaired Student's t-test and are indicated with asterisks (*=p<0.05; **=p<0.01, ***=p<0.001). Statistically significant differences between temperatures are indicated with a number sign (#=p<0.05; ##=p<0.01; ###=p<0.001). The exact p-value for each significant difference can be found in Appendix Table S2.


mTORC2 in adipose tissue is not required for cold - induced lipid droplet mobilization . ( A ) Representative H & amp ; amp ; E staining of sWAT sections from AdRiKO and control mice ( n = 5 / group ) .

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mTORC2 in adipose tissue is not required for cold-induced lipid droplet mobilization. (A) Representative H&amp;E staining of sWAT sections from AdRiKO and control mice (n=5/group).


( B ) Non - esterified fatty acids ( NEFAs ) in plasma of AdRiKO and control mice ( n = 6 / group ) .

(B) Non-esterified fatty acids (NEFAs) in plasma of AdRiKO and control mice (n=6/group).


( C ) Glycerol in plasma of AdRiKO and control mice ( n = 6 / group ) .

(C) Glycerol in plasma of AdRiKO and control mice (n=6/group).


( D ) Representative H & amp ; amp ; E staining of BAT sections from AdRiKO and control mice ( n = 5 / group ) .

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(D) Representative H&amp;E staining of BAT sections from AdRiKO and control mice (n=5/group).


( E ) Triglycerides ( TGs ) in BAT of AdRiKO and control mice housed at 22 ° C or 4 ° C for 8h ( n = 6 / group ) .

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(E) Triglycerides (TGs) in BAT of AdRiKO and control mice housed at 22°C or 4°C for 8h (n=6/group).


( F ) NEFAs in BAT of AdRiKO and control mice ( n = 6 / group ) . Data represent mean ± SEM . Statistically significant differences between AdRiKO and control mice were determined with unpaired Student ' s t - test and are indicated with asterisks ( * = p < 0 . 05 ; * * = p < 0 . 01 , * * * = p < 0 . 001 ) . Statistically significant differences between temperatures were determined with unpaired Student ' s t - test and are indicated with a number sign ( # = p < 0 . 05 ; # # = p < 0 . 01 ; # # # = p < 0 . 001 ) . The exact p - value for each significant difference can be found in Appendix Table S2 .

(F) NEFAs in BAT of AdRiKO and control mice (n=6/group). Data represent mean ± SEM. Statistically significant differences between AdRiKO and control mice were determined with unpaired Student's t-test and are indicated with asterisks (*=p<0.05; **=p<0.01, ***=p<0.001). Statistically significant differences between temperatures were determined with unpaired Student's t-test and are indicated with a number sign (#=p<0.05; ##=p<0.01; ###=p<0.001). The exact p-value for each significant difference can be found in Appendix Table S2.


mTORC2 in adipose tissue is not required for cold - induced mitochondrial uncoupling and β - oxidation . ( A ) mRNA levels of the indicated genes in BAT of AdRiKO and control mice housed at 22 ° C or at 4 ° C for 8h ( n = 6 / group ) .

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mTORC2 in adipose tissue is not required for cold-induced mitochondrial uncoupling and β-oxidation. (A) mRNA levels of the indicated genes in BAT of AdRiKO and control mice housed at 22°C or at 4°C for 8h (n=6/group).


( B ) Immunoblot analysis of BAT from AdRiKO and control mice housed at 22 ° C or at 4 ° C for 8h for the indicated proteins ( n = 6 / group , each lane represents a mix of 3 mice ) .

(B) Immunoblot analysis of BAT from AdRiKO and control mice housed at 22°C or at 4°C for 8h for the indicated proteins (n=6/group, each lane represents a mix of 3 mice).


( C ) mRNA levels of the indicated genes in BAT of AdRiKO and control mice housed at 22 ° C or at 4 ° C for 8h ( n = 6 / group , each lane represents a mix of 3 mice ) .

(C) mRNA levels of the indicated genes in BAT of AdRiKO and control mice housed at 22°C or at 4°C for 8h (n=6/group, each lane represents a mix of 3 mice).


( D ) Immunoblot analysis of BAT from AdRiKO and control mice housed at 22 ° C or at 4 ° C for 8h for the indicated proteins ( n = 6 / group , each lane represents a mix of 3 mice ) .

(D) Immunoblot analysis of BAT from AdRiKO and control mice housed at 22°C or at 4°C for 8h for the indicated proteins (n=6/group, each lane represents a mix of 3 mice).


( E ) Mitochondrial DNA content of BAT from AdRiKO and control mice housed at 22 ° C or at 4 ° C for 8h ( n = 6 / group ) .

(E) Mitochondrial DNA content of BAT from AdRiKO and control mice housed at 22°C or at 4°C for 8h (n=6/group).


( F ) Representative electronmicrographs of BAT from AdRiKO and control mice housed at 22 ° C or at 4 ° C for 4h ( n = 3 / group ) .

(F) Representative electronmicrographs of BAT from AdRiKO and control mice housed at 22°C or at 4°C for 4h (n=3/group).


( G ) Oxygen consumption rate ( OCR ) of BAT explants from AdRiKO and control mice housed at 22 ° C or at 4 ° C for 4h ( n = 7 / group ) .

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(G) Oxygen consumption rate (OCR) of BAT explants from AdRiKO and control mice housed at 22°C or at 4°C for 4h (n=7/group).


H ) Maximal respiration ( VO2 max ) of AdRiKO and control mice housed at 22 ° C or at 4 ° C for 8h ( n = 9 ( control 22 ° C ) , n = 7 ( AdRiKO 22 ° C ) , n = 8 ( control 4 ° C ) , n = 8 ( AdRiKO 4 ° C ) .

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H) Maximal respiration (VO2 max) of AdRiKO and control mice housed at 22°C or at 4°C for 8h (n=9 (control 22°C), n=7 (AdRiKO 22°C), n=8 (control 4°C), n=8 (AdRiKO 4°C).


( I ) Respiration ( VO2 ) of AdRiKO and control mice upon cold exposure ( n = 8 / group ) . Data represent mean ± SEM . Statistically significant differences between AdRiKO and control mice were determined with unpaired Student ' s t - test and are indicated with asterisks ( * = p < 0 . 05 ; * * = p < 0 . 01 , * * * = p < 0 . 001 ) . Statistically significant differences between temperatures were determined with unpaired Student ' s t - test and are indicated with a number sign ( # = p < 0 . 05 ; # # = p < 0 . 01 ; # # # = p < 0 . 001 ) . The exact p - value for each significant difference can be found in Appendix Table S2 .

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(I) Respiration (VO2) of AdRiKO and control mice upon cold exposure (n=8/group). Data represent mean ± SEM. Statistically significant differences between AdRiKO and control mice were determined with unpaired Student's t-test and are indicated with asterisks (*=p<0.05; **=p<0.01, ***=p<0.001). Statistically significant differences between temperatures were determined with unpaired Student's t-test and are indicated with a number sign (#=p<0.05; ##=p<0.01; ###=p<0.001). The exact p-value for each significant difference can be found in Appendix Table S2.


mTORC2 in adipose tissue is required for cold - induced glucose uptake and glycolysis ( A ) 2 - Deoxyglucose - 6 - phosphate ( 2DG6P ) accumulation in BAT of AdRiKO and control mice housed at 22 ° C or at 4 ° C for 4h ( n = 6 / group ) . (

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mTORC2 in adipose tissue is required for cold-induced glucose uptake and glycolysis (A) 2-Deoxyglucose-6-phosphate (2DG6P) accumulation in BAT of AdRiKO and control mice housed at 22°C or at 4°C for 4h (n=6/group). (


( B ) Extracellular acidification rate ( ECAR ) of BAT explants from AdRiKO and control mice housed at 22 ° C or at 4 ° C for 4h ( n = 7 / group ) .

(B) Extracellular acidification rate (ECAR) of BAT explants from AdRiKO and control mice housed at 22°C or at 4°C for 4h (n=7/group).


( C ) Immunoblot analysis of BAT from AdRiKO and control mice housed at 22 ° C or at 4 ° C for 8h for the indicated proteins ( n = 6 / group , each lane represents a mix of 3 mice ) .

(C) Immunoblot analysis of BAT from AdRiKO and control mice housed at 22°C or at 4°C for 8h for the indicated proteins (n=6/group, each lane represents a mix of 3 mice).


( D ) Immunoblot analysis of isolated plasma membranes from BAT of AdRiKO and control mice housed at 22 ° C or at 4 ° C for 8h for the indicated proteins ( n = 6 / group , each lane represents a mix of 3 mice ) .

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(D) Immunoblot analysis of isolated plasma membranes from BAT of AdRiKO and control mice housed at 22°C or at 4°C for 8h for the indicated proteins (n=6/group, each lane represents a mix of 3 mice).


( E ) Immunoblot analysis of mitochondrial and cytosolic fractions from BAT of AdRiKO and control mice housed at 22 ° C or at 4 ° C for 4h for the indicated proteins ( n = 6 / group , each lane represents a mix of 3 mice ) .

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(E) Immunoblot analysis of mitochondrial and cytosolic fractions from BAT of AdRiKO and control mice housed at 22°C or at 4°C for 4h for the indicated proteins (n=6/group, each lane represents a mix of 3 mice).


( F ) Cytosolic hexokinase activity in BAT of AdRiKO and control mice housed at 22 ° C or at 4 ° C for 4h ( n = 5 ( control 22 ° C ) , n = 5 ( AdRiKO 22 ° C ) , n = 7 ( control 4 ° C ) , n = 7 AdRiKO 4 ° C ) ) .

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(F) Cytosolic hexokinase activity in BAT of AdRiKO and control mice housed at 22°C or at 4°C for 4h (n=5 (control 22°C), n=5 (AdRiKO 22°C), n=7 (control 4°C), n=7 AdRiKO 4°C)).


( G ) Mitochondrial hexokinase activity in BAT of AdRiKO and control mice housed at 22 ° C or at 4 ° C for 4h ( n = 5 ( control 22 ° C ) , n = 5 ( AdRiKO 22 ° C ) , n = 7 ( control 4 ° C ) , n = 7 AdRiKO 4 ° C ) ) . Data represent mean ± SEM . Statistically significant differences between AdRiKO and control mice were determined with unpaired Student ' s t - test and are indicated with asterisks ( * = p < 0 . 05 ; * * = p < 0 . 01 , * * * = p < 0 . 001 ) . Statistically significant differences between temperatures were determined with unpaired Student ' s t - test and are indicated with a number sign ( # = p < 0 . 05 ; # # = p < 0 . 01 ; # # # = p < 0 . 001 ) . The exact p - value for each significant difference can be found in Appendix Table S2 .

O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Mitochondrial hexokinase activity in BAT of AdRiKO and control mice housed at 22°C or at 4°C for 4h (n=5 (control 22°C), n=5 (AdRiKO 22°C), n=7 (control 4°C), n=7 AdRiKO 4°C)). Data represent mean ± SEM. Statistically significant differences between AdRiKO and control mice were determined with unpaired Student's t-test and are indicated with asterisks (*=p<0.05; **=p<0.01, ***=p<0.001). Statistically significant differences between temperatures were determined with unpaired Student's t-test and are indicated with a number sign (#=p<0.05; ##=p<0.01; ###=p<0.001). The exact p-value for each significant difference can be found in Appendix Table S2.


Restoration of glucoseuptake or Akt signaling suppresses the thermogenic defect in AdRiKO mice . ( A ) HKII mRNA expression level in BAT of AdRiKO and control mice infected with either AAV9 - HKII or AAV9 - empty ( n = 8 / group ) .

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Restoration of glucoseuptake or Akt signaling suppresses the thermogenic defect in AdRiKO mice. (A) HKII mRNA expression level in BAT of AdRiKO and control mice infected with either AAV9-HKII or AAV9-empty (n=8/group).


( B ) Cold - induced 2 - Deoxyglucose - 6 - phosphate ( 2DG6P ) accumulation in BAT of AdRiKO and control mice infected with either AAV9 - HKII or AAV9 - empty housed at 4 ° C for 4h ( n = 8 / group ) .

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(B) Cold-induced 2-Deoxyglucose-6-phosphate (2DG6P) accumulation in BAT of AdRiKO and control mice infected with either AAV9-HKII or AAV9-empty housed at 4°C for 4h (n=8/group).


( C ) Body temperature of AdRiKO and control mice infected with either AAV9 - HKII or AAV9 - empty housed at 22 ° C ( n = 8 / group ) .

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(C) Body temperature of AdRiKO and control mice infected with either AAV9-HKII or AAV9-empty housed at 22°C (n=8/group).


( D ) Body temperature upon cold exposure of AdRiKO and control mice infected with either AAV9 - HKII or AAV9 - empty . The left panel represents body temperature after each hour of cold exposure while the right panel represents body temperature as a bar graph for the 3h cold exposure time point ( n = 8 / group ) . a = significant difference between AdRiKO and control mice infected with AAV9 - empty , b = significant difference between AdRiKO and control mice infected with AAV9 - HKII , d = significant difference between AdRiKO mice infected with AAV9 - empty and AAV9 - HKII .

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(D) Body temperature upon cold exposure of AdRiKO and control mice infected with either AAV9-HKII or AAV9-empty. The left panel represents body temperature after each hour of cold exposure while the right panel represents body temperature as a bar graph for the 3h cold exposure time point (n=8/group). a=significant difference between AdRiKO and control mice infected with AAV9-empty, b= significant difference between AdRiKO and control mice infected with AAV9-HKII, d= significant difference between AdRiKO mice infected with AAV9-empty and AAV9-HKII.


( E ) Immunoblot analysis of BAT from AdRiKO and control mice infected with either AAV8 - Akt2S474D or AAV8 - empty ( n = 6 / group , each lane represents a mix of 3 mice ) .

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(E) Immunoblot analysis of BAT from AdRiKO and control mice infected with either AAV8-Akt2S474D or AAV8-empty (n=6/group, each lane represents a mix of 3 mice).


( F ) Body temperature of AdRiKO and control mice infected with either AAV8 - Akt2S474D or AAV8 - empty housed at 22 ° C ( n = 11 / group ) .

(F) Body temperature of AdRiKO and control mice infected with either AAV8-Akt2S474D or AAV8-empty housed at 22°C (n=11/group).


( G ) Body temperature upon cold exposure of AdRiKO and control mice infected with either AAV8 - Akt2S474D or AAV8 - empty . The left panel represents body temperature after each hour of cold exposure while the right panel represents body temperature as a bar graph for the 3h cold exposure time point ( n = 11 / group ) . a = significant difference between AdRiKO and control mice infected with AAV8 - empty , b = significant difference between AdRiKO and control mice infected with AAV8 - Akt2S474D , d = significant difference between AdRiKO mice infected with AAV8 - empty and AAV8 - Akt2S474D .

(G) Body temperature upon cold exposure of AdRiKO and control mice infected with either AAV8-Akt2S474D or AAV8-empty. The left panel represents body temperature after each hour of cold exposure while the right panel represents body temperature as a bar graph for the 3h cold exposure time point (n=11/group). a=significant difference between AdRiKO and control mice infected with AAV8-empty, b= significant difference between AdRiKO and control mice infected with AAV8-Akt2S474D, d= significant difference between AdRiKO mice infected with AAV8-empty and AAV8-Akt2S474D.


( H ) Cold - induced 2 - Deoxyglucose - 6 - phosphate ( 2DG6P ) accumulation in BAT of AdRiKO and control mice infected with either AAV8 - Akt2S474D or AAV8 - empty housed at 4 ° C for 4h ( n = 7 ( control AAV8 - null ) , n = 6 ( AdRiKO AAV8 - null ) , n = 6 ( control AAV8 - AktS474D ) , n = 6 ( AdRiKO AAV8 - AktS474D ) ) . Data represent mean ± SEM . Statistically significant differences between AdRiKO and control mice were determined with unpaired Student ' s t - test and are indicated with asterisks ( * = p < 0 . 05 ; * * = p < 0 . 01 , * * * = p < 0 . 001 ) . Statistically significant differences between viruses were determined with unpaired Student ' s t - test and are indicated with a number sign ( # = p < 0 . 05 ; # # = p < 0 . 01 ; # # # = p < 0 . 001 ) . The exact p - value for each significant difference can be found in Appendix Table S2 .

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(H) Cold-induced 2-Deoxyglucose-6-phosphate (2DG6P) accumulation in BAT of AdRiKO and control mice infected with either AAV8-Akt2S474D or AAV8-empty housed at 4°C for 4h (n=7 (control AAV8-null), n=6 (AdRiKO AAV8-null), n=6 (control AAV8-AktS474D), n=6 (AdRiKO AAV8-AktS474D)). Data represent mean ± SEM. Statistically significant differences between AdRiKO and control mice were determined with unpaired Student's t-test and are indicated with asterisks (*=p<0.05; **=p<0.01, ***=p<0.001). Statistically significant differences between viruses were determined with unpaired Student's t-test and are indicated with a number sign (#=p<0.05; ##=p<0.01; ###=p<0.001). The exact p-value for each significant difference can be found in Appendix Table S2.


B , C . Distribution of the m6A peaks across host mRNAs ( B ) and EBV mRNAs ( C ) was analyzed based on the MeRIP - seq data .

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B, C. Distribution of the m6A peaks across host mRNAs (B) and EBV mRNAs (C) was analyzed based on the MeRIP-seq data.


B . The BZLF1 m6A levels were measured using MeRIP - qPCR in CNE2EBV , Raji and B95 - 8 cells with latent EBV infection or lytic reactivation . TPA ( 30 ng / ml ) and NaB ( 2 mM ) were used to treat the cells for 24 h to reactivate EBV . The cellular RNA was harvested for MeRIP - qPCR assays . The fold enrichment was determined by calculating the 2 - ΔCt of the MeRIP sample relative to the input sample . The mean value of the results in mock - treated cells was normalized to 1 .

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B. The BZLF1 m6A levels were measured using MeRIP-qPCR in CNE2EBV, Raji and B95-8 cells with latent EBV infection or lytic reactivation. TPA (30 ng/ml) and NaB (2 mM) were used to treat the cells for 24 h to reactivate EBV. The cellular RNA was harvested for MeRIP-qPCR assays. The fold enrichment was determined by calculating the 2-ΔCt of the MeRIP sample relative to the input sample. The mean value of the results in mock-treated cells was normalized to 1.


B . The EBV infection efficiency in HK1 cells transfected with the indicated siRNAs was analyzed by FACS . The cells were infected with EBV - GFP virus at 24 h after siRNA transfection . The ratio of GFP - positive cells was quantified by FACS at 24 hpi . The mean value of the GFP - positive rates in the siNC transfected HK1 cells was normalized to 1 .

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B. The EBV infection efficiency in HK1 cells transfected with the indicated siRNAs was analyzed by FACS. The cells were infected with EBV-GFP virus at 24 h after siRNA transfection. The ratio of GFP-positive cells was quantified by FACS at 24 hpi. The mean value of the GFP-positive rates in the siNC transfected HK1 cells was normalized to 1.


M . The relative EBV production in C666 cells transfected with YTHDF1 - specific siRNAs ( siY1 - 1 and siY1 - 2 ) or the siNC control was measured by qPCR . The DNA from the cell - free culture supernatant was harvested at 24 h after siRNA transfection . The number of EBV copies in the supernatant of the cells was quantified by qPCR using specific primers targeting BamHI - W fragment region of EBV . The mean value of EBV copies in the siNC cells was normalized to 1 .

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M. The relative EBV production in C666 cells transfected with YTHDF1-specific siRNAs (siY1-1 and siY1-2) or the siNC control was measured by qPCR. The DNA from the cell-free culture supernatant was harvested at 24 h after siRNA transfection. The number of EBV copies in the supernatant of the cells was quantified by qPCR using specific primers targeting BamHI-W fragment region of EBV. The mean value of EBV copies in the siNC cells was normalized to 1.


B . The relative mRNA levels of BZLF1 and BRLF1 in the latently infected or reactivated ( 24 h post NaB treatment ) CNE2EBV cells transfected with the YTHDF1 - specific siRNAs ( siY1 - 1 and siY1 - 2 ) or the siNC control . After 24 h of siRNA transfection , CNE2EBV cells were treated with NaB ( 2 mM ) or mock for 24 h , followed by qRT - PCR analysis . All the gene expression levels were normalized to the housekeeping gene ACTB .

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B. The relative mRNA levels of BZLF1 and BRLF1 in the latently infected or reactivated (24 h post NaB treatment) CNE2EBV cells transfected with the YTHDF1-specific siRNAs (siY1-1 and siY1-2) or the siNC control. After 24 h of siRNA transfection, CNE2EBV cells were treated with NaB (2 mM) or mock for 24 h, followed by qRT-PCR analysis. All the gene expression levels were normalized to the housekeeping gene ACTB.


F . The protein level of YTHDF1 , BZLF1 and ACTB in CNE2EBV cells following EBV reactivation ( 24 h post - NaB treatment ) , EBV infected HK1 or NPEC2 - Bmi1 cells at 24 hpi . The whole - cell protein was extracted at same time as the RNA .

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F. The protein level of YTHDF1, BZLF1 and ACTB in CNE2EBV cells following EBV reactivation (24 h post-NaB treatment), EBV infected HK1 or NPEC2-Bmi1 cells at 24 hpi. The whole-cell protein was extracted at same time as the RNA.


D . The relative enrichment of the BZLF1 and BRLF1 mRNA in CNE2EBV cells with transfected with siYTHDF2 , siYTHDF3 or siNC siRNA following EBV reactivation . After 24 h of siRNA transfection , CNE2EBV cells were treated with TPA ( 30 ng / ml ) and NaB ( 2 mM ) for 24 h . The RIP assays were performed using YTHDF1 - specific antibody or IgG control in these cells . Fold enrichment was determined by calculating the 2 - ΔCt of the RIP sample relative to the input sample .

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D. The relative enrichment of the BZLF1 and BRLF1 mRNA in CNE2EBV cells with transfected with siYTHDF2, siYTHDF3 or siNC siRNA following EBV reactivation. After 24 h of siRNA transfection, CNE2EBV cells were treated with TPA (30 ng/ml) and NaB (2 mM) for 24 h. The RIP assays were performed using YTHDF1-specific antibody or IgG control in these cells. Fold enrichment was determined by calculating the 2-ΔCt of the RIP sample relative to the input sample.