( d ) Western blot on total extracted from hiwΔN , wild - type ( C155 - Gal4 / + ) Rae1 expression ( C155 - Gal4 / + ; UAS - NTAP - Rae1 / + ) larval brains . Full - blots are presented in Supplementary Figure 12 . ( e ) of Hiw in wild - type Rae1 expression larval brains ( * P 0 . 001 , on seven independent experiments ) . Arrows indicate a 38 - kDa endogenous Rae1 arrowheads indicate 58 - kDa transgenic NTAP - Rae1 . Error bars denote s . e . m .

O O O B-EXP_ASSAY I-EXP_ASSAY O O O O B-GENEPROD O O O O O O O B-GENEPROD O O O B-GENEPROD O O O O B-GENEPROD O O O O O B-GENEPROD O B-GENEPROD O O O O B-TISSUE O O O O O O O O O O O O O O O B-GENEPROD O O O O B-GENEPROD O O B-TISSUE O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O

(d) Western blot analysis on total proteins extracted from hiwΔN, wild-type (C155-Gal4/+) and Rae1 expression (C155-Gal4/+; UAS-NTAP-Rae1/+) larval brains. Full-length blots are presented in Supplementary Figure 12. (e) Quantification of Hiw protein levels in the wild-type and the Rae1 expression larval brains (*P 0.001, based on seven independent experiments). Arrows indicate a 38-kDa endogenous Rae1 protein and the arrowheads indicate the 58-kDa transgenic NTAP-Rae1 protein. Error bars denote s.e.m.


( a ) Representative confocal of segment A3 muscle 6 / 7 synapses stained for both DVGLUT ( green ) FasII ( red ) , in wild - type , Rae1EX28 , Rae1EX28 ; atg1 / + , Rae1EX28 ; atg2 / + Rae1EX28 ; atg18 / + wandering larvae . Scale bar represents 10 μm . ( b ) of number of boutons in wild - type , atg1 / + , atg2 / + , atg18 / + , Rae1EX28 , Rae1EX28 ; atg1 / + , Rae1EX28 ; atg2 / + Rae1EX28 ; atg18 / + larval NMJs ( segment A3 muscle 6 / 7 ; n = 12 , 12 , 12 , 12 , 12 , 16 , 12 21 , respectively ) .

O O O O B-EXP_ASSAY O O O B-TISSUE O O O B-SUBCELLULAR O O O B-GENEPROD O O O B-GENEPROD O O O O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O B-GENEPROD O B-GENEPROD O O O B-ORGANISM O O O O O O O O O O O O O B-SUBCELLULAR O O O O O B-GENEPROD O O O B-GENEPROD O O O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O B-GENEPROD O B-GENEPROD O O O B-SUBCELLULAR O O O B-TISSUE O O O O O O O O O O O O O O O O O O O O O O O O

(a) Representative confocal images of segment A3 muscle 6/7 synapses stained for both DVGLUT (green) and FasII (red), in wild-type, Rae1EX28, Rae1EX28; atg1/+, Rae1EX28; atg2/+ and Rae1EX28; atg18/+ wandering larvae. Scale bar represents 10 μm. (b) Quantification of the number of boutons in wild-type, atg1/+, atg2/+, atg18/+, Rae1EX28, Rae1EX28; atg1/+, Rae1EX28; atg2/+ and Rae1EX28; atg18/+ larval NMJs (segment A3 muscle 6/7; n = 12, 12, 12, 12, 12, 16, 12 and 21, respectively).


( c ) Western blot on total extracted from wild - type , Rae1EX28 , Rae1EX28 ; atg1 / + , Rae1EX28 ; atg2 / + Rae1EX28 ; atg18 / + larval brains . neural form of β - catenin ( 82 kDa , see Online Methods ) was used as an internal control of neuronal . Full - blots are presented in Supplementary Figure 13 . ( d ) of Hiw in wild - type , Rae1EX28 , Rae1EX28 ; atg1 / + , Rae1EX28 ; atg2 / + Rae1EX28 ; atg18 / + larval brains ( on six independent experiments ) .

O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O B-GENEPROD O B-GENEPROD O O O B-TISSUE O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O 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-GENEPROD O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O B-GENEPROD O B-GENEPROD O O O B-TISSUE O O O O O O O

(c) Western blot analysis on total proteins extracted from wild-type, Rae1EX28, Rae1EX28; atg1/+, Rae1EX28; atg2/+ and Rae1EX28; atg18/+ larval brains. The neural form of β-catenin (82 kDa, see Online Methods) was used as an internal control of neuronal proteins. Full-length blots are presented in Supplementary Figure 13. (d) Quantification of Hiw protein levels in wild-type, Rae1EX28, Rae1EX28; atg1/+, Rae1EX28; atg2/+ and Rae1EX28; atg18/+ larval brains (based on six independent experiments).


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

O O O O B-EXP_ASSAY O B-TISSUE O O O O B-GENEPROD O O O B-GENEPROD O O O O O B-ORGANISM O O O O O O O B-GENEPROD O O O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O O O O O B-GENEPROD O B-GENEPROD O O O O O B-GENEPROD O O O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O O O O O B-GENEPROD O B-GENEPROD O O O O O B-GENEPROD O O O O O O O O O O O O O O O O B-SUBCELLULAR O B-TISSUE O O O O O O B-GENEPROD O O O O B-GENEPROD O O O O O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD O B-GENEPROD I-GENEPROD O B-GENEPROD O B-ORGANISM O O O O O O O O O O O O O O O O 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 in hepatocytes untreated ( None ) or treated with 3 - methyladenine ( 3MA ) cultured in regular medium ( RM ) or oleate ( OL ) ( * P 0 . 02 , * * P 0 . 002 , n = 3 ) .

O O B-SMALL_MOLECULE O B-CELL_TYPE O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O B-SMALL_MOLECULE 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 in vector - infected ( VEC ) siAtg5 in RM , OL or in MCDM ( * P 0 . 001 , n = 5 ) . OL values are in mM .

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

O O B-SMALL_MOLECULE O O O O O O O O B-GENEPROD O B-CELL_LINE I-CELL_LINE I-CELL_LINE O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O

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

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

O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O 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 ) MCDM ( d ) ( * P 0 . 05 , * * P 0 . 01 , * * * P 0 . 001 as compared to VEC , n = 3 7 ) .

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

O O B-SMALL_MOLECULE O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O B-SMALL_MOLECULE O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE O O B-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O B-SMALL_MOLECULE 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 B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O B-GENEPROD O O O O B-GENEPROD O O O O B-CELL_TYPE 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 stained as labelled . Arrows indicate co - localization .

O O O O O O O B-CELL_TYPE O O O O O O O O O B-SMALL_MOLECULE 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 of cultured hepatocytes . dm , double - vesicles . Arrows indicate membranes in LDs . Insets show double in nucleus ( N ) , lipid - containing vesicles ( L ) mitochondria ( mit ) , but not in LDs .

O O B-EXP_ASSAY O O B-CELL_TYPE O O O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O O O O O B-SUBCELLULAR O O O O O B-SUBCELLULAR O O O O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O O O B-SUBCELLULAR O O O O O O O B-SUBCELLULAR O

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 ) LC3 - labelled bilayer membranes ( ) . c , Percentages of autophagic vacuoles ( AVs ) containing only lipid ( Lipids , * ) , other cargo ( Other ) or mixed cargo ( Mixed ) in treated as in b ( * P 0 . 01 , * * P 0 . 001 with in RM , n = 4 6 ) . Left : representative examples . Error bars , s . e . m .

O O B-ORGANISM B-TISSUE B-GENEPROD O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O B-GENEPROD O O O O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 homogenates ( Hom ) LDs from fed ( F ) or 24 - h starved ( S ) mice . IκB , inhibitor of factor of kappa polypeptide gene enhancer ; GPDH , glyceraldehyde - 3 - phosphate dehydrogenase .

O O B-EXP_ASSAY O O O O O B-SUBCELLULAR O O O O O O O O O O O O O B-ORGANISM O B-GENEPROD O O O O O O O O O O B-GENEPROD 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 . Arrows : Lipid - containing double vesicles . b , Electron of autophagic vacuoles ( AVs ) isolated from mice fed or starved for indicated time periods . percentages of double vesicles containing lipid in isolated AVs from 3 - 4 mice per condition are shown ( * P < 0 . 01 as compared to fractions from fed ) . ( * ) indicate lipid )

O O O O B-SUBCELLULAR I-SUBCELLULAR O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O 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-SMALL_MOLECULE O O O O B-ORGANISM O O O O O O B-SUBCELLULAR I-SUBCELLULAR O O O O O B-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR O B-SMALL_MOLECULE O O O O O O O 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-SMALL_MOLECULE 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 lysosomes ( Lys ) . L - 1 , LAMP1 .

O O B-EXP_ASSAY O O O B-SUBCELLULAR I-SUBCELLULAR O O O O O O O O B-GENEPROD O

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


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

O O B-EXP_ASSAY O B-TISSUE O B-SUBCELLULAR O O O O B-ORGANISM O

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


e , Liver homogenate immunoblots . PDI , disulphide isomerase .

O O B-TISSUE O B-EXP_ASSAY O B-GENEPROD O O O O

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


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

O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE 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).


O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O

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


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

O O O B-SMALL_MOLECULE O B-SUBCELLULAR 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 isolated LDs . Error bars , s . e . m .

O O B-EXP_ASSAY O O O B-SUBCELLULAR O O O O O O O O O O

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


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

O O B-SMALL_MOLECULE O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O B-CELL_LINE B-ORGANISM B-CELL_TYPE I-CELL_TYPE O O O O O O O O O O O B-GENEPROD O O B-GENEPROD O O O O B-ORGANISM 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 ) lactate ( j ) , rate of 14C - lactate appearance in blood from acutely injected [ U - 14C ] glucose in tail vein ( k ) of WT Pink1 KO mice were analysed . Data are expressed as mean ± s . e . m . * P0 . 05 ( Student ' s t - ; n = 3 - 4 independent experiments ) .

B-TISSUE O O B-SMALL_MOLECULE O O O B-SMALL_MOLECULE O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O B-TISSUE O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O B-TISSUE I-TISSUE O O O O O B-GENEPROD O B-ORGANISM O O O O O O O O O O O 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 of relative mRNA abundances of Glut1 , Glut3 , Hk2 Gapdh in WT Pink1 KO miceMEF ( a ) ß - Actin mRNA was used for normalization , Pink1 KO data are expressed as change relative to WT .

B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD I-GENEPROD O O B-GENEPROD O B-ORGANISM O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O B-GENEPROD 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 of abundances of PINK1 , GLUT1 , GLUT3 ( not detectable in MEF ) , HK2 , GAPDH HIF1α in WT Pink1 - null mice MEF ( b ) ß - Actin was used as loading control .

B-EXP_ASSAY I-EXP_ASSAY O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O B-CELL_LINE O O B-GENEPROD O B-GENEPROD I-GENEPROD O O B-GENEPROD O O B-ORGANISM B-CELL_LINE O O O B-GENEPROD I-GENEPROD I-GENEPROD 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 of relative mRNA abundances of Glut1 , Glut3 , Hk2 Gapdh in WT Pink1 KO neurons ( c ) . ß - Actin mRNA was used for normalization , Pink1 KO data are expressed as change relative to WT .

B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD I-GENEPROD O O B-GENEPROD O B-CELL_TYPE O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O B-GENEPROD 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 of abundances of PINK1 , GLUT1 , GLUT3 ( not detectable in MEF ) , HK2 , GAPDH HIF1α in WT Pink1 - null neurons ( d ) ; ß - Actin was used as loading control .

B-EXP_ASSAY I-EXP_ASSAY O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O B-GENEPROD O B-GENEPROD I-GENEPROD O O B-GENEPROD O O B-CELL_TYPE O O O O B-GENEPROD I-GENEPROD I-GENEPROD 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 neurons (d); ß-Actin was used as loading control.


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

O O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O B-GENEPROD O B-GENEPROD I-GENEPROD O O B-GENEPROD O B-ORGANISM B-TISSUE I-TISSUE O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O 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-GENEPROD O O O O O O O O O O O O O O O O O O O O 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 of Hif1α mRNA abundance in MEF transfected with siHif1α , or siControl ( 100 nM ) , after 72 h .

O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O O B-CELL_LINE 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-GENEPROD O O O O O O B-GENEPROD O O O O O O O O B-GENEPROD O O O O O B-ORGANISM B-CELL_LINE 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 of relative mRNA abundances of Glut1 , Glut3 , Hk2 Gapdh in WT Pink1 KO mice MEF transfected with siControl or siHif1α . ß - Actin mRNA was used for normalization , data are expressed as change relative to WT - siControl .

O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD I-GENEPROD O O B-GENEPROD O B-ORGANISM B-CELL_LINE O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD 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 in Pink1 KO mice MEF transfected with siHif1α or siControl shows efficacy of siHif1α at knocking down endogenous HIF1α ; ß - actin was used as loading control . As shown , siHif1α partially prevented increase in GLUT1 , HK2 GAPDH abundances .

O O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O O B-GENEPROD O B-ORGANISM B-CELL_LINE O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD I-GENEPROD 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.


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

O O O B-SMALL_MOLECULE O O O O O O O O O O B-SMALL_MOLECULE O O O O O O B-GENEPROD O B-CELL_LINE 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 endogenous increased HIF1α ( g ) , as well as increased glycolytic flux ( h ) observed in Pink1 KO neurons . Only representative western blots are shown ; replicates semi - quantitative estimation of band intensities are shown in Supplementary Information .

O O O O O O O O B-GENEPROD O O O O O O O O B-SMALL_MOLECULE O O O O O O B-GENEPROD O B-CELL_TYPE O O O B-EXP_ASSAY I-EXP_ASSAY 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 ) , ( b ) mitochondrial inner potential ( Δψm ) in WT Pink1 KO mice MEF .

O O O B-SUBCELLULAR O O O O O O O B-SUBCELLULAR I-SUBCELLULAR O O O O O O O B-SUBCELLULAR I-SUBCELLULAR O O O O O O B-GENEPROD O B-ORGANISM B-CELL_LINE 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 HIF1α in WT Pink1 KO MEF transfected with siHif1α ( or its siControl ) ; ß - actin was used as loading control .

O O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD I-GENEPROD O O B-GENEPROD O B-CELL_LINE O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD 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 activity in WT Pink1 KO MEF transfected with siHif1α or siControl .

O O B-SUBCELLULAR O O O B-GENEPROD O B-CELL_LINE O O O O O O

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


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

O O O O B-SMALL_MOLECULE O O O B-SUBCELLULAR B-SMALL_MOLECULE O O O O O O O O B-GENEPROD O

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


( g ) Detection of mitochondrial O2 · − using mitochondrial - specific probe MitoSox in MEF incubated in absence ( − ) or in presence ( + ) of plasma 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 B-SUBCELLULAR B-SMALL_MOLECULE O O O B-SUBCELLULAR O O O B-EXP_ASSAY O B-CELL_LINE O O O O O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O B-SUBCELLULAR O O O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O O B-GENEPROD O O O O O O O O O O O O O B-GENEPROD O O B-GENEPROD O O O O O O O O O O B-SUBCELLULAR B-SMALL_MOLECULE 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 abundance by treatments that remove mitochondrial ROS ; α - TUBULIN was used as loading control . Only representative western blots are shown ; replicates semi - quantitative estimation of band intensities are shown in Supplementary Information . Data are expressed as mean ± s . e . m . * P0 . 05 ( Student ' s t - ; n = 3 - 4 independent experiments ) .

O O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O B-GENEPROD O B-CELL_LINE O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD O O O O O B-SUBCELLULAR B-SMALL_MOLECULE O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O 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 cycle phases , ( b ) growth in WT Pink1 KO MEF after 72 h in absence ( denoted as - 2DG ) or in presence ( + 2DG ) of glucose - metabolizing inhibitor , 2 - deoxyglucose ( 2DG , 1 mM ) .

O O O O O B-EXP_ASSAY O O O O O O O O B-EXP_ASSAY O O B-GENEPROD O B-CELL_LINE O O O O O O O O O O B-SMALL_MOLECULE O O O O O O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE 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 cycle phases , ( d ) growth in WT Pink1 KO MEF 72 h after transfection with siHif1α ( + siHif1α ) or siControl ( denoted as - siHif1α ) .

O O O O O B-EXP_ASSAY O O O O O O O O B-EXP_ASSAY O O B-GENEPROD O B-CELL_LINE 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 DAPI immunostaining in subventricular zone of WT Pink1 KO mouse brain , corresponding . * P0 . 05 ; # P0 . 05 versus other conditions ( of variance followed by Bonferroni ; n = 3 - 4 independent experiments ) . data are expressed as mean ± s . e . m . Scale bar , 100 μm .

O O O B-GENEPROD O B-GENEPROD B-EXP_ASSAY I-EXP_ASSAY O B-TISSUE I-TISSUE O O B-GENEPROD O B-ORGANISM B-TISSUE O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Pink1 KO mice neurons .

O O O O B-SUBCELLULAR B-SMALL_MOLECULE O O O O O B-GENEPROD O B-ORGANISM B-CELL_TYPE O

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


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

O O O O O B-EXP_ASSAY O O O O B-GENEPROD O B-CELL_TYPE O

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


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

O O O O O O O O B-GENEPROD O B-CELL_TYPE O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O 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 ) SYCP3 ( red ) on spread spermatocytes at ( A ) zygotene ( Zyg . ) , ( B ) pachytene ( Pac . ) , ( C ) diplotene ( Dip . ) , ( D ) late diplotene ( L . dip . ) , ( E ) prometaphase I ( ProM I ) .

O O O O O B-EXP_ASSAY O B-GENEPROD O O O B-GENEPROD O O O O O B-CELL_TYPE O O O O O O O O O O O O O O O O O O O O O O O O O O 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-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 ) SYCP1 ( pseudocoloured in pink ) on spread spermatocytes at ( F ) zygotene , ( G ) pachytene , ( H ) diplotene .

O O O O O B-EXP_ASSAY O B-GENEPROD O O O B-GENEPROD O O O O O O O B-CELL_TYPE O O O O O O O O O O O O O O O O

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 ) HORMAD1 ( pseudocoloured in blue ) on spread spermatocytes at ( I ) zygotene , ( J ) pachytene , ( K ) diplotene . sex bivalent ( XY ) their pseudoautosomal regions ( arrows ) , as well as round ( arrowheads ) are indicated . Scale bar : 10 µm .

O O O O O B-EXP_ASSAY O B-GENEPROD O O O B-GENEPROD O O O O O O O B-CELL_TYPE O O O O O O O O O O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O

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

O O O O O B-EXP_ASSAY O B-GENEPROD O O O B-GENEPROD O O O O O B-CELL_TYPE O O O O O 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-EXP_ASSAY O B-GENEPROD O O O O O B-GENEPROD O O O O O B-CELL_TYPE 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 ) REC8 - myc ( pseudocoloured in purple ) on a spread mid zygotene spermatocyte . sex univalents ( X , Y ) are indicated . Scale bar : 10 µm .

O O O B-EXP_ASSAY O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O B-CELL_TYPE O O O O B-SUBCELLULAR O B-SUBCELLULAR 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 ) SYCP3 ( red ) on spread pachytene - like ( Pac . - like ) 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 B-EXP_ASSAY O B-GENEPROD O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O B-CELL_TYPE O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR O B-SUBCELLULAR O B-SUBCELLULAR O O O O O O B-SUBCELLULAR O O B-SUBCELLULAR I-SUBCELLULAR 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 ) SYCP3 ( red ) , counterstaining of 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 ) , ( J ) metaphase II ( M II ) . sex bivalent ( XY ) is indicated . Arrows indicate centriolar at poles . Arrowheads indicate Sororin labeling at centromere of Y chromosome . B - F Selected metaphase I autosomal ( B C ) sex ( E F ) bivalents . sex chromosomes ( X , Y ) are indicated . K , L Enlarged metaphase II centromeres . Scale bar : 10 µm .

O O B-EXP_ASSAY O B-GENEPROD O O O B-GENEPROD O O O O O O O B-EXP_ASSAY O O O O O B-CELL_TYPE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-SUBCELLULAR O B-SUBCELLULAR O O O B-GENEPROD O O B-SUBCELLULAR O B-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O O O O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR O B-SUBCELLULAR O B-SUBCELLULAR O O O O O O O O O O B-SUBCELLULAR 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 ) kinetochores ( ACA serum , red ) , counterstaining of with DAPI ( blue ) on spread spermatocytes at ( A ) metaphase I ( M I ) , ( B ) metaphase II ( M II ) , ( C ) anaphase II ( A II ) . Arrowheads indicate poles . centromere of Y chromosome chromatids ( Y ) is indicated . Scale bar : 10 µm .

O O B-EXP_ASSAY O B-GENEPROD O O O B-SUBCELLULAR O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O B-EXP_ASSAY O O O O O B-CELL_TYPE O O O O O O O O O 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-SUBCELLULAR O B-SUBCELLULAR O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O B-SUBCELLULAR 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 ) SYCP3 ( red ) , counterstaining of with DAPI ( blue ) on spread Sgo2 - / - spermatocytes at ( A ) pachytene ( Pac . ) , ( B ) diplotene ( Dip . ) , ( C ) metaphase I ( M I ) , ( D ) metaphase II - like ( M II - like ) . sex bivalent ( XY ) is indicated . Arrowheads indicate round bodies . Arrows in ( A ) indicate pseudoautomal region in sex bivalent , centriolar at poles in ( C , D ) . Scale bar : 10 µm .

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

O O B-EXP_ASSAY O B-GENEPROD O O O B-GENEPROD O O O O O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O B-SMALL_MOLECULE O B-GENEPROD O O O O B-GENEPROD O O O O B-SMALL_MOLECULE O B-CELL_TYPE O O O O O O O B-CELL_TYPE 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-SUBCELLULAR O O O O O O B-CELL_TYPE O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O 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-GENEPROD O O O O B-CELL_TYPE O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O 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-SUBCELLULAR O O B-SUBCELLULAR I-SUBCELLULAR O O O O O B-SUBCELLULAR I-SUBCELLULAR O B-SUBCELLULAR O B-SUBCELLULAR O 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 on ( C ) single sequencing , ( D ) ImageStream ( E ) or automated FISH of RPE - 1 in untreated condition ( blue circles ) or treated with Auxin for 48 h ( red squares ) . Error bars represent on number of analyzed Dashed lines indicate means of aneuploidy rates in untreated ( blue ) or Auxin - treated ( red ) condition . Red asterisks ( IAA ) Blue ( Untreated ) indicate significance over respective mean using a binomial . * 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 mean ) using indicated methods to measure chromosome mis - segregation rate . Bold numbers represent a statistically significant difference from mean for each method . Orange to red gradient highlights chromosomes that mis - segregate ( with at least one method ) at a significantly higher rate compared to mean level ( weak chromosomes ) . green to green gradient highlights chromosomes that mis - segregate ( with at least one method ) at a significantly lower rate compared to 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 sequencing .

O O O O O O B-SUBCELLULAR B-EXP_ASSAY O O O O O O O O O O B-SUBCELLULAR B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O B-SUBCELLULAR O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O B-SUBCELLULAR O O O O O O O B-SUBCELLULAR O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY O B-EXP_ASSAY I-EXP_ASSAY O

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

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

O O O B-EXP_ASSAY I-EXP_ASSAY O B-CELL_LINE I-CELL_LINE I-CELL_LINE O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY B-SUBCELLULAR O B-SMALL_MOLECULE O O O O O O B-SUBCELLULAR O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-SUBCELLULAR O O B-SUBCELLULAR I-SUBCELLULAR O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD 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 proportion of chromosome mis - segregation observed in both lines after auxin addition . Only movies in which dCas9 mScarlet - I dots were clearly visible during whole division were taken into consideration for ( N = 6 - 10 ) .

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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 of mitotic errors leading to aneuploidy in fixed 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 ( G ) proportion of chromosome mis - segregation ( H ) type of different chromosome mis - segregations observed . Error bars represent of two replicates . n > 66 mis - segregation events . Paired t lagging chromosomes vs mis - alignment chromosomes , * p = 0 . 0275 .

O O O O O O O O B-EXP_ASSAY O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-SMALL_MOLECULE O O B-SUBCELLULAR O B-EXP_ASSAY O O O O O O B-SUBCELLULAR O O O B-EXP_ASSAY O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O B-SUBCELLULAR B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O B-SUBCELLULAR B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O B-SUBCELLULAR O O O O B-SUBCELLULAR O O O O O O O O

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

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

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

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

O O O O O O O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O O O 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-SUBCELLULAR O O O O O O O O O O O O O B-SUBCELLULAR O O O O O 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. 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 mean of ( F ) centromere ( n = 4 ) mis - segregation rate ( from Fig . 1F ) ( r = Spearman rank coefficient ) . Lines represent linear regression with a 95 % confidence interval . Data from chr 13 , 14 , 21 22 were excluded from .

O O O O O O O O O O O O O O O O O B-SUBCELLULAR O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O 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-SUBCELLULAR 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 mean of ( G ) centromere CENP - B boxes FISH signal ( n > 50 ) mis - segregation rate , respectively ( r = Spearman rank coefficient ) . Lines represent linear regression with a 95 % confidence interval . Data from chr 13 , 14 , 21 22 were excluded from .

O O O O O O O O O O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR B-EXP_ASSAY O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O B-SUBCELLULAR 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 ) to measure CENP - B at specific chromosome in untreated . 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 normalized CENP - B at every chromosome over mean on each metaphase spread ( n > 37 per chromosome ) ± . Acrocentric chromosomes 13 , 14 , 21 22 were not analyzed are marked by a . One - way ANOVA with post - hoc Tukey ' s multiple comparison shows high diversity between chromosomes . t - against mean was used to estimate statistical significance for each chromosome . * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 ; * * * * p < 0 . 0001 .

O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O B-SUBCELLULAR O O O O O O O O O O O B-SUBCELLULAR O O O B-SUBCELLULAR I-SUBCELLULAR O O O O O O O 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-SUBCELLULAR O O O O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O 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 mean of CENP - B IF - FISH signal ( n > 37 ) mis - segregation rate ( r = Spearman rank coefficient ) . 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 sum of normalized reads of different HOR arrays representing CENP - C binding following CUT & RUN , sequencing centromere mapping . were treated for 6 h with IAA to deplete CENP - A . Error bars represent of three independent experiments dashed represents mean . Acrocentric chromosomes 13 - 14 , 21 22 were marked by a . t - against mean was used to estimate 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 B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY B-SUBCELLULAR B-EXP_ASSAY O O O O O O O B-SMALL_MOLECULE O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O B-SUBCELLULAR O 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 mean of CENP - C reads ( n = 3 ) CENP - B reads ( n = 3 ) ( r = Spearman rank coefficient ) . Data from chr 1 , 5 19 were excluded from in addition to chr 13 , 14 , 21 22 to better assess correlation without FISH correction lines represent linear regression with a 95 % confidence interval .

O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O

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 mean of CENP - C reads ( n = 3 ) mis - segregation rate ( r = Spearman rank coefficient ) . lines represent linear regression with 95 % confidence interval . Data from chr 13 , 14 , 21 22 were excluded from . lines represent linear regression with a 95 % confidence interval .

O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O

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 ) 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 of chromosome mis - segregation in indicated lines with without IAA addition for 24 h . Error bars represent of three independent experiments ( n > 200 per experiment ) . Dunnett ' s multiple comparisons was used to compare conditions . Grey lines separate independent experiments . Fold changes between grey red bars are also indicated .

O O O B-EXP_ASSAY O B-SUBCELLULAR I-SUBCELLULAR O O O B-SUBCELLULAR I-SUBCELLULAR O O O B-EXP_ASSAY O B-CELL_LINE I-CELL_LINE I-CELL_LINE B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O B-SUBCELLULAR B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

B. Representative 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 centromere strength model with microtubule destabilizer BAL27862 compound . D . Representative image of centromere 17 ( red ) centromere X ( green ) FISH on RPE - 1 treated with 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 frequency of chromosome mis - segregation into micronuclei after 28 h treatment with BAL27862 drug at indicated . Error bars represent of three independent experiments , dashed represents mean . n > 40 micronuclei were analyzed . Dunnett ' s multiple comparisons , * : p < 0 . 05 . DMSO was used at same 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 mFISH stained RPE - 1 metaphase spread . Chromosome 3 homologs are indicated by arrows with single CENP - B box chromosomes signal magnified in insets . Scale bar represents 5 µm .

O O O B-EXP_ASSAY O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O B-SUBCELLULAR I-SUBCELLULAR O O O O O O O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O O O

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 ) centromere 3 ( green ) FISH on RPE - 1 interphase nucleus after 48 h IAA treatment . CENP - B box signal at 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 of CENP - B box FISH at chromosome 3 homolog contained in main nucleus versus homolog present in micronucleus Error bars represent of three independent experiments . n = 69 . Unpaired t , * * * * 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 ( 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 show a micronucleus or main nucleus containing Y chromosome ( red ) detected by FISH in no - doxycycline or doxycycline treated 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 mean ( n ≥ 3 ) frequency of micronuclei containing chromosome Y or chromosome X ± doxycycline treatment for 72 h IAA for 48 h ± . n > 200 with a micronucleus . Mann - Whitney . * 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 mean ) using indicated methods to measure chromosome mis - segregation rate . Bold numbers represent significant statistical difference from mean for each method . Orange to red gradient highlights chromosomes that mis - segregate ( with at least one method ) at a significantly higher rate compared to mean level ( weak chromosomes ) . green to green gradient highlights chromosomes that mis - segregate ( with at least one method ) at a significantly lower rate compared to 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 karyotype distribution of DLD - 1 RPE - 1 in untreated or Auxin - treated

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

O O O B-EXP_ASSAY O O B-SUBCELLULAR O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O B-EXP_ASSAY O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR B-EXP_ASSAY O O O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O

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 indicated features in DLD - 1 ( n = 1 ) . lines represent linear regression with 95 % confidence band . Data from chr 13 , 14 , 21 , 22 , Y SN were excluded from .

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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 of three independent experiments ( n > 170 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 ) after siRNA against CENP - A / HJURP for 48 h . CENP - B box signals at micronuclei are magnified in insets . Scale bar represent 5 µm . ( Right ) Bar graph represents mean of CENP - B box FISH contained in main nucleus versus one present in micronucleus ± . Color dots represent independent experiments . ( n > 43 micronuclei 840 main nucleus ) Mann - Whitney U : * * * p = 0 . 0001 .

O O O O O O B-EXP_ASSAY O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O O O O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR B-EXP_ASSAY O O O B-SUBCELLULAR O O O O B-SUBCELLULAR O O O O O O O O O O O O B-SUBCELLULAR O O B-SUBCELLULAR O O O O O O O O O O O O O O O

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 mis - segregation rate or CENP - C ( n = 3 ) reads over chromosome mis - segregation rate ( r = Spearman rank coefficient ) . lines represent linear regression with 95 % confidence interval . Data from chr 13 , 14 , 21 22 were excluded from . 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 Epac1 . ( A ) Immunoblot of BAT stimulated with norepinephrine ( NE ) for indicated .

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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 of BAT stimulated with NE for 5 minutes in presence of Rapamycin ( Rapa ) , Torin or Wortmannin ( Wrtm ) for indicated .

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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 of BAT stimulated with 8 - Br - cAMP for 5 minutes in presence of Rapa , Torin or Wrtm for indicated .

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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 of BAT stimulated with NE for 5 minutes in presence of Wrtm , H89 or ESI - 09 for indicated .

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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 of BAT stimulated with 8 - Br - cAMP for 5 minutes in presence of Wrtm , H89 or ESI - 09 for indicated . 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 cold activate mTORC2 in vivo . ( A ) Immunoblot of BAT from control mice treated with either norepinephrine ( NE ) or vehicle for 30 minutes for indicated ( 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 of BAT from AdRiKO control mice treated with either norepinephrine ( NE ) or vehicle for 30 minutes for indicated ( 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 of BAT from AdRiKO control mice housed at either 22 ° C or 4 ° C for 2h for indicated ( 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 ) of AdRiKO control mice housed at 22 ° C ( n = 11 ( control ) , n = 9 ( AdRiKO ) ) .

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


( E ) of AdRiKO control mice housed at 30 ° C for 2 ( 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 ) loss upon cold exposure of AdRiKO control mice ( n = 20 ( control ) , n = 17 ( AdRiKO ) ) . Data represent mean ± . Statistically significant differences between AdRiKO control mice were determined with unpaired Student ' s t - 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 ) . 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 of sWAT sections from AdRiKO 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 ( NEFAs ) in plasma of AdRiKO control mice ( n = 6 / group ) .

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


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

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


( D ) Representative H & amp ; amp ; E of BAT sections from AdRiKO 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 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 control mice ( n = 6 / group ) . Data represent mean ± . Statistically significant differences between AdRiKO control mice were determined with unpaired Student ' s t - 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 - are indicated with a number sign ( # = p < 0 . 05 ; # # = p < 0 . 01 ; # # # = p < 0 . 001 ) . exact p - value for each significant difference can be found in Appendix Table S2 .

O O O B-SMALL_MOLECULE O B-TISSUE O B-ORGANISM O B-ORGANISM O O O O O O O O O O O O O O O O O B-ORGANISM O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) 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 β - oxidation . ( A ) mRNA of indicated genes in BAT of AdRiKO 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 of BAT from AdRiKO control mice housed at 22 ° C or at 4 ° C for 8h for indicated ( 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 of indicated genes in BAT of AdRiKO control mice housed at 22 ° C or at 4 ° C for 8h ( n = 6 / group , each lane represents a mix of 3 mice ) .

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

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(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 control mice housed at 22 ° C or at 4 ° C for 4h ( n = 3 / group ) .

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(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 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 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 control mice upon cold exposure ( n = 8 / group ) . Data represent mean ± . Statistically significant differences between AdRiKO control mice were determined with unpaired Student ' s t - 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 - are indicated with a number sign ( # = p < 0 . 05 ; # # = p < 0 . 01 ; # # # = p < 0 . 001 ) . 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 glycolysis ( A ) 2 - Deoxyglucose - 6 - phosphate ( 2DG6P ) accumulation in BAT of AdRiKO 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 control mice housed at 22 ° C or at 4 ° C for 4h ( n = 7 / group ) .

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(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 of BAT from AdRiKO control mice housed at 22 ° C or at 4 ° C for 8h for indicated ( 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 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 of isolated plasma membranes from BAT of AdRiKO control mice housed at 22 ° C or at 4 ° C for 8h for indicated ( 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 of mitochondrial fractions from BAT of AdRiKO control mice housed at 22 ° C or at 4 ° C for 4h for indicated ( 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 ) hexokinase activity in BAT of AdRiKO 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 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 ± . Statistically significant differences between AdRiKO control mice were determined with unpaired Student ' s t - 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 - are indicated with a number sign ( # = p < 0 . 05 ; # # = p < 0 . 01 ; # # # = p < 0 . 001 ) . exact p - value for each significant difference can be found in Appendix Table S2 .

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(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 thermogenic defect in AdRiKO mice . ( A ) HKII mRNA expression level in BAT of AdRiKO 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 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 ) of AdRiKO 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).