( A ) Survival of Drosophila melanogaster ( W1118 ) larvae fed with 2 µmol NAT / g - diet 2 d after heat stress at 38 . 5oC for 60 min ( data are means ± SEM ; n = 10 ) . * * P < 0 . 01 vs . zero time .

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

(A) Survival of Drosophila melanogaster (W1118) larvae fed with 2 µmol NAT/g-diet 2 d after heat stress at 38.5oC for 60 min (data are means ± SEM; n=10). **P<0.01 vs. zero time.


( B ) Effects of NAT feeding on expression of antioxidant enzymes , catalase , SOD1 , and SOD2 ( data are means ± SEM ; n = 10 ) . * P < 0 . 05 vs . zero time .

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

(B) Effects of NAT feeding on expression of antioxidant enzymes, catalase, SOD1, and SOD2 (data are means ± SEM; n=10). *P<0.05 vs. zero time.


( C ) Representative images of nuclei ( left ) and FoxO ( middle ) in the fat body of Drosophila larvae before and after NAT feeding for 12 h . White signals in the merged image ( right ) show the overlap of both signals . Scale bar : 50 µm . ( D ) Relative whiteness intensities before and after NAT feeding ( data are means ± SEM ; n = 15 ) . * * P < 0 . 01 vs . zero time .

O 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 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

(C) Representative images of nuclei (left) and FoxO (middle) in the fat body of Drosophila larvae before and after NAT feeding for 12 h. White signals in the merged image (right) show the overlap of both signals. Scale bar: 50 µm. (D) Relative whiteness intensities before and after NAT feeding (data are means ± SEM; n=15). **P<0.01 vs. zero time.


( E ) Effects of NAT feeding on FoxO expression in the fat body of Drosophila larvae ( data are means ± SEM ; n = 8 ) . * P < 0 . 05 , * * P < 0 . 01 vs . zero time .

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

(E) Effects of NAT feeding on FoxO expression in the fat body of Drosophila larvae (data are means ± SEM; n=8). *P<0.05, **P<0.01 vs. zero time.


( F ) Effects of N - acetyltransferase ( Dat ) knockdown in gut enteroendocrine cells ( TK - Gut - Gal4 > UAS - dsDat ) on survival of Drosophila larvae ( data are means ± SEM ; n = 15 ) . * * P < 0 . 01 vs . without NAT .

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(F) Effects of N-acetyltransferase (Dat) knockdown in gut enteroendocrine cells (TK-Gut-Gal4>UAS-dsDat) on survival of Drosophila larvae (data are means ± SEM; n=15). **P<0.01 vs. without NAT.


( A ) Survival of Drosophila S2 cells treated with preheating , 100 µM NAT , and 100 µM NAT with 10 mM 2 - aminobicyclo - ( 2 , 2 , 1 ) - heptane - 2 - carboxylic acid ( BCH ) after heat stress at 42oC for 60 min ( data are means ± SEM ; n = 10 ) . After pretreatment with chemicals for 12 h , those were removed by changing the medium . * P < 0 . 05 , * * P < 0 . 01 vs . PBS .

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

(A) Survival of Drosophila S2 cells treated with preheating, 100 µM NAT, and 100 µM NAT with 10 mM 2-aminobicyclo-(2,2,1)-heptane-2-carboxylic acid (BCH) after heat stress at 42oC for 60 min (data are means ± SEM; n=10). After pretreatment with chemicals for 12 h, those were removed by changing the medium. *P<0.05, **P<0.01 vs. PBS.


( B ) Survival of Drosophila S2 cells pretreated with 100 µM NAT , 100 µM NAT with 30 µg / ml antimycin A , and preheating with or without 30 µg / ml antimycin A 48 h after heat stress at 42oC for 60 min ( data are means ± SEM ; n = 10 ) . After pretreatment with chemicals for 12 h , they were removed by changing the medium . * P < 0 . 05 vs . PBS .

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(B) Survival of Drosophila S2 cells pretreated with 100 µM NAT, 100 µM NAT with 30 µg/ml antimycin A, and preheating with or without 30 µg/ml antimycin A 48 h after heat stress at 42oC for 60 min (data are means ± SEM; n=10). After pretreatment with chemicals for 12 h, they were removed by changing the medium. *P<0.05 vs. PBS.


( C ) Effects of 100 µM NAT on MitoRed localization in mitochondria of S2 cells . Upper : representative images of time - dependent changes in MitoRed localization in mitochondria after adding NAT . Scale bar : 20 µm . Lower : relative intensities of MitoRed fluorescent signals in mitochondria were quantified by microplate reader . Different letters above bars represent significant differences [ P < 0 . 05 ( data are means ± SEM , n = 12 ) ] .

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(C) Effects of 100 µM NAT on MitoRed localization in mitochondria of S2 cells. Upper: representative images of time-dependent changes in MitoRed localization in mitochondria after adding NAT. Scale bar: 20 µm. Lower: relative intensities of MitoRed fluorescent signals in mitochondria were quantified by microplate reader. Different letters above bars represent significant differences [P <0.05 (data are means ± SEM, n=12)].


( D ) ROS concentrations in S2 cells after adding 100 µM NAT ( data are means ± SEM ; n = 12 ) . * P < 0 . 05 vs . zero time .

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

(D) ROS concentrations in S2 cells after adding 100 µM NAT (data are means ± SEM; n=12). *P<0.05 vs. zero time.


( F ) Effects of dsRNA targeting of FoxO on NAT - dependent expression of Keap1 ( data are means ± SEM ; n = 12 ) . * P < 0 . 05 , * * P < 0 . 01 vs . without NAT . Note that NAT feeding did not increase Keap1 expression in FoxO knockdown flies .

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(F) Effects of dsRNA targeting of FoxO on NAT-dependent expression of Keap1 (data are means ± SEM; n=12). *P<0.05, **P<0.01 vs. without NAT. Note that NAT feeding did not increase Keap1 expression in FoxO knockdown flies.


( G ) Effect of NAT on growth of HCT116 colon cancer cells in mice ( BALB / cSlc - nu / nu , 6 wks old , female ) ( data are means ± SEM ; n = 5 ) . * P < 0 . 05 , * * P < 0 . 01 vs . without NAT . Test mice began to freely drink 5 mg / ml NAT solution for 7 d before transplantation of the tumor cells on day 0 : the average amount of NAT consumed by each mouse was about 8 . 8 mg / day ( 0 . 4 - 0 . 5 g / kg weight / day ) . HCT116 cells ( 5 . 0 x 106 cells ) were transplanted into each test mouse using a 1 ml syringe with a 27G needle .

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

(G) Effect of NAT on growth of HCT116 colon cancer cells in mice (BALB/cSlc-nu/nu, 6 wks old, female) (data are means ± SEM; n=5). *P<0.05, **P<0.01 vs. without NAT. Test mice began to freely drink 5 mg/ml NAT solution for 7 d before transplantation of the tumor cells on day 0: the average amount of NAT consumed by each mouse was about 8.8 mg/day (0.4 - 0.5 g/kg weight/day). HCT116 cells (5.0 x 106 cells) were transplanted into each test mouse using a 1 ml syringe with a 27G needle.


a , Percentage of ciliated cells in control ( Ctrl ) and IFT20 − MEFs ( * P = 0 . 030 , n = 3 ) and wild - type ( WT ) and Ift88 − / − KECs ( * * P = 0 . 003 , 25 cells each , n = 3 ) after 24 h of serum removal . Arrows , cilia .

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a, Percentage of ciliated cells in control (Ctrl) and IFT20− MEFs (*P = 0.030, n = 3) and wild-type (WT) and Ift88−/− KECs (**P = 0.003, 25 cells each, n = 3) after 24 h of serum removal. Arrows, cilia.


b , LC3 immunoblot ( left ) for autophagic flux quantification ( right ) in Ctrl and IFT20 − MEFs ( top , * * P = 0 . 0003 , n = 7 ) , and in WT and Ift88 − / − KECs ( bottom , * P = 0 . 035 , n = 9 ) . PI , protease inhibitors ; S , serum .

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b, LC3 immunoblot (left) for autophagic flux quantification (right) in Ctrl and IFT20− MEFs (top, **P = 0.0003, n = 7), and in WT and Ift88−/− KECs (bottom, *P = 0.035, n = 9). PI, protease inhibitors; S, serum. c


c , Autophagic flux in WT and Ift88 − / − KECs by mCherry - GFP - LC3 puncta quantification . Puncta : yellow , autophagosomes ; red only , autophagolysosomes ; total , both together ( * * P = 0 . 008 , * P = 0 . 025 ) .

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c, Autophagic flux in WT and Ift88−/−KECs by mCherry-GFP-LC3 puncta quantification. Puncta: yellow, autophagosomes; red only, autophagolysosomes; total, both together (**P = 0.008, *P = 0.025).


. d , LC3 immunoblot ( left ) for quantification of autophagosome formation ( right ) . ( * P = 0 . 048 , n = 4 ) .

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. d, LC3 immunoblot (left) for quantification of autophagosome formation (right). (*P = 0.048, n = 4).


e , Electron microscopy images ( left ) and morphometric quantification of autophagic vacuoles ( AV , right ) in Ctrl and IFT20 − MEFs ( top , * * P = 0 . 001 , 10 fields in 2 experiments ) and KECs WT and Ift88 − / − ( bottom , * P = 0 . 037 , 5 fields ) . Arrows , autophagic vacuoles ( AV , black ) and endosomal compartments ( yellow ) .

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e, Electron microscopy images (left) and morphometric quantification of autophagic vacuoles (AV, right) in Ctrl and IFT20− MEFs (top, **P = 0.001, 10 fields in 2 experiments) and KECs WT and Ift88−/− (bottom, *P = 0.037, 5 fields). Arrows, autophagic vacuoles (AV, black) and endosomal compartments (yellow).


f , Percentage of ciliated MEFs after induction of cilia resorption with 50 ng ml − 1 platelet - derived growth factor ( PDGF ) treatment . ( * * P = 0 . 001 , * * * P = 0 . 0009 , 121 , 230 and 162 cells , n = 5 ) .

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f, Percentage of ciliated MEFs after induction of cilia resorption with 50 ng ml−1 platelet-derived growth factor (PDGF) treatment. (**P = 0.001, ***P = 0.0009, 121, 230 and 162 cells, n = 5).


g , LC3immunoblot ( top ) for autophagic flux quantification ( bottom ) upon PDGF treatment ( * P = 0 . 005 , n = 3 ) . Scale bars in a , 10 µm . n . s . , statistically non - significant . Mean ± s . d . in b , d and mean ± s . e . m . in other panels .

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g, LC3immunoblot (top) for autophagic flux quantification (bottom) upon PDGF treatment (*P = 0.005, n = 3). Scale bars in a, 10 µm. n.s., statistically non-significant. Mean ± s.d. in b, d and mean ± s.e.m. in other panels.


a , GFP - LC3 puncta pattern ( left ) and quantification ( right ) in MEFs treated with purmorphamine ( Purmo ; top , * P = 0 . 028 , n = 4 ) , in Ptc − / − MEFs ( middle , * P = 0 . 015 , n = 4 ) and after myc - GLI1 overexpression ( bottom , * P = 0 . 012 , n = 3 ) .

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a, GFP-LC3 puncta pattern (left) and quantification (right) in MEFs treated with purmorphamine (Purmo; top, *P = 0.028, n = 4), in Ptc−/− MEFs (middle, *P = 0.015, n = 4) and after myc-GLI1 overexpression (bottom, *P = 0.012, n = 3).


b , Autophagic flux by mCherry - GFP - LC3 puncta quantification upon purmorphamine treatment in MEFs in the presence of serum . Yellow , autophagosomes ; red only , autophagolysosomes ; total , both compartments . ( * P = 0 . 0323 , 40 fields ) .

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

b, Autophagic flux by mCherry-GFP-LC3 puncta quantification upon purmorphamine treatment in MEFs in the presence of serum. Yellow, autophagosomes; red only, autophagolysosomes; total, both compartments. (*P = 0.0323, 40 fields).


c , LC3 immunoblot ( left ) for autophagic flux quantification ( right ) in Ctrl ( * * P = 0 . 001 ) and MEFs knocked down for SMO ( SMO − ) ( * * P = 0 . 0001 ) upon purmorphamine treatment ( * P = 0 . 009 , n = 4 ) .

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c, LC3 immunoblot (left) for autophagic flux quantification (right) in Ctrl (**P = 0.001) and MEFs knocked down for SMO (SMO−) (**P = 0.0001) upon purmorphamine treatment (*P = 0.009, n = 4).


d , Autophagic flux by mCherry - GFP - LC3 puncta quantification in WT and Ift88 − / − KECs upon purmorphamine treatment ( * P = 0 . 046 , * * P = 0 . 008 , 40 fields ) .

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d, Autophagic flux by mCherry-GFP-LC3 puncta quantification in WT and Ift88−/− KECs upon purmorphamine treatment (*P = 0.046, **P = 0.008, 40 fields).


. e , LC3 immunoblot ( left ) for autophagic flux quantification ( right ) in Ift88 − / − KECs overexpressing myc - GLI1 ( * * P = 0 . 00006 , n = 3 ) .

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. e, LC3 immunoblot (left) for autophagic flux quantification (right) in Ift88−/− KECs overexpressing myc-GLI1 (**P = 0.00006, n = 3).


f , Autophagic flux by mCherry - GFP - LC3puncta quantification in MEFs treated with cyclopamine upon serum removal ( * P = 0 . 011 , * * P = 0 . 013 , 25 fields ) . Scale bars , 10 µm . Mean ± s . d . in c , f and mean ± s . e . m . in other panels .

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f, Autophagic flux by mCherry-GFP-LC3puncta quantification in MEFs treated with cyclopamine upon serum removal (*P = 0.011, **P = 0.013, 25 fields). Scale bars, 10 µm. Mean ± s.d. in c, f and mean ± s.e.m. in other panels.


a , Co - immunostaining ( top ) and 3D reconstruction ( middle and bottom ) for the indicated autophagy - related proteins ( ATGs , green ) and acetylated tubulin ( red , ciliary axoneme marker ) in KECs maintained in the absence of serum for 24 h . Yellow arrows , colocalization .

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a, Co-immunostaining (top) and 3D reconstruction (middle and bottom) for the indicated autophagy-related proteins (ATGs, green) and acetylated tubulin (red, ciliary axoneme marker) in KECs maintained in the absence of serum for 24 h. Yellow arrows, colocalization.


b , Schematic representation of the ATGs associated with the axoneme ( top left ) . Co - immunostaining for ATGs and acetylated tubulin in cilia isolated by peeling off . Individual channels and merged images are shown .

b, Schematic representation of the ATGs associated with the axoneme (top left). Co-immunostaining for ATGs and acetylated tubulin in cilia isolated by peeling off. Individual channels and merged images are shown.


c , Co - immunostaining ( bottom ) and 3D reconstruction ( top ) for ATGs ( green ) and gamma - tubulin ( red , basal body marker ) in the same cells and conditions . Arrows , colocalization ( yellow ) , no colocalization ( white ) .

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c, Co-immunostaining (bottom) and 3D reconstruction (top) for ATGs (green) and gamma-tubulin (red, basal body marker) in the same cells and conditions. Arrows, colocalization (yellow), no colocalization (white).


d - f , Percentage of cells with colocalizing ATGs in the basal body ( BB ) in WT and Ift88 − / − KECs in the presence or absence of serum . d , Serum - and IFT - dependent association of ATGs with the BB ( VPS34 , † P = 0 . 04 , * * P = 0 . 002 , n = 4 ; ATG16L , † † P = 0 . 0003 , * * P = 0 . 0009 , 15 cells each per experiment , n = 7 ) . e , IFT - dependent but serum - independent association of ATGs with the BB ( * * P = 0 . 001 , * * * P = 9 . 33768 × 10 − 6 , n = 4 ; * P = 0 . 018 , 15 cells each per experiment , n = 5 ) . f , BB association of ATGs independent on serum or IFT ( ATG5 , n = 5 ; LC3 , n = 8 ; 15 cells each per experiment ) . Scale bars , 10 & amp ; amp ; mgr ; m . Mean ± s . e . m .

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d-f, Percentage of cells with colocalizing ATGs in the basal body (BB) in WT and Ift88−/− KECs in the presence or absence of serum. d, Serum- and IFT-dependent association of ATGs with the BB (VPS34, †P = 0.04, **P = 0.002, n = 4; ATG16L, ††P = 0.0003, **P = 0.0009, 15 cells each per experiment, n = 7). e, IFT-dependent but serum-independent association of ATGs with the BB (**P = 0.001, ***P = 9.33768 × 10−6, n = 4; *P = 0.018, 15 cells each per experiment, n = 5). f, BB association of ATGs independent on serum or IFT (ATG5, n = 5; LC3, n = 8; 15 cells each per experiment). Scale bars, 10 &amp;mgr;m. Mean ± s.e.m.


a , b , Immunoblot for the indicated proteins of WT and Ift88 − / − KECs subjected to co - immunoprecipitation of IFT20 ( a ) and ATG16L ( b ) . Inp , 1 / 10 input ; IP , immunoprecipitate ; FT , 1 / 10 flow - through .

a, b, Immunoblot for the indicated proteins of WT and Ift88−/− KECs subjected to co-immunoprecipitation of IFT20 (a) and ATG16L (b). Inp, 1/10 input; IP, immunoprecipitate; FT, 1/10 flow-through.


c , Immunoblot for ATG16L and clathrin in MEF homogenate ( Hom ) , and the pellets from 1 h centrifugation at 100 , 000g ( organelles ( Org ) ) , 300 , 000g ( 300k ) and 500 , 000g ( vesicles , 500k ) .

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c, Immunoblot for ATG16L and clathrin in MEF homogenate (Hom), and the pellets from 1 h centrifugation at 100,000g (organelles (Org)), 300,000g (300k) and 500,000g (vesicles, 500k).


d , Immunoblot for IFT20 and ATG16L in the same fractions isolated from Ctrl , IFT20 − and IFT88 − MEFs in the presence ( top ) and absence ( bottom ) of serum .

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d, Immunoblot for IFT20 and ATG16L in the same fractions isolated from Ctrl, IFT20− and IFT88− MEFs in the presence (top) and absence (bottom) of serum.


e , Immunogold electron microscopy for ATG16L ( 15 - nm particles ) and IFT20 ( 10 - nm particles ) in vesicles from MEFs isolated by the same method as in c . Right , quantification of IFT20 and ATG16L presence in the vesicles ( 11 fields ) . Mean ± s . e . m .

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e, Immunogold electron microscopy for ATG16L (15-nm particles) and IFT20 (10-nm particles) in vesicles from MEFs isolated by the same method as in c. Right, quantification of IFT20 and ATG16L presence in the vesicles (11 fields). Mean ± s.e.m.


a , b , Ciliated Atg5 − / − MEFs in serum + ( * P = 0 . 038 , 25 cells each per experiment , n = 3 ) ( a ) or serum − ( * P = 0 . 006 , nonlinear fit regression , 25 cells each per experiment , n = 3 ) ( b ) .

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a, b, Ciliated Atg5−/− MEFs in serum+ (*P = 0.038, 25 cells each per experiment, n = 3) (a) or serum− (*P = 0.006, nonlinear fit regression, 25 cells each per experiment, n = 3) (b).


c , Scanning electron microscopy ( SEM ) of primary cilia . Arrows , cilia - associated vesicles ; arrowheads , ciliary pocket .

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c, Scanning electron microscopy (SEM) of primary cilia. Arrows, cilia-associated vesicles; arrowheads, ciliary pocket.


. d , Cilia length quantification from SEM ( * P = 0 . 027 n = 28 ; * * P = 0 . 001 , † † P = 0 . 0001 , n = 23 ) .

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. d, Cilia length quantification from SEM (*P = 0.027 n = 28; **P = 0.001, ††P = 0.0001, n = 23).


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e, f, GLI1 (*P = 0.04, n = 3) (e) and GLI2 (***P = 0.0005, **P = 0.0046, †P = 0.0132, n = 3) (f) messenger RNA expression in Atg5−/− MEFs.


g , h , IFT20 immunoblot in Atg5 − / − ( g ) and WT MEFs with lysosomal inhibitors ( NL ) ( h ) .

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g, h, IFT20 immunoblot in Atg5−/− (g) and WT MEFs with lysosomal inhibitors (NL) (h).


i , IFT20 immunoblot in cytosol ( Cyt ) , homogenate ( Hom ) and autophagosomes ( APG ) . j , IFT20 protein levels in Atg5 − / − MEFs ( * P = 0 . 038 , n = 6 ) . k , IFT20 immunoblot in Atg5 − / − MEFs . n . s . , statistically non - significant . Mean ± s . d . in d and j and mean ± s . e . m . in other panels .

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i, IFT20 immunoblot in cytosol (Cyt), homogenate (Hom) and autophagosomes (APG). j, IFT20 protein levels in Atg5−/− MEFs (*P = 0.038, n = 6). k, IFT20 immunoblot in Atg5−/− MEFs. n.s., statistically non-significant. Mean ± s.d. in d and j and mean ± s.e.m. in other panels.


( A ) Breast cancer cell lines were sorted using CD44 for luminal MCF - 7 cells ( left panel ) or EpCAM for MDA - MB - 231 cells ( right panel ) . Serial mammosphere assays were performed using 4000 cells for each cell fraction , counting spheres after 3 weeks . Plots show the mean ± SEM of mammospheres formed with 4000 cells from 3 independent experiments performed in triplicate .

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(A) Breast cancer cell lines were sorted using CD44 for luminal MCF-7 cells (left panel) or EpCAM for MDA-MB-231 cells (right panel). Serial mammosphere assays were performed using 4000 cells for each cell fraction, counting spheres after 3 weeks. Plots show the mean ± SEM of mammospheres formed with 4000 cells from 3 independent experiments performed in triplicate.


( B ) Extreme limiting dilution assay ( ELDA ) of MCF - 7 cells . Sorted cells were serially diluted ( 1250 , 625 , 312 , 156 and 78 cells ) and then seeded in 96 - well plates . After one week , the cells were fixed , stained and counted . The lower limit for well repopulation in CD44 + cells was 312 cells , compared with 615 cells for both unsorted and CD44 - populations . The data obtained were evaluated using the ELDA software . p values that test the single - hit hypothesis using the likelihood ratio test are shown . The curves are depicted in Fig

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(B) Extreme limiting dilution assay (ELDA) of MCF-7 cells. Sorted cells were serially diluted (1250, 625, 312, 156 and 78 cells) and then seeded in 96-well plates. After one week, the cells were fixed, stained and counted. The lower limit for well repopulation in CD44+ cells was 312 cells, compared with 615 cells for both unsorted and CD44- populations. The data obtained were evaluated using the ELDA software. p values that test the single-hit hypothesis using the likelihood ratio test are shown. The curves are depicted in Fig EV1


( C ) Relative expression of the stemness factors SOX2 and OCT4 and the EMT markers SNAIL , Vimentin and TWIST in MCF - 7 / CD44 + cells . The fold change was calculated using GAPDH as an internal control using the 2 - ΔΔCT method comparing stem or non - stem cells versus the total cell line . The box and whiskers graph shows the range , median and quartile interval values from 3 independent experiments .

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(C) Relative expression of the stemness factors SOX2 and OCT4 and the EMT markers SNAIL, Vimentin and TWIST in MCF-7/CD44+ cells. The fold change was calculated using GAPDH as an internal control using the 2-ΔΔCT method comparing stem or non-stem cells versus the total cell line. The box and whiskers graph shows the range, median and quartile interval values from 3 independent experiments.


( D ) miR - 10b overexpression affecting stemness and EMT factors . Relative quantification of miR - 10b ( relative to RNU6B ) , HOXD10 and RHOC ( relative to GAPDH ) was performed in MCF - 7 / CD44 + fractions compared to non - enriched cells . Box plots show the range , median and quartile interval values from 3 independent experiments . A two - tailed Student ' s t test was used to compare the two groups ( p < 0 . 05 ) . SEM , standard error of the mean ; TLDA , TaqMan Low Density Arrays ; and EMT , epithelial - mesenchymal transition .

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(D) miR-10b overexpression affecting stemness and EMT factors. Relative quantification of miR-10b (relative to RNU6B), HOXD10 and RHOC (relative to GAPDH) was performed in MCF-7/CD44+ fractions compared to non-enriched cells. Box plots show the range, median and quartile interval values from 3 independent experiments. A two-tailed Student's t test was used to compare the two groups (p < 0.05). SEM, standard error of the mean; TLDA, TaqMan Low Density Arrays; and EMT, epithelial-mesenchymal transition.


( E ) Relative expression of stemness factors SOX2 and OCT4 and EMT markers SNAIL and Vimentin in MDA - MB - 231 / EpCAM + cells . The fold change was calculated using GAPDH as an internal control using the 2 - ΔΔCT method comparing stem or non - stem cells versus the total cells . The box and whiskers graph shows the range , median and quartile interval values from 3 independent experiments .

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(E) Relative expression of stemness factors SOX2 and OCT4 and EMT markers SNAIL and Vimentin in MDA-MB-231/EpCAM+ cells. The fold change was calculated using GAPDH as an internal control using the 2-ΔΔCT method comparing stem or non-stem cells versus the total cells. The box and whiskers graph shows the range, median and quartile interval values from 3 independent experiments.


( F ) Extreme Limiting Dilution Assay ( ELDA ) of MDA - MB - 231 cells . Sorted cells were serially diluted ( 1250 , 625 , 312 , 156 and 78 cells ) and then seeded in 96 - well plates . After one week , the cells were fixed , stained and counted . The lower limit for well repopulation in EpCAM + cells was 156 cells compared with 625 cells for both unsorted and EpCAM - populations . The data obtained were evaluated using the ELDA software . p values that test the single - hit hypothesis using the likelihood ratio test are shown . The curves are depicted in Fig

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Extreme Limiting Dilution Assay (ELDA) of MDA-MB-231 cells. Sorted cells were serially diluted (1250, 625, 312, 156 and 78 cells) and then seeded in 96-well plates. After one week, the cells were fixed, stained and counted. The lower limit for well repopulation in EpCAM+ cells was 156 cells compared with 625 cells for both unsorted and EpCAM- populations. The data obtained were evaluated using the ELDA software. p values that test the single-hit hypothesis using the likelihood ratio test are shown. The curves are depicted in Fig EV1


( G ) Nine miRNAs are commonly regulated in both CD44 + and EpCam + breast cancer cells . Quantitative RT - PCR of miRNAs was performed on sorted MCF - 7 and MDA - MB - 231 cell populations using TaqMan Low Density Arrays ( TLDA ) . Results were plotted as a heat map comparing CD44 + to CD44 - cells ( MCF - 7 ) and EpCam + to EpCam - cells . The scale is shown below the map .

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(G) Nine miRNAs are commonly regulated in both CD44+ and EpCam+ breast cancer cells. Quantitative RT-PCR of miRNAs was performed on sorted MCF-7 and MDA-MB-231 cell populations using TaqMan Low Density Arrays (TLDA). Results were plotted as a heat map comparing CD44+ to CD44- cells (MCF-7) and EpCam+ to EpCam- cells. The scale is shown below the map.


( A ) Upper panel , left : MCF - 7 / CD44 + cells were transfected with anti - miR - 10b or a scrambled control , and qRT - PCR was performed . Upper panel , right : An aliquot of these cells was subjected to mammosphere - forming assays . The graph shows the mean ± SEM of the number of formed mammospheres per 4000 seeded cells in 3 independent experiments . Lower panel , left : Expression of miR - 10b relative to RNU6B in MCF - 7 cells stably transfected with a plasmid containing the miR - 10b gene versus an empty vector . Results are the mean ± SEM values from 3 independent experiments . Lower panel , right : miR - 10b - overexpressing MCF - 7 cells analyzed by serial mammosphere assays . The mean ± SEM of the number of formed mammospheres per 4000 seeded cells in 3 independent experiments was plotted on each bar .

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(A) Upper panel, left: MCF-7/CD44+ cells were transfected with anti-miR-10b or a scrambled control, and qRT-PCR was performed. Upper panel, right: An aliquot of these cells was subjected to mammosphere-forming assays. The graph shows the mean ± SEM of the number of formed mammospheres per 4000 seeded cells in 3 independent experiments. Lower panel, left: Expression of miR-10b relative to RNU6B in MCF-7 cells stably transfected with a plasmid containing the miR-10b gene versus an empty vector. Results are the mean ± SEM values from 3 independent experiments. Lower panel, right: miR-10b-overexpressing MCF-7 cells analyzed by serial mammosphere assays. The mean ± SEM of the number of formed mammospheres per 4000 seeded cells in 3 independent experiments was plotted on each bar.


( B ) Upper panel : Mammosphere formation was performed by seeding 20 , 000 MDA - MB - 231 / EpCAM + cells , and after 3 weeks , the cells were disaggregated , counted , and reseeded . Stable miR - 10b - overexpressing cells from 3 independent clones were tested in 3 passages , and cell number was plotted as the mean ± SEM .

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(B) Upper panel: Mammosphere formation was performed by seeding 20,000 MDA-MB-231/EpCAM+ cells, and after 3 weeks, the cells were disaggregated, counted, and reseeded. Stable miR-10b-overexpressing cells from 3 independent clones were tested in 3 passages, and cell number was plotted as the mean ± SEM.


( C ) In vivo limiting dilution assays . miR - 10 - OE or control cells were subcutaneously injected in the backs of mice at 3 doses ( 1 , 000 , 10 , 000 and 50 , 000 cells for MCF - 7 and 5000 , 10 000 and 20 000 for SKBR - 3 ) . Mice were sacrificed after eight weeks , and tumor burden was analyzed using the ELDA software . Plots are depicted in Fig EV2 . The tables show the number of mice with tumors versus the total number of mice injected . Below the tables , the estimated frequency ( freq ) of stem cells for each group and p is shown . Stem cell frequency for MCF - 7 cells was 1 in 34252 ( range 94888 - 12364 ) in miR - 10b - OE cells versus 1 in 238965 ( Range 1817801 - 31414 ) in miR - scr cells . Stem cell frequency for SKBR3 cells was 1 in 5013 ( Range 11135 - 2257 ) in miR - 10b - OE cells versus 1 in 18778 ( Range 45397 - 7767 ) in miR - scr cells .

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(C) In vivo limiting dilution assays. miR-10-OE or control cells were subcutaneously injected in the backs of mice at 3 doses (1,000, 10,000 and 50,000 cells for MCF-7 and 5000, 10 000 and 20 000 for SKBR-3). Mice were sacrificed after eight weeks, and tumor burden was analyzed using the ELDA software. Plots are depicted in Fig EV2. The tables show the number of mice with tumors versus the total number of mice injected. Below the tables, the estimated frequency (freq) of stem cells for each group and p is shown. Stem cell frequency for MCF-7 cells was 1 in 34252 (range 94888-12364) in miR-10b-OE cells versus 1 in 238965 (Range 1817801-31414) in miR-scr cells. Stem cell frequency for SKBR3 cells was 1 in 5013 (Range 11135-2257) in miR-10b-OE cells versus 1 in 18778 (Range 45397-7767) in miR-scr cells.


( D ) Stemness and EMT marker mRNA levels following miR - 10b modulation . miR - 10b , OCT4 / 3 , SNAIL and Vimentin expression was quantified in breast cancer cell lines using transient transfection of miR - 10b versus a miR - Scr vector ( left ) or anti - miR - 10b versus anti - miR - ctl ( right ) . Log - scale plots show the mean and SEM for the relative expression of each gene from triplicate independent transient transfections , each performed three times . A Mann - Whitney U test was used to compare expression between groups . SOX2 , OCT4 and Vimentin were significantly modulated by up - or down - regulation of miR - 10b ( p = 0 . 0043 , p = 0 . 0054 and p = 0 . 000908 , respectively ) . SEM , standard error of the mean .

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(D) Stemness and EMT marker mRNA levels following miR-10b modulation. miR-10b, OCT4/3, SNAIL and Vimentin expression was quantified in breast cancer cell lines using transient transfection of miR-10b versus a miR-Scr vector (left) or anti-miR-10b versus anti-miR-ctl (right). Log-scale plots show the mean and SEM for the relative expression of each gene from triplicate independent transient transfections, each performed three times. A Mann-Whitney U test was used to compare expression between groups. SOX2, OCT4 and Vimentin were significantly modulated by up- or down-regulation of miR-10b (p=0.0043, p=0.0054 and p=0.000908, respectively). SEM, standard error of the mean.


( B ) MCF - 7 , SKBR - 3 and T47 - D breast cancer cells were transfected with gain - or loss - of - function constructs / shRNAs and their respective controls ( a miR - 10b - OE plasmid or anti - miR - 10b ) . MREs of the validated target HOXD10 and of the putative targets PTEN and PIK3CA were cloned into pMIR - Report , and dual luciferase reporter assays were performed . A mutated control miR - 10b ( MRE MUT PTEN ) was also included . Bars show the mean ± SEM from 6 independent transfections .

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(B) MCF-7, SKBR-3 and T47-D breast cancer cells were transfected with gain- or loss-of-function constructs/shRNAs and their respective controls (a miR-10b-OE plasmid or anti-miR-10b). MREs of the validated target HOXD10 and of the putative targets PTEN and PIK3CA were cloned into pMIR-Report, and dual luciferase reporter assays were performed. A mutated control miR-10b (MRE MUT PTEN) was also included. Bars show the mean ± SEM from 6 independent transfections.


( C ) PTEN and PIK3CA expression after miR - 10b modulation . Left panel shows miR - 10b , PTEN and PIK3CA expression by qRT - PCR in MCF - 7 cells with ectopic miR - 10b expression . Right panel shows miR - 10b , PTEN and PIK3CA expression by qRT - PCR in MDA - MB - 231 cells after antagomiR - 10b transfection . Data are from three independent transient transfections performed by triplicate .

(C) PTEN and PIK3CA expression after miR-10b modulation. Left panel shows miR-10b, PTEN and PIK3CA expression by qRT-PCR in MCF-7 cells with ectopic miR-10b expression. Right panel shows miR-10b, PTEN and PIK3CA expression by qRT-PCR in MDA-MB-231 cells after antagomiR-10b transfection. Data are from three independent transient transfections performed by triplicate.


( D ) To further confirm PTEN targeting , we performed gain - or loss - of - function experiments in breast cancer cell lines ( MCF - 7 , MCF - 7 / CD44 + , SKBR - 3 and T47D ) by overexpressing miR - 10b or inhibiting it with a miR - 10b - OE plasmid or anti - miR - 10b shRNAs . PTEN and miR - 10b levels were measured by qRT - PCR . The log2 - fold change of PTEN ( left panel ) and PIK3CA ( right panel ) mRNA levels obtained through the 2 - ΔΔCT method were plotted against their respective miR - 10b expression levels . Correlation analysis was performed [ PTEN ( r = - 0 . 80 ) , PI3KCA ( r = 0 . 43 ) ] . A two - tailed Student ' s t test was used to compare the two groups ( * p < 0 . 05 ) . SEM , standard error of the mean .

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(D) To further confirm PTEN targeting, we performed gain- or loss-of-function experiments in breast cancer cell lines (MCF-7, MCF-7/CD44+, SKBR-3 and T47D) by overexpressing miR-10b or inhibiting it with a miR-10b-OE plasmid or anti-miR-10b shRNAs. PTEN and miR-10b levels were measured by qRT-PCR. The log2-fold change of PTEN (left panel) and PIK3CA (right panel) mRNA levels obtained through the 2-ΔΔCT method were plotted against their respective miR-10b expression levels. Correlation analysis was performed [PTEN (r=-0.80), PI3KCA (r=0.43)]. A two-tailed Student's t test was used to compare the two groups (*p < 0.05). SEM, standard error of the mean.


( E ) PTEN protein modulation by miR - 10b . Upper panel : PTEN protein levels in SKBR - 3 , and MCF - 7 cells were analyzed by Western blot . GAPDH was used as a loading control with cells cultured as in ( B ) . Lower panel : Densitometric analysis of Western blot assays performed with three biological replicates .

(E) PTEN protein modulation by miR-10b. Upper panel: PTEN protein levels in SKBR-3, and MCF-7 cells were analyzed by Western blot. GAPDH was used as a loading control with cells cultured as in (B). Lower panel: Densitometric analysis of Western blot assays performed with three biological replicates.


( F ) PTEN immunohistochemistry analysis of MCF - 7 - miR - 10b - OE or control MCF - 7 - miR - Scr - OE tumorxenografts .

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(F) PTEN immunohistochemistry analysis of MCF-7-miR-10b-OE or control MCF-7-miR-Scr-OE tumorxenografts.


( G ) miR - 10b overexpression does not increase mammosphere formation in PTEN - depleted cells . MCF - 7 - miR - Scr - OE cells ( left panels ) and MCF - 7 - miR - 10b - OE ( right panels ) were transfected with a Dicer - substrate RNA directed toward PTEN , and after 24 h , quantitative RT - PCR assays for PTEN , SOX2 , OCT4 , SNAIL and Vimentin were performed . Additionally , the same cells were analyzed for mammosphere formation . The graph shows the mean and standard error of the relative increase of each marker or number of mammospheres in PTEN - depleted cells ( black bars ) and in cells transfected with a scrambled control ( white bars ) in three independent biological experiments performed by triplicate .

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(G) miR-10b overexpression does not increase mammosphere formation in PTEN-depleted cells. MCF-7-miR-Scr-OE cells (left panels) and MCF-7-miR-10b-OE (right panels) were transfected with a Dicer-substrate RNA directed toward PTEN, and after 24 h, quantitative RT-PCR assays for PTEN, SOX2, OCT4, SNAIL and Vimentin were performed. Additionally, the same cells were analyzed for mammosphere formation. The graph shows the mean and standard error of the relative increase of each marker or number of mammospheres in PTEN-depleted cells (black bars) and in cells transfected with a scrambled control (white bars) in three independent biological experiments performed by triplicate.


( H ) T47 - D cells were transfected with antagomiR - 10b , a dsi - RNA for PTEN or both and mammospheres produced as described in material and methods . Bars show mean and SEM from three independent experiments . * p = 0 . 003 by Student ´ s T test , after ANOVA showing statistical differences in groups .

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(H) T47-D cells were transfected with antagomiR-10b, a dsi-RNA for PTEN or both and mammospheres produced as described in material and methods. Bars show mean and SEM from three independent experiments. * p=0.003 by Student´s T test, after ANOVA showing statistical differences in groups.


( I ) PTEN overexpression compensates for miR - 10b overexpression . Left panel : MDA - 468 cells were transfected with a plasmid overexpressing wild - type PTEN or a version containing a mutated MRE in its 3 ' - UTR , with or without a plasmid containing miR10b and PTEN mRNA expression was measured by end - point RT - PCR . Middle panel : Relative expression of stemness factor SNAIL . The plots show the relative expression from biological triplicate analyses ( mean and SE shown ) . Right panel : Relative expression of stemness factor SOX2 . The plots show the relative expression from biological triplicate analyses ( mean and SE shown ) .

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

(I) PTEN overexpression compensates for miR-10b overexpression. Left panel: MDA-468 cells were transfected with a plasmid overexpressing wild-type PTEN or a version containing a mutated MRE in its 3'-UTR, with or without a plasmid containing miR10b and PTEN mRNA expression was measured by end-point RT-PCR. Middle panel: Relative expression of stemness factor SNAIL. The plots show the relative expression from biological triplicate analyses (mean and SE shown). Right panel: Relative expression of stemness factor SOX2. The plots show the relative expression from biological triplicate analyses (mean and SE shown).


( A ) AKT phosphorylation in CSCs from MCF - 7 cells sorted by CD44 . GAPDH protein levels were used as a loading control .

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(A) AKT phosphorylation in CSCs from MCF-7 cells sorted by CD44. GAPDH protein levels were used as a loading control.


( B ) Upper panel . Cultures were serum - starved for 18 h and then stimulated with insulin or exposed to the PI3K pathway inhibitors Wortmannin or LY294002 . PTEN , PI3KCA , GSK3 and phospho - AKT Ser - 473 protein levels were analyzed by Western blot . GAPDH was used as a loading control . AKT kinase activity was analyzed in these cells by immunoprecipitating phosphorylated AKT proteins with a p - AKT - Ser - 473 antibody and using chimeric GSK - 3 as a substrate . A two - tailed Student ' s t test was used to compare the two groups ( p < 0 . 05 ) . SEM , standard error of the mean . Lower panel . SKBR3 cells were transiently transfected with a plasmid containing an open reading frame of PTEN , miR - 10b or both and phospho - AKT , AKT or tubulin levels analyzed by Western Blot assays . Control is empty vector - transfected cells .

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 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Upper panel. Cultures were serum-starved for 18 h and then stimulated with insulin or exposed to the PI3K pathway inhibitors Wortmannin or LY294002. PTEN, PI3KCA, GSK3 and phospho-AKT Ser-473 protein levels were analyzed by Western blot. GAPDH was used as a loading control. AKT kinase activity was analyzed in these cells by immunoprecipitating phosphorylated AKT proteins with a p-AKT-Ser-473 antibody and using chimeric GSK-3 as a substrate. A two-tailed Student's t test was used to compare the two groups (p<0.05). SEM, standard error of the mean. Lower panel. SKBR3 cells were transiently transfected with a plasmid containing an open reading frame of PTEN, miR-10b or both and phospho-AKT, AKT or tubulin levels analyzed by Western Blot assays. Control is empty vector-transfected cells.


( C ) Clonogenic ability of miR - 10b - depleted CD44 + MCF - 7 cells after PI3K pathway inhibition . Cultures were serum - starved for 18 h and then stimulated with insulin or exposed to the PI3K pathway inhibitors wortmannin or LY294002 . Cells transfected with anti - miR - 10b or a scrambled control ( anti - miR - ctl ) were seeded in soft agar at clonal density and exposed to insulin or PIK3CA inhibitors . After 3 weeks , colonies were quantified . Bars show the mean ± SEM for 3 independent experiments .

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(C) Clonogenic ability of miR-10b-depleted CD44+ MCF-7 cells after PI3K pathway inhibition. Cultures were serum-starved for 18 h and then stimulated with insulin or exposed to the PI3K pathway inhibitors wortmannin or LY294002. Cells transfected with anti-miR-10b or a scrambled control (anti-miR-ctl) were seeded in soft agar at clonal density and exposed to insulin or PIK3CA inhibitors. After 3 weeks, colonies were quantified. Bars show the mean ± SEM for 3 independent experiments.


( D ) miR - 10b overexpression does not induce additional changes in cell colony numbers in response to insulin or PI3K inactivation . MCF - 7 stable miR - Scr - OE or miR - 10b - OE cells were seeded in soft agar at clonal density and exposed to insulin or PIK3CA inhibitors . After 3 weeks , colonies were quantified . Bars show the mean ± SEM for 3 independent experiments .

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 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

(D) miR-10b overexpression does not induce additional changes in cell colony numbers in response to insulin or PI3K inactivation. MCF-7 stable miR-Scr-OE or miR-10b-OE cells were seeded in soft agar at clonal density and exposed to insulin or PIK3CA inhibitors. After 3 weeks, colonies were quantified. Bars show the mean ± SEM for 3 independent experiments.


Module - trait relationships were established by WGCNA using eight independent microarray analyses comprising 375 human prostate samples . Gene modules ( y axis ) are denoted by an arbitrary color name . Bins show the Pearson correlation value between gene expression levels of each module within the noted phenotype / disease stage ( x axis ) and p values . A value of 1 ( red ) quantifies the strongest positive correlation ( genes are upregulated ) , - 1 ( blue ) the strongest negative correlation ( genes are downregulated ) , and 0 ( white ) no correlation .

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

Module-trait relationships were established by WGCNA using eight independent microarray analyses comprising 375 human prostate samples. Gene modules (y axis) are denoted by an arbitrary color name. Bins show the Pearson correlation value between gene expression levels of each module within the noted phenotype/disease stage (x axis) and p values. A value of 1 (red) quantifies the strongest positive correlation (genes are upregulated), -1 (blue) the strongest negative correlation (genes are downregulated), and 0 (white) no correlation.


Microarray analysis was performed following doxycycline - regulated specific AR - V7 depletion ( using a tet - plKO backbone ) in 22Rv1 PC cells compared to doxycycline - treated shGFP controls . The genes that were significantly regulated by shAR - V7 ( in either direction , p value < 0 . 05 ) were distributed among the gene modules defined by WGCNA in panel A . Upregulated genes ( red ) are those in which expression decreased following AR - V7 depletion and conversely downregulated genes ( blue ) are those that increased following AR - V7 depletion .

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

Microarray analysis was performed following doxycycline-regulated specific AR-V7 depletion (using a tet-plKO backbone) in 22Rv1 PC cells compared to doxycycline-treated shGFP controls. The genes that were significantly regulated by shAR-V7 (in either direction, p value < 0.05) were distributed among the gene modules defined by WGCNA in panel A. Upregulated genes (red) are those in which expression decreased following AR-V7 depletion and conversely downregulated genes (blue) are those that increased following AR-V7 depletion.


Hornberg et al . , 2011 gene expression profiling array data was analyzed to determine the expression levels of the seven genes in human CRPC bone metastases , grouped by their relative levels of AR - VS , mainly AR - V7 . ( High - levels of AR - V7 ( top quartile ) or lower levels of AR - V7 ( quartiles 1 - 3 ) . Data are plotted as the mean ± s . e . m . . Non - parametric Mann - Whitney test was performed ( two - tailed ) . Note that BUB1B expression was not measured in these microarrays . * * Significant at a p value < 0 . 05 . N ( AR - V7 low ) = 20 ; N ( AR - V7 high ) = 10 .

Hornberg et al., 2011 gene expression profiling array data was analyzed to determine the expression levels of the seven genes in human CRPC bone metastases, grouped by their relative levels of AR-VS, mainly AR-V7. (High-levels of AR-V7 (top quartile) or lower levels of AR-V7 (quartiles 1-3). Data are plotted as the mean ± s.e.m.. Non-parametric Mann-Whitney test was performed (two-tailed). Note that BUB1B expression was not measured in these microarrays. ** Significant at a p value < 0.05. N (AR-V7 low) = 20; N (AR-V7 high) = 10.


The Kaplan - Meier curves for Disease - Free Survival ( DFS ) and overall survival were built using the Prostate Adenocarcinoma TCGA dataset ( 465 samples ) ( upper graphs ) . The black curves denote cases with normal expression of the gene set , and red represents cases where the mRNA levels of the seven genes were upregulated ( z - score threshold ≤ 1 . 96 ) . For DFS : p - value = 0 . 0009 ; for death : p - value = 0 . 026 . An independent dataset was analyzed ( Prostate Adenocarcinoma MSKCC , Cancer Cell 2010 , 123 samples ) ( lower graphs ) . The black curves denote cases with normal expression of the gene set , and red represents cases where the mRNA levels of at least five genes of the gene set were upregulated ( z - score threshold ≤ 1 . 96 ) . For DFS : p - value = 0 . 0007 ; for death : p - value = 0 . 00546 .

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

The Kaplan-Meier curves for Disease-Free Survival (DFS) and overall survival were built using the Prostate Adenocarcinoma TCGA dataset (465 samples) (upper graphs). The black curves denote cases with normal expression of the gene set, and red represents cases where the mRNA levels of the seven genes were upregulated (z-score threshold ≤ 1.96). For DFS: p-value = 0.0009; for death: p-value = 0.026. An independent dataset was analyzed (Prostate Adenocarcinoma MSKCC, Cancer Cell 2010, 123 samples) (lower graphs). The black curves denote cases with normal expression of the gene set, and red represents cases where the mRNA levels of at least five genes of the gene set were upregulated (z-score threshold ≤ 1.96). For DFS: p-value = 0.0007; for death: p-value = 0.00546.


Cell proliferation was examined in the CRPC cell line 22Rv1 following individual depletion of mRNAs for the seven genes or shGFP controls , using shRNA against the coding region for each gene ( shRNA # 2 ) . Cell number was measured using a non - perturbing nuclear restricted dye and quantified after 72 hours using Incucyte Zoom System . Data shown are mean ± s . e . m . of 8 to 12 replicates normalized to their shGFP control . Kruskal - Wallis test ( p value < 0 . 0001 , two - tailed ) and Dunn ' s multiple comparisons test were performed .

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

Cell proliferation was examined in the CRPC cell line 22Rv1 following individual depletion of mRNAs for the seven genes or shGFP controls, using shRNA against the coding region for each gene (shRNA #2). Cell number was measured using a non-perturbing nuclear restricted dye and quantified after 72 hours using Incucyte Zoom System. Data shown are mean ± s.e.m. of 8 to 12 replicates normalized to their shGFP control. Kruskal-Wallis test (p value < 0.0001, two-tailed) and Dunn's multiple comparisons test were performed.


Representative images of 22Rv1 stably depleted of BUB1B , or control ( shGFP ) are shown .

Representative images of 22Rv1 stably depleted of BUB1B, or control (shGFP) are shown.


22Rv1 stably depleted of each of the seven genes were transfected with a dual plasmid luciferase reporter system which quantifies AR activity and basal transcription . The assay was conducted in 2 % CSS to measure AR ligand - independent transcriptional activity . Data represent two independent experiments performed in triplicate , showing the mean ± s . e . m . , and normalized to their shGFP controls . Kruskal - Wallis test ( p value < 0 . 0001 , two - tailed ) and Dunn ' s multiple comparisons test were performed . * Significant at a p value < 0 . 05 , * * p value < 0 . 001 .

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

22Rv1 stably depleted of each of the seven genes were transfected with a dual plasmid luciferase reporter system which quantifies AR activity and basal transcription. The assay was conducted in 2% CSS to measure AR ligand-independent transcriptional activity. Data represent two independent experiments performed in triplicate, showing the mean ± s.e.m., and normalized to their shGFP controls. Kruskal-Wallis test (p value < 0.0001, two-tailed) and Dunn's multiple comparisons test were performed. * Significant at a p value < 0.05, ** p value < 0.001.


The expression of FKBP5 and UBE2C determined by RT - qPCR analysis and normalized to GAPDH mRNA levels was examined in 22Rv1 cells stably expressing shRNA for each of the seven genes . Cells were cultured in 2 % CSS . Data represent two independent experiments performed in duplicate or triplicate , showing the mean ± s . e . m . , and normalized to their shGFP controls . Kruskal - Wallis test ( p value < 0 . 0001 , two - tailed ) and Dunn ' s multiple comparisons test were performed . * Significant at a p value < 0 . 05 , * * p value < 0 . 001 .

The expression of FKBP5 and UBE2C determined by RT-qPCR analysis and normalized to GAPDH mRNA levels was examined in 22Rv1 cells stably expressing shRNA for each of the seven genes. Cells were cultured in 2% CSS. Data represent two independent experiments performed in duplicate or triplicate, showing the mean ± s.e.m., and normalized to their shGFP controls. Kruskal-Wallis test (p value < 0.0001, two-tailed) and Dunn's multiple comparisons test were performed. * Significant at a p value < 0.05, ** p value < 0.001.


The CRPC cell line 22Rv1 was cultured in 2 % CSS media and was treated for 72 hours with vehicle ( DMSO ) , doxorubicin ( DOX ) , N9 - isopropylolomoucine ( N - 9 ) , or the combination of DOX and N - 9 at different concentrations . Cell confluence was monitored using Incucyte Zoom System and the experiments were done with eight replicates each . The data were analyzed using Compusyn software , and a normalized isobologram was built . The table shows the Combination Index ( CI ) for the different drug combinations . CI = 1 represents additivity , CI < 1 synergism , and CI > 1 antagonistic effects .

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

The CRPC cell line 22Rv1 was cultured in 2% CSS media and was treated for 72 hours with vehicle (DMSO), doxorubicin (DOX), N9-isopropylolomoucine (N-9), or the combination of DOX and N-9 at different concentrations. Cell confluence was monitored using Incucyte Zoom System and the experiments were done with eight replicates each. The data were analyzed using Compusyn software, and a normalized isobologram was built. The table shows the Combination Index (CI) for the different drug combinations. CI=1 represents additivity, CI<1 synergism, and CI>1 antagonistic effects.


The non - tumorigenic prostate epithelial cell line RWPE1 , the AR - null PC cell line PC3 , the androgen dependent cell line LNCaP , and the CRPC cell lines C4 - 2B and 22Rv1 were treated for 72 hours with vehicle ( DMSO ) , DOX ( 100 ng / mL [ 184 nM ] ) , N - 9 ( 200 ng / mL [ 613 nM ] ) , or the combination of DOX ( 100 ng / mL [ 184 nM ] ) and N - 9 ( 200 ng / mL [ 613 nM ] ) . C4 - 2B and 22Rv1 cells were kept in 10 % CSS media , and the other cell lines in 10 % FBS . Cell confluence was monitored using the Incucyte Zoom System . Data represent two independent experiments , with four to six replicates each , showing the mean ± s . e . m . , and normalized to vehicle controls ( Kruskal - Wallis test , P value < 0 . 0001 , two - tailed ) . * Significant at a p value < 0 . 05 , * * p value < 0 . 01 , * * * p value < 0 . 001 .

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

The non-tumorigenic prostate epithelial cell line RWPE1, the AR-null PC cell line PC3, the androgen dependent cell line LNCaP, and the CRPC cell lines C4-2B and 22Rv1 were treated for 72 hours with vehicle (DMSO), DOX (100 ng/mL [184 nM]), N-9 (200 ng/mL [613 nM]), or the combination of DOX (100 ng/mL [184 nM]) and N-9 (200 ng/mL [613 nM]). C4-2B and 22Rv1 cells were kept in 10% CSS media, and the other cell lines in 10% FBS. Cell confluence was monitored using the Incucyte Zoom System. Data represent two independent experiments, with four to six replicates each, showing the mean ± s.e.m., and normalized to vehicle controls (Kruskal-Wallis test, P value <0.0001, two-tailed). *Significant at a p value < 0.05, ** p value < 0.01, *** p value < 0.001.


The non - tumorigenic prostate cell line RWPE - 1 and the CRPC cell line 22Rv1 were treated for 72 hours with vehicle ( DMSO ) or the combination of DOX and N - 9 at 100 ng / mL and N - 9 200 ng / mL , respectively . Cell confluence was monitored using the Incucyte Zoom System .

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

The non-tumorigenic prostate cell line RWPE-1 and the CRPC cell line 22Rv1 were treated for 72 hours with vehicle (DMSO) or the combination of DOX and N-9 at 100 ng/mL and N-9 200 ng/mL, respectively. Cell confluence was monitored using the Incucyte Zoom System.


( A ) Distribution of antimycin A resistance scores for wild isolates , compared to standard laboratory strain ( red vertical line ) . Resistance scores are the ratio of fitness on rich glucose media with vs without 500 µg / L antimycin A . Fitness was estimated based on colony size on solid media , corrected for spatial and plate effects After quality control , we obtained quantitative fitness scores for 154 strains , with a signal - to - noise ratio of 29 . 8 and an unexplained variance of 0 . 12 .

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(A) Distribution of antimycin A resistance scores for wild isolates, compared to standard laboratory strain (red vertical line). Resistance scores are the ratio of fitness on rich glucose media with vs without 500 µg/L antimycin A. Fitness was estimated based on colony size on solid media, corrected for spatial and plate effects After quality control, we obtained quantitative fitness scores for 154 strains, with a signal-to-noise ratio of 29.8 and an unexplained variance of 0.12.


( B ) Volcano plot of a genome - wide association using mixed - model linear regression of antimycin A resistance for 118 , 527 genetic variants . Variants with moderate or high impact are shown in blue . Red dot : the variant at locus I : 3845516 , which causes a T343A change in the Pyk1 amino - acid sequence . This variant was among the top scoring ( effect size = - 0 . 645 , rank 34 ; p = 0 . 0008 , rank 70 ) .

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(B) Volcano plot of a genome-wide association using mixed-model linear regression of antimycin A resistance for 118,527 genetic variants. Variants with moderate or high impact are shown in blue. Red dot: the variant at locus I:3845516, which causes a T343A change in the Pyk1 amino-acid sequence. This variant was among the top scoring (effect size=-0.645, rank 34; p=0.0008, rank 70).


( C ) Boxplot showing antimycin A resistance for 154 strains grouped by the two alleles at the pyk1 locus . Strains carrying a C at this genomic locus ( orange box ) generally have higher antimycin resistance than strains carrying the reference allele T ( blue box ) . As is standard , this boxplot and all other boxplots in this manuscript show the median of the data as central line , the quartiles as box and the extend of the rest of the distribution as whiskers . Points which are 1 . 5 times the inter - quartile range beyond the high and low quartiles are considered outliers and shown individually .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Boxplot showing antimycin A resistance for 154 strains grouped by the two alleles at the pyk1 locus. Strains carrying a C at this genomic locus (orange box) generally have higher antimycin resistance than strains carrying the reference allele T (blue box). As is standard, this boxplot and all other boxplots in this manuscript show the median of the data as central line, the quartiles as box and the extend of the rest of the distribution as whiskers. Points which are 1.5 times the inter-quartile range beyond the high and low quartiles are considered outliers and shown individually.


( B ) Left boxplot : two glycolytic intermediates directly upstream of PYK were strongly depleted in the A - strain ( nT - strain = 9 , nA - strain = 8 ) . Right barplot : PYK activity was directly measured using a lactate dehydrogenase - coupled colorimetric enzyme activity assay , showing the mean and standard deviation of substrate conversion rate for 3 biological replicates , each measured in technical duplicates . Data information : Significance keys : * p < 0 . 05 , * * p < 0 . 005 , * * * p < 0 . 0005 ( Welch ' s t - test )

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(B) Left boxplot: two glycolytic intermediates directly upstream of PYK were strongly depleted in the A-strain (nT-strain=9, nA-strain=8). Right barplot: PYK activity was directly measured using a lactate dehydrogenase-coupled colorimetric enzyme activity assay, showing the mean and standard deviation of substrate conversion rate for 3 biological replicates, each measured in technical duplicates. Data information: Significance keys: * p<0.05, ** p<0.005, *** p<0.0005 (Welch's t-test)


( C ) Concentrations of fructose - 1 , 6 - bisphosphate , which correlate with glycolytic flux , are significantly higher in A - strain ( ratio = 1 . 35 , nT - strain = 9 , nA - strain = 8 ) . Data information : Significance keys : * p < 0 . 05 , * * p < 0 . 005 , * * * p < 0 . 0005 ( Welch ' s t - test )

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(C) Concentrations of fructose-1,6-bisphosphate, which correlate with glycolytic flux, are significantly higher in A-strain (ratio = 1.35, nT-strain=9, nA-strain=8). Data information: Significance keys: * p<0.05, ** p<0.005, *** p<0.0005 (Welch's t-test)


( D ) Boxplots for selected metabolomics data indicate differences in energy and redox status between T - and A - strains ( nT - strain = 9 , nA - strain = 8 ) . Data information : Significance keys : * p < 0 . 05 , * * p < 0 . 005 , * * * p < 0 . 0005 ( Welch ' s t - test )

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(D) Boxplots for selected metabolomics data indicate differences in energy and redox status between T- and A-strains (nT-strain=9, nA-strain=8). Data information: Significance keys: * p<0.05, ** p<0.005, *** p<0.0005 (Welch's t-test)


( E ) Heatmap of the 432 genes that are differentially expressed at the RNA level between the T - and A - strains ( FDR < 10 % ) and are measured in all four conditions ( columns ) . First column : genes ordered by increasing fold - changes for RNAs ( computed as log2 [ T ] - log2 [ A ] ) . Second column : fold - changes for proteins ( computed as log2 [ T ] - log2 [ A ] ) . Third column : fold - changes for RNAs in cells treated with rapamycin & caffeine ( TORC1 inhibition ; computed as log2 [ treatment ] - log2 [ control ] ) ( data from ref . ( Rallis et al , 2013 ) ) . Fourth column : fold - changes for RNAs in cells treated with H2O2 ( oxidative stress ; computed as log2 [ treatment ] - log2 [ control ] ) ( data from Chen et al , 2003 ) . Log2 fold - changes are capped at absolute values of 1 for all columns .

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

(E) Heatmap of the 432 genes that are differentially expressed at the RNA level between the T- and A-strains (FDR <10%) and are measured in all four conditions (columns). First column: genes ordered by increasing fold-changes for RNAs (computed as log2[T]-log2[A]). Second column: fold-changes for proteins (computed as log2[T]-log2[A]). Third column: fold-changes for RNAs in cells treated with rapamycin & caffeine (TORC1 inhibition; computed as log2[treatment]-log2[control]) (data from ref. (Rallis et al, 2013)). Fourth column: fold-changes for RNAs in cells treated with H2O2 (oxidative stress; computed as log2[treatment]-log2[control]) (data from Chen et al, 2003). Log2 fold-changes are capped at absolute values of 1 for all columns.


( A ) Growth measured by optical density ( OD ) at 600nm for 3 biological replicates each of the T - and A - strains over 6 hrs of exponential growth in YES media . The x - axis refers to the time since inoculation of the culture . ( B ) The same data as in ( A ) plotted on a log2 scale ( solid lines ) with lines fitted ( dotted lines ) . The doubling time is the inverse of the slope .

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(A) Growth measured by optical density (OD) at 600nm for 3 biological replicates each of the T- and A-strains over 6 hrs of exponential growth in YES media. The x-axis refers to the time since inoculation of the culture. (B) The same data as in (A) plotted on a log2 scale (solid lines) with lines fitted (dotted lines). The doubling time is the inverse of the slope.


( C ) Media samples were taken from the same cultures ( n = 3 for both strains ) at the 8 hr timepoint and the remaining glucose was quantified . The consumed glucose was calculated based on the amount of glucose measured in the same , fresh media and normalised to the OD of each culture at the time of sampling .

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(C) Media samples were taken from the same cultures (n=3 for both strains) at the 8 hr timepoint and the remaining glucose was quantified. The consumed glucose was calculated based on the amount of glucose measured in the same, fresh media and normalised to the OD of each culture at the time of sampling.


( D ) Oxygen consumption rates in A - and T - strains ( ratio of means = 0 . 65 , p = 0 . 0002 , Welch ' s T - test , 3 biological replicates , each measured in technical duplicates ) . Data information : Vertical bars show the mean of the data . Error bars and shaded areas in all cases denote standard deviation . Significance keys : * p < 0 . 05 , * * * p < 0 . 0005 ( Welch ' s t - test ) .

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(D) Oxygen consumption rates in A- and T-strains (ratio of means=0.65, p=0.0002, Welch's T-test, 3 biological replicates, each measured in technical duplicates). Data information: Vertical bars show the mean of the data. Error bars and shaded areas in all cases denote standard deviation. Significance keys: * p<0.05, *** p<0.0005 (Welch's t-test).


( E ) Culture dry weight after 24 hrs was measured reported as a fraction of the weight of glucose put into the media for 3 biological replicates per strain . Data information : Vertical bars show the mean of the data . Error bars and shaded areas in all cases denote standard deviation . Significance keys : * p < 0 . 05 , * * * p < 0 . 0005 ( Welch ' s t - test ) .

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

(E) Culture dry weight after 24 hrs was measured reported as a fraction of the weight of glucose put into the media for 3 biological replicates per strain. Data information: Vertical bars show the mean of the data. Error bars and shaded areas in all cases denote standard deviation. Significance keys: * p<0.05, *** p<0.0005 (Welch's t-test).


( A ) Spot assays on solid media from a three - fold dilution series of exponential cultures at the same cell density in 96 - well plates ( 3 biological replicates of each strain ) and spotted in 16 technical replicates ( each dilution in 4x4 square ) . The A - strain is more resistant to antimycin A but less resistant to oxidative stress triggered by H2O2 or diamide . A control without toxin ( left ) was included in each batch of spot assays performed and a representative image is shown here .

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(A) Spot assays on solid media from a three-fold dilution series of exponential cultures at the same cell density in 96-well plates (3 biological replicates of each strain) and spotted in 16 technical replicates (each dilution in 4x4 square). The A-strain is more resistant to antimycin A but less resistant to oxidative stress triggered by H2O2 or diamide. A control without toxin (left) was included in each batch of spot assays performed and a representative image is shown here.


( B ) Fitness ( approximated by maximum slope of smoothed growth curves ) of A - and T - strains on 12 carbon sources , with either yeast extract ( YE ) or ammonium ( NH4 ) , with or without 0 . 1 % priming glucose to support initial growth . For all 48 conditions , two biological replicates of A - and T - strains were grown in technical quadruplicates each . Dotted lines in panels mark a fitness ratio of 1 ( i . e . , same fitness ) .

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(B) Fitness (approximated by maximum slope of smoothed growth curves) of A- and T-strains on 12 carbon sources, with either yeast extract (YE) or ammonium (NH4), with or without 0.1% priming glucose to support initial growth. For all 48 conditions, two biological replicates of A- and T-strains were grown in technical quadruplicates each. Dotted lines in panels mark a fitness ratio of 1 (i.e., same fitness).


( C ) Fitness of A - and T - strains on 95 nitrogen sources on Biolog Phenotype MicroArrays . Conditions with no substantial growth were excluded ( black circles , maximum slope < 0 . 015 ) . Red lines show arbitrary significance cut - off , put at | log2 ( A - strain / T - strain ) | > 0 . 75 .

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(C) Fitness of A- and T-strains on 95 nitrogen sources on Biolog Phenotype MicroArrays. Conditions with no substantial growth were excluded (black circles, maximum slope <0.015). Red lines show arbitrary significance cut-off, put at |log2(A-strain/T-strain)|>0.75.


( D ) Fitness of A - and T - strains on 72 different drugs and toxins , at 4 concentrations each , on Biolog Phenotype MicroArrays . ( Results for benzamidine were inconclusive , with both strains comparatively resistant in one concentration each . )

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(D) Fitness of A- and T-strains on 72 different drugs and toxins, at 4 concentrations each, on Biolog Phenotype MicroArrays. (Results for benzamidine were inconclusive, with both strains comparatively resistant in one concentration each.)


( E ) Spot assays to validate selected results Several assays were performed with both h - and h90 strains , and no mating - type specific differences were evident between the two sets of allele - swap strains .

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(E) Spot assays to validate selected results Several assays were performed with both h- and h90 strains, and no mating-type specific differences were evident between the two sets of allele-swap strains.


( F ) Chronological lifespan of A - and T - strains , i . e . the proportion of non - dividing cells in stationary phase that maintain proliferative potential after refeeding . The data show colony forming units ( CFUs ) per ml of culture over 7 days of stationary phase in glucose - depleted rich media . Three biological repeats were carried out for both strains , with each repeat measured as technical triplicates . Error bars represent standard error of the biological replicates .

(F) Chronological lifespan of A- and T-strains, i.e. the proportion of non-dividing cells in stationary phase that maintain proliferative potential after refeeding. The data show colony forming units (CFUs) per ml of culture over 7 days of stationary phase in glucose-depleted rich media. Three biological repeats were carried out for both strains, with each repeat measured as technical triplicates. Error bars represent standard error of the biological replicates.


( G ) Boxplot showing resistance to 3mM H2O2 grouped by pyk1 allele for 156 strains from our collection . The T - strains had a higher mean fitness in H2O2 than the A - strains ( 1 . 01 ± 0 . 06 vs 0 . 95 ± 0 . 11 ; p = 0 . 0021 , Welch ' s t - test ) . The resistance score was obtained as for antimycin A .

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(G) Boxplot showing resistance to 3mM H2O2 grouped by pyk1 allele for 156 strains from our collection. The T-strains had a higher mean fitness in H2O2 than the A-strains (1.01±0.06 vs 0.95±0.11; p=0.0021, Welch's t-test). The resistance score was obtained as for antimycin A.


( H ) Boxplot showing growth fitness on rich media , grouped by pyk1 allele for 158 S . pombe strains ( 0 . 88 ± 0 . 16 vs 0 . 94 ± 0 . 24 ; p = 0 . 18 , Welch ' s t - test ) .

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(H) Boxplot showing growth fitness on rich media, grouped by pyk1 allele for 158 S. pombe strains (0.88±0.16 vs 0.94±0.24; p=0.18, Welch's t-test).


a , Starvation ( 3 h ) - induced autophagy results in a downregulation of H4K16ac in histone extracts of MEF cells .

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a, Starvation (3 h)-induced autophagy results in a downregulation of H4K16ac in histone extracts of MEF cells.


b , Upon rapamycin treatment ( 300 nM ) LC3 conversion and downregulation of H4K16ac are observed in WT MEF cells but not in the autophagy - deficient atg5 − / − and atg7 − / − MEF cells .

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b, Upon rapamycin treatment (300 nM) LC3 conversion and downregulation of H4K16ac are observed in WT MEF cells but not in the autophagy-deficient atg5−/− and atg7−/− MEF cells.


c , Rapamycin treatment increased the LC3 - II / LC3 - I ratio and promoted H4K16ac decrease in sirt1 − / − and WT MEF cells .

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c, Rapamycin treatment increased the LC3-II/LC3-I ratio and promoted H4K16ac decrease in sirt1−/− and WT MEF cells.


d , Rapamycin - induced autophagy led to downregulation of H4K16ac at 48 h in histone extracts of HeLa and U20S cells , and after 6 h in U1810 cells .

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d, Rapamycin-induced autophagy led to downregulation of H4K16ac at 48 h in histone extracts of HeLa and U20S cells, and after 6 h in U1810 cells.


e , Quantification of H4K16 acetylation by immunoblotting is depicted for rapamycin - treated cells . Data are expressed as mean ± s . e . m . ( n = 3 - 5 ) ; * P 0 . 05 ; * * P 0 . 01 .

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e, Quantification of H4K16 acetylation by immunoblotting is depicted for rapamycin-treated cells. Data are expressed as mean ± s.e.m. (n = 3-5); *P 0.05; **P 0.01.


Rapamycin treatment ( 48 h ) promoted the downregulation of the H4K16 histone acetyltransferase hMOF expression level in MEF ( a )

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Rapamycin treatment (48 h) promoted the downregulation of the H4K16 histone acetyltransferase hMOF expression level in MEF (a)


Rapamycin treatment ( 48 h ) promoted the downregulation of the H4K16 histone acetyltransferase hMOF expression level in transfected HeLa cells ( b ) .

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Rapamycin treatment (48 h) promoted the downregulation of the H4K16 histone acetyltransferase hMOF expression level in transfected HeLa cells (b).


VPA ( 1 mM ) treatment counteracted rapamycin - induced H4K16ac downregulation ( c ) and decreased the LC3 ratio ( d ) .

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VPA (1 mM) treatment counteracted rapamycin-induced H4K16ac downregulation (c) and decreased the LC3 ratio (d).


e , Co - treatment with chloroquine ( CQ , 10 µM ) showed that the decrease in LC3 ratio was a result of an increase in autophagic flux .

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e, Co-treatment with chloroquine (CQ, 10 µM) showed that the decrease in LC3 ratio was a result of an increase in autophagic flux.


f , Inhibition of autophagy by CQ after hMOF overexpression shows that hMOF does not inhibit autophagic flux .

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f, Inhibition of autophagy by CQ after hMOF overexpression shows that hMOF does not inhibit autophagic flux.


g , The yeast homologue of hMOF , Sas2 , tagged with 3 × HA showed a complete disappearance of HA signal upon autophagy induction after 3 h .

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g, The yeast homologue of hMOF, Sas2, tagged with 3× HA showed a complete disappearance of HA signal upon autophagy induction after 3 h.


h , Overexpression of Sas2 repressed the downregulation of H4K16ac upon rapamycin treatment .

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h, Overexpression of Sas2 repressed the downregulation of H4K16ac upon rapamycin treatment.


a , VPA increases the autophagic flux in rapamycin - treated HeLa cells transfected with the mRFP - GFP - LC3 tandem reporter construct which allows distinction between autophagosomes ( GFP + / RFP + yellow puncta ) and autolysosomes ( GFP − / RFP + red puncta ) .

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a, VPA increases the autophagic flux in rapamycin-treated HeLa cells transfected with the mRFP-GFP-LC3 tandem reporter construct which allows distinction between autophagosomes (GFP+/RFP+ yellow puncta) and autolysosomes (GFP−/RFP+ red puncta).


b , Confocal microscopy image of a cell treated with rapamycin and VPA depicting a high ratio of red to green LC3 puncta indicating an increase in autophagic flux .

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b, Confocal microscopy image of a cell treated with rapamycin and VPA depicting a high ratio of red to green LC3 puncta indicating an increase in autophagic flux.


c , Co - treatment with VPA and rapamycin led to increased cell death .

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c, Co-treatment with VPA and rapamycin led to increased cell death.


d , Co - treatment with CQ abrogated VPA + rapamycin - induced cell death .

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d, Co-treatment with CQ abrogated VPA + rapamycin-induced cell death.


e , f , Increasing H4K16ac levels by either overexpression of hMOF , inhibition of SIRT1 by siRNA or the chemical inhibitor Ex527 promoted cell death upon rapamycin treatment .

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e, f, Increasing H4K16ac levels by either overexpression of hMOF, inhibition of SIRT1 by siRNA or the chemical inhibitor Ex527 promoted cell death upon rapamycin treatment.


Co - treatment with the autophagy inhibitor bafilomycin A1 ( BafA , 40 nM ) ( g ) or 3 - methyladenine ( 3MA , 5 mM ) ( h ) abrogated VPA + rapamycin - induced cell death .

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Co-treatment with the autophagy inhibitor bafilomycin A1 (BafA, 40 nM) (g) or 3-methyladenine (3MA, 5 mM) (h) abrogated VPA + rapamycin-induced cell death.


Treatment of HeLa cells upon amino - acid starvation with VPA induced cell death ( i ) , which was rescued when cells were co - treated with CQ ( j ) . Data are expressed as mean ± s . e . m . ( n = 3 ) .

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Treatment of HeLa cells upon amino-acid starvation with VPA induced cell death (i), which was rescued when cells were co-treated with CQ (j). Data are expressed as mean ± s.e.m. (n = 3).


A sWGS performed on sorted CD3 + / Vβ + malignant T - cells isolated from the blood of P1 , shown as a representative example . Copy number profiles and ploidy estimates were calculated using ichorCNA . Blue represents balanced genomic regions , green indicates loss / µdeletion , and red indicates gain / amplification of regions on chromosome 17 . Darker color shades indicate higher log - 2 ratios .

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A sWGS performed on sorted CD3+/Vβ+ malignant T-cells isolated from the blood of P1, shown as a representative example. Copy number profiles and ploidy estimates were calculated using ichorCNA. Blue represents balanced genomic regions, green indicates loss/µdeletion, and red indicates gain/amplification of regions on chromosome 17. Darker color shades indicate higher log-2 ratios.


B Heatmap of extracted CNA [ log2 ratios ] of STAT genes on chr17 ( i . e . , STAT3 , STAT5A and STAT5B ) , and other relevant tumor suppressors ( i . e , STAT1 , SOCS1 and TP53 ) and oncogenes ( MYC ) . Red indicates gain / amplification , blue indicates loss / deletion . The balance threshold was set to + / - 0 . 1 . P33 presented with two malignant clones in the blood and both were sequenced ( P33 _ 1 ( Clone 1 ) , P33 _ 2 ( Clone 2 ) ) . * Tetraploid malignant clone .

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B Heatmap of extracted CNA [log2 ratios] of STAT genes on chr17 (i.e., STAT3, STAT5A and STAT5B), and other relevant tumor suppressors (i.e, STAT1, SOCS1 and TP53) and oncogenes (MYC). Red indicates gain/amplification, blue indicates loss/deletion. The balance threshold was set to +/- 0.1. P33 presented with two malignant clones in the blood and both were sequenced (P33_1 (Clone 1), P33_2 (Clone 2)). *Tetraploid malignant clone.


A - C Spearman correlation analysis using CNA log2 ratios and the percentage of clonal CD3 + cells detected in patients for ( A ) STAT3 , ( B ) STAT5A and ( C ) STAT5B .

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A-C Spearman correlation analysis using CNA log2 ratios and the percentage of clonal CD3+ cells detected in patients for (A) STAT3, (B) STAT5A and (C) STAT5B.


D - F RT - qPCR expression data of ( C ) STAT3 , ( D ) STAT5A , and ( E ) STAT5B genes from whole PBMCs isolated from patient blood ( n = 5 ) or healthy controls ( n = 9 ) . GAPDH expression was used for normalization . Statistical significance was calculated using two - tailed unpaired t - test . P - value : < 0 . 01 ( * * ) , < 0 . 0001 ( * * * * ) . One experiment was performed in technical triplicates . The bold dashed line in the middle of the violin plot denotes the median value , while the thin dotted lines denote the interquartile range .

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D-F RT-qPCR expression data of (C) STAT3, (D) STAT5A, and (E) STAT5B genes from whole PBMCs isolated from patient blood (n = 5) or healthy controls (n = 9). GAPDH expression was used for normalization. Statistical significance was calculated using two-tailed unpaired t-test. P-value: <0.01 (**), <0.0001 (****). One experiment was performed in technical triplicates. The bold dashed line in the middle of the violin plot denotes the median value, while the thin dotted lines denote the interquartile range.


G Immunoblotting for total STAT3 / 5 protein isolated from the respective whole PBMCs from patient blood ( n = 3 ) , or from healthy controls ( n = 3 ) . HSC70 was used as loading control . One experiment was performed .

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G Immunoblotting for total STAT3/5 protein isolated from the respective whole PBMCs from patient blood (n = 3), or from healthy controls (n = 3). HSC70 was used as loading control. One experiment was performed.


H Representative image of H & E , CD3 , pY - STAT5 single staining and CD3 / pY - STAT5 double staining on skin biopsy tissue from P3 ( n = 1 ) , which demonstrates infiltration of CD3 + pY - STAT5 + ( 13 . 45 % ) cells into the dermal and subcutaneous layer of the skin . Arrows indicate pY - STAT5 + single and CD3 + pY - STAT5 + cells .

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H Representative image of H&E, CD3, pY-STAT5 single staining and CD3/pY-STAT5 double staining on skin biopsy tissue from P3 (n = 1), which demonstrates infiltration of CD3+ pY-STAT5+ (13.45%) cells into the dermal and subcutaneous layer of the skin. Arrows indicate pY-STAT5+ single and CD3+ pY-STAT5+ cells.


I - M Quantification of IHC staining from skin - biopsy sections of L - CTCL patients ( n = 6 ) and healthy individuals ( n = 5 ) As the number of STAT5A / B positive cells was negligible ( less than 0 . 03 / per mm2 ) in healthy skin biopsy sections , healthy control samples were considered negative for pY - STAT5 , and levels were set to 0 . Statistical significance was calculated using an unpaired Welch ' s t - test . P - value : < 0 . 05 ( * ) . The bold dashed line in the middle of the violin plot denotes the median value , while the thin dotted lines denote the interquartile range .

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I-M Quantification of IHC staining from skin-biopsy sections of L-CTCL patients (n = 6) and healthy individuals (n = 5) As the number of STAT5A/B positive cells was negligible (less than 0.03/per mm2) in healthy skin biopsy sections, healthy control samples were considered negative for pY-STAT5, and levels were set to 0. Statistical significance was calculated using an unpaired Welch's t-test. P-value: <0.05 (*).The bold dashed line in the middle of the violin plot denotes the median value, while the thin dotted lines denote the interquartile range.


A - C Graphic depicts the aberration patterns on ( A ) chromosome 17 ( STAT3 / 5 ) , ( B ) chromosome 2 ( STAT1 ) and ( C ) chromosome 16 ( SOCS1 ) in the CTCL cell lines used in the study . Aberration patterns are represented as the log2 ratio of the fluorescence intensity of the tumor DNA vs . reference DNA and were generated by aCGH . Immunoblotting for total protein STAT1 / 3 / 5 and activation levels : phospo - Tyr ( 701 ) - STAT1 / phospho - Tyr ( 705 ) - STAT3 / phospho - Tyr ( 694 / 699 ) - STAT5 , and SOCS1 protein levels . The cytokine - dependent cell line , SeAx , was collected 30 min after 5 ng / ml IL - 2 and IL - 4 cytokine addition . HSC70 was used as loading control . One representative out of three independent experiments is shown .

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A-C Graphic depicts the aberration patterns on (A) chromosome 17 (STAT3/5), (B) chromosome 2 (STAT1) and (C) chromosome 16 (SOCS1) in the CTCL cell lines used in the study. Aberration patterns are represented as the log2 ratio of the fluorescence intensity of the tumor DNA vs. reference DNA and were generated by aCGH. Immunoblotting for total protein STAT1/3/5 and activation levels: phospo-Tyr (701)-STAT1/phospho-Tyr (705)-STAT3/phospho-Tyr (694/699)-STAT5, and SOCS1 protein levels. The cytokine-dependent cell line, SeAx, was collected 30 min after 5 ng/ml IL-2 and IL-4 cytokine addition. HSC70 was used as loading control. One representative out of three independent experiments is shown.


A Heatmap of IC50 values calculated from drug response analysis using CellTiter - Glo viability assays upon 48h drug treatment . One representative , out of three independent experiments performed in triplicates , is shown .

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A Heatmap of IC50 values calculated from drug response analysis using CellTiter-Glo viability assays upon 48h drug treatment. One representative, out of three independent experiments performed in triplicates, is shown.