A , MCF10A WT , cGAS KO , STING KO , MAVS KO were treated with poly ( dA - dT ) / LyoVec at indicated concentrations for 24 hours . Immunoblotting was performed with indicated antibodies .

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

A, MCF10A WT, cGAS KO, STING KO, and MAVS KO cells were treated with poly(dA-dT)/LyoVec at indicated concentrations for 24 hours. Immunoblotting was performed with indicated antibodies.


B , 4T1 WT , Sting KO , Mavs KO , Irf3 KO were treated with poly ( dA - dT ) / LyoVec at indicated concentrations for 24 hours . Immunoblotting was performed with indicated antibodies .

B, 4T1 WT, Sting KO, Mavs KO, and Irf3 KO cells were treated with poly(dA-dT)/LyoVec at indicated concentrations for 24 hours. Immunoblotting was performed with indicated antibodies.


C , END - seq was used to map double - strand breaks ( DSBs ) associated with TA dinucleotide repeats in MCF10A with indicated treatments . ATR inhibitor ( ATRi ) AZD6738 ( 250nM ) was used with ionizing radiation ( IR , 10 Gy ) for 6 hours . NT , not treated .

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

C, END-seq was used to map double-strand breaks (DSBs) associated with TA dinucleotide repeats in MCF10A cells with the indicated treatments. ATR inhibitor (ATRi) AZD6738 ( 250nM) was used with ionizing radiation (IR, 10 Gy) for 6 hours. NT, not treated.


D , MCF10A WT , MDA5 KO , RIG - I KO , MAVS KO were transfected with AT - rich oligos ( 2 μg / ml ) with LyoVec for 24 hours . Immunoblotting was performed with indicated antibodies .

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

D, MCF10A WT, MDA5 KO, RIG-I KO, and MAVS KO cells were transfected with AT-rich oligos (2 μg/ml) with LyoVec for 24 hours. Immunoblotting was performed with indicated antibodies.


E , MCF10A WT STING KO were transfected with AT - rich non - AT - rich oligos ( 2 μg / ml ) with lipofectamine 3000 for 4 hours . Immunoblotting was performed with indicated antibodies .

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

E, MCF10A WT and STING KO cells were transfected with AT-rich and non-AT-rich oligos (2 μg/ml) with lipofectamine 3000 for 4 hours. Immunoblotting was performed with indicated antibodies.


F , 4T1 WT , Sting KO , Irf3 KO were transfected with AT - rich non - AT - rich oligos ( 2 μg / ml ) with LyoVec for 24 hours . Immunoblotting was performed with indicated antibodies .

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

F, 4T1 WT, Sting KO, and Irf3 KO cells were transfected with AT-rich and non-AT-rich oligos (2 μg/ml) with LyoVec for 24 hours. Immunoblotting was performed with indicated antibodies.


( a - f ) HepG2 were treated with bovine serum albumin ( Con ) , 500 μM PA or 100 nM rapamycin ( Rap ) for 9 h subjected to following analyses . ( a , c - f ) were stained with ubiquitin ( Ub ) , p62 , LC3 LAMP1 antibodies DAPI ( blue ) . Boxed areas are magnified in right - most panels ( c , d ) . Co - localization between LAMP1 LC3 ( e ) was quantified ( f ; n = 3 ) .

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

(a-f) HepG2 cells were treated with bovine serum albumin (Con), 500 μM PA or 100 nM rapamycin (Rap) for 9 h and subjected to following analyses. (a,c-f) Cells were stained with ubiquitin (Ub), p62, LC3 and LAMP1 antibodies and DAPI (blue). Boxed areas are magnified in right-most panels (c,d). Co-localization between LAMP1 and LC3 staining (e) was quantified (f; n=3).


( b ) were subjected to serial extraction ( solubility fractionation ) with indicated of Triton X - 100 ( TX100 ) or sodium dodecyl sulphate ( SDS ) analysed by immunoblotting with indicated antibodies .

O O O O O O O O O O O O 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) Cells were subjected to serial protein extraction (solubility fractionation) with indicated concentration of Triton X-100 (TX100) or sodium dodecyl sulphate (SDS) and analysed by immunoblotting with indicated antibodies.


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

(a-f) HepG2 cells were treated with bovine serum albumin (Con), 500 μM PA or 100 nM rapamycin (Rap) for 9 h and subjected to following analyses. (a,c-f) Cells were stained with ubiquitin (Ub), p62, LC3 and LAMP1 antibodies and DAPI (blue). Boxed areas are magnified in right-most panels (c,d). Co-localization between LAMP1 and LC3 staining (e) was quantified (f; n=3).


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


( g , h ) HepG2 stably transduced with mCherry ( mCh ) - GFP - LC3 - expressing retroviruses were treated with Con , Rap or PA for 9 h examined under a live confocal microscope ( g ) . Yellow dots represent autophagosomes while red dots indicate autolysosomes in which GFP signal was faded out . Number of autolysosomes was quantified ( h ; n = 7 ) . Scale bar , 5 μm . data are shown as mean ± s . e . m . * * * P0 . 001 ( Student ' s t - ) . Molecular markers are indicated in kDa .

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

(g,h) HepG2 cells stably transduced with mCherry (mCh)-GFP-LC3-expressing retroviruses were treated with Con, Rap or PA for 9 h and examined under a live confocal microscope (g). Yellow dots represent autophagosomes while red dots indicate autolysosomes in which GFP signal was faded out. Number of autolysosomes was quantified (h; n=7). Scale bar, 5 μm. All data are shown as mean±s.e.m. ***P0.001 (Student's t-test). Molecular weight markers are indicated in kDa.


HepG2 were treated with bovine serum albumin ( Con or ( − ) ) , PA ( 500 μM ) , PA + verapamil ( Ver , 50 μM ) or PA + nicardipine ( Nic , 100 μM ) for 9 h . ( a - d ) After each treatment , were loaded with a calcium indicator X - Rhod - 1 - AM ( a , b ) or Fura - 2 - AM ( c , d ) . Calcium were visualized by confocal microscopy ( a ) or by dual microscopy ( c , 340 / 380 nm image ) quantified ( b , d ; n = 8 30 , respectively ) .

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

HepG2 cells were treated with bovine serum albumin (Con or (−)), PA (500 μM), PA+verapamil (Ver, 50 μM) or PA+nicardipine (Nic, 100 μM) for 9 h. (a-d) After each treatment, cells were loaded with a calcium indicator X-Rhod-1-AM (a,b) or Fura-2-AM (c,d). Calcium levels were visualized by laser confocal microscopy (a) or by dual fluorescent microscopy (c, 340/380 nm ratio image) and quantified (b,d; n=8 and 30, respectively).


( e - h ) with indicated treatments were subjected to solubility fractionation . 1 % Triton X - 100 - insoluble fractions were dissolved in 2 % SDS , analysed by immunoblotting ( e , f ) quantified ( g , h ; n = 3 ) .

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

(e-h) Cells with indicated treatments were subjected to solubility fractionation. 1% Triton X-100-insoluble fractions were dissolved in 2% SDS, analysed by immunoblotting (e,f) and quantified (g,h; n=3).


( i - l ) with indicated treatments were subjected to immunostaining with indicated antibodies ( i , k ) . was stained with DAPI ( blue ) . Amount of aggregated was quantified ( j ; n = 10 ) . Co - localization between LAMP1 LC3 was quantified ( l ; n = 4 ) . Boxed areas in are magnified in right - most panels ( i , k ) . Scale bar , 10 μm ( a ) , 20 μm ( c ) , 5 μm ( i , k ) . data are shown as mean ± s . e . m . * P0 . 05 , * * P0 . 01 , * * * P0 . 001 ( Student ' s t - ) . Molecular markers are indicated in kDa .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-l) Cells with indicated treatments were subjected to immunostaining with indicated antibodies (i,k). DNA was stained with DAPI (blue). Amount of aggregated proteins was quantified (j; n=10). Co-localization between LAMP1 and LC3 was quantified (l; n=4). Boxed areas in fluorescence images are magnified in right-most panels (i,k). Scale bar, 10 μm (a), 20 μm (c), 5 μm (i,k). All data are shown as mean±s.e.m. *P0.05, **P0.01, ***P0.001 (Student's t-test). Molecular weight markers are indicated in kDa.


Four - month - old C57BL / 6 mice kept on HFD for 2 months were subjected to daily administration of phosphate - buffered saline ( Con , n = 4 ) or verapamil ( Ver , 25 mg per kg , i . p . , n = 3 ) for 10 days . Low - fat diet ( LFD ) - kept mice ( n = 5 ) of same were used as a negative control . ( a ) was daily monitored during injection period . ( b ) Daily food consumption was measured during injection period .

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

Four-month-old C57BL/6 male mice kept on HFD for 2 months were subjected to daily administration of phosphate-buffered saline (Con, n=4) or verapamil (Ver, 25 mg per kg body weight, i.p., n=3) for 10 days. Low-fat diet (LFD)-kept mice (n=5) of same age were used as a negative control. (a) Body weight was daily monitored during injection period. (b) Daily food consumption was measured during injection period.


( c ) Livers were collected from indicated mice photographed . ( d , e ) Total liver mass ( d ) were measured from indicated mice .

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

(c) Livers were collected from indicated mice and photographed. (d,e) Total liver mass (d) were measured from indicated mice.


( c ) Livers were collected from indicated mice photographed . ( d , e ) Total liver mass ( d ) total epididymal adipose tissue ( eWAT ) mass ( e ) were measured from indicated mice .

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

(c) Livers were collected from indicated mice and photographed. (d,e) Total liver mass (d) and total epididymal white adipose tissue (eWAT) mass (e) were measured from indicated mice.


( f - h ) Relative mRNA expression was analysed from eWAT of indicated mice through quantitative reverse transcription - PCR .

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

(f-h) Relative mRNA expression was analysed from eWAT of indicated mice through quantitative reverse transcription-PCR.


( i , j ) Liver sections were analysed by hematoxylin eosin ( HE , upper panels ) Oil Red O ( ORO , lower panels ) ( i ) . ORO densities were quantified ( j ) .

(i,j) Liver sections were analysed by hematoxylin and eosin (HE, upper panels) and Oil Red O (ORO, lower panels) staining (i). ORO densities were quantified (j).


( k , l ) Calcium - induced CaMKII autophosphorylation in livers was analysed by immunoblotting ( k ) quantified ( l ) .

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

(k,l) Calcium-induced CaMKII autophosphorylation in livers was analysed by immunoblotting (k) and quantified (l).


( m ) hepatocytes from 2 - month - old C57BL / 6 mice kept on LFD were treated with bovine serum albumin ( Con ) , PA ( 500 μM ) , PA + verapamil ( Ver , 50 μM ) or PA + nicardipine ( Nic , 100 μM ) for 12 h . After each treatment , were loaded with a calcium indicator Fura - 2 - AM . Calcium were visualized by dual microscopy at 340 / 380 nm , of intensities of Fura - 2 - AM at 340 nm over 380 nm was quantified ( n = 17 ) . Scale bar , 1 cm ( c ) , 200 μm ( i ) . data are shown as mean ± s . e . m . * P0 . 05 , * * P0 . 01 , * * * P0 . 001 ( Student ' s t - ) . Molecular markers are indicated in kDa .

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

(m) Primary hepatocytes from 2-month-old C57BL/6 mice kept on LFD were treated with bovine serum albumin (Con), PA (500 μM), PA+verapamil (Ver, 50 μM) or PA+nicardipine (Nic, 100 μM) for 12 h. After each treatment, cells were loaded with a calcium indicator Fura-2-AM. Calcium levels were visualized by dual fluorescent microscopy at 340/380 nm, and ratio of fluorescence intensities of Fura-2-AM at 340 nm over 380 nm was quantified (n=17). Scale bar, 1 cm (c), 200 μm (i). All data are shown as mean±s.e.m. *P0.05, **P0.01, ***P0.001 (Student's t-test). Molecular weight markers are indicated in kDa.


Four - month - old C57BL / 6 mice kept on HFD for 2 months were subjected to daily administration of phosphate - buffered saline ( Con , n = 4 ) or verapamil ( Ver , 25 mg per kg , i . p . , n = 3 ) for 10 days . Low - fat diet - kept mice ( n = 5 ) of same were used as a negative control . ( a - d ) Livers were subjected to solubility fractionation . 1 % Triton X - 100 - soluble ( a ) - insoluble ( b ) fractions were analysed by immunoblotting ( a , b ) quantified ( c , d ) .

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

Four-month-old C57BL/6 male mice kept on HFD for 2 months were subjected to daily administration of phosphate-buffered saline (Con, n=4) or verapamil (Ver, 25 mg per kg body weight, i.p., n=3) for 10 days. Low-fat diet-kept mice (n=5) of same age were used as a negative control. (a-d) Livers were subjected to solubility fractionation. 1% Triton X-100-soluble (a) and -insoluble (b) fractions were analysed by immunoblotting (a,b) and quantified (c,d).


( e , f ) Paraffin sections of indicated livers were subjected to p62 immunostaining hematoxylin counterstaining ( e ) . p62 - positive areas were quantified ( f ) . Scale bar , 200 μm ( e ) . data are shown as mean ± s . e . m . * P0 . 05 , * * P0 . 01 , * * * P0 . 001 ( Student ' s t - ) . Molecular markers are indicated in kDa .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Paraffin sections of indicated livers were subjected to p62 immunostaining and hematoxylin counterstaining (e). p62-positive areas were quantified (f). Scale bar, 200 μm (e). All data are shown as mean±s.e.m. *P0.05, **P0.01, ***P0.001 (Student's t-test). Molecular weight markers are indicated in kDa.


Four - month - old C57BL / 6 mice kept on HFD for 2 months were subjected to daily administration of phosphate - buffered saline ( Con , n = 4 ) or verapamil ( Ver , 25 mg per kg , i . p . , n = 3 ) for 10 days . Low - fat diet - kept mice ( n = 5 ) of same were used as a negative control . ( a , b ) of LC3 - II from 1 % Triton X - 100 - insoluble fraction of livers were analysed by immunoblotting ( a ) quantified ( b ) .

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

Four-month-old C57BL/6 male mice kept on HFD for 2 months were subjected to daily administration of phosphate-buffered saline (Con, n=4) or verapamil (Ver, 25 mg per kg body weight, i.p., n=3) for 10 days. Low-fat diet-kept mice (n=5) of same age were used as a negative control. (a,b) Levels of LC3-II from 1% Triton X-100-insoluble fraction of livers were analysed by immunoblotting (a) and quantified (b).


( c , d ) Frozen sections of indicated livers were subjected to LC3 / LAMP1 immunostaining DAPI counterstaining ( c ) . Co - localization between LC3 LAMP1 was quantified ( d ) . Boxed areas in are magnified in right - most panels ( c ) . Scale bar , 5 μm ( c ) . data are shown as mean ± s . e . m . * * * P0 . 001 ( Student ' s t - ) . Molecular markers are indicated in kDa .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Frozen sections of indicated livers were subjected to LC3/LAMP1 immunostaining and DAPI counterstaining (c). Co-localization between LC3 and LAMP1 was quantified (d). Boxed areas in fluorescence images are magnified in right-most panels (c). Scale bar, 5 μm (c). All data are shown as mean±s.e.m. ***P0.001 (Student's t-test). Molecular weight markers are indicated in kDa.


Four - month - old C57BL / 6 mice kept on HFD for 2 months were subjected to daily administration of phosphate - buffered saline ( Con , n = 4 ) or verapamil ( Ver , 25 mg per kg , i . p . , n = 3 ) for 10 days . Low - fat diet - kept mice of same ( n = 5 ) were used as a negative control . ( a , b ) Liver sections were subjected to F4 / 80 immunostaining , which visualizes macrophage infiltration hematoxylin counterstaining ( a ) . F4 / 80 - positive areas were quantified ( b ) .

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

Four-month-old C57BL/6 male mice kept on HFD for 2 months were subjected to daily administration of phosphate-buffered saline (Con, n=4) or verapamil (Ver, 25 mg per kg body weight, i.p., n=3) for 10 days. Low-fat diet-kept mice of same age (n=5) were used as a negative control. (a,b) Liver sections were subjected to F4/80 immunostaining, which visualizes macrophage infiltration and hematoxylin counterstaining (a). F4/80-positive areas were quantified (b).


( c - f ) Glucose tolerance ( GTT , c , d ) insulin tolerance ( ITT , e , f ) were conducted using indicated mice ( c , e ) . under curve ( AUC ) was quantified from GTT ITT data ( d , f ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-f) Glucose tolerance tests (GTT, c,d) and insulin tolerance tests (ITT, e,f) were conducted using indicated mice (c,e). Area under the curve (AUC) was quantified from GTT and ITT data (d,f).


( g ) Serum insulin were measured from indicated mice before ( Basal ) 10 min after ( Glucose stimulated ) glucose injection ( n = 4 ) .

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

(g) Serum insulin levels were measured from indicated mice before (Basal) and 10 min after (Glucose stimulated) glucose injection (n=4).


( h , i ) Pancreas sections were analysed by hematoxylin eosin ( HE ) ( h ) . Islet areas were quantified ( i ; n = 4 ) . Scale bar , 200 μm ( a ) , 100 μm ( h ) . data are shown as mean ± s . e . m . * P0 . 05 , * * P0 . 01 , * * * P0 . 001 ( Student ' s t - ) .

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

(h,i) Pancreas sections were analysed by hematoxylin and eosin (HE) staining (h). Islet areas were quantified (i; n=4). Scale bar, 200 μm (a), 100 μm (h). All data are shown as mean±s.e.m. *P0.05, **P0.01, ***P0.001 (Student's t-test).


( a - g ) Four - month - old C57BL / 6 mice kept on HFD for 2 months were subjected to daily administration of phosphate - buffered saline ( Con , n = 4 ) or verapamil ( Ver , 25 mg per kg , i . p . , n = 4 ) for 10 days . Systolic blood pressure ( a ) , LV wall mass ( b ) , diastolic LV volume ( c ) , systolic LV volume ( d ) , ejection fraction ( e , % EF ) , stroke volume ( f , SV ) cardiac output ( g , CO ) were analysed by tail - cuff method ( a ) or echocardiography ( b - g ) . data are shown as mean ± s . e . m . * P0 . 05 ( Student ' s t - ) .

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

(a-g) Four-month-old C57BL/6 male mice kept on HFD for 2 months were subjected to daily administration of phosphate-buffered saline (Con, n=4) or verapamil (Ver, 25 mg per kg body weight, i.p., n=4) for 10 days. Systolic blood pressure (a), LV wall mass (b), diastolic LV volume (c), systolic LV volume (d), ejection fraction (e, % EF), stroke volume (f, SV) and cardiac output (g, CO) were analysed by tail-cuff method (a) or echocardiography (b-g). All data are shown as mean±s.e.m. *P0.05 (Student's t-test).


A . Expression of endothelial Notch target genes in microvascular endothelial isolated from skeletal muscle of mice kept on control diet ( CD , 10 % fat , 70 % carbohydrates ) or high - fat diet ( HFD , 60 % fat , 20 % carbohydrates ) or high - fat sugar diet ( HFS , 60 % fat , 20 % carbohydrates 42 g / l sucrose in drinking water ) . n = 4 , data represent mean ± , unpaired t - .

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

A. Expression of endothelial Notch target genes in microvascular endothelial cells isolated from skeletal muscle of mice kept on control diet (CD, 10% fat, 70% carbohydrates) or high-fat diet (HFD, 60% fat, 20% carbohydrates) or high-fat and sugar diet (HFS, 60% fat, 20% carbohydrates and 42 g/l sucrose in drinking water). n=4, data represent mean ± SEM, unpaired t-test.


B . Expression of endothelial Notch target genes in microvascular endothelial isolated from skeletal muscle of mice fasted for 24 hours ( Fasted group ) then refed for 4 hours ( Fasted + Refed group ) normalized to mice fed for 24 hours ( Fed group ) . n = 5 , data represent mean ± , unpaired t - .

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

B. Expression of endothelial Notch target genes in microvascular endothelial cells isolated from skeletal muscle of mice fasted for 24 hours (Fasted group) and then refed for 4 hours (Fasted+Refed group) normalized to mice fed for 24 hours (Fed group). n=5, data represent mean ± SEM, unpaired t-test.


C . Blood glucose of control ( n = 5 ) NICDiOE - EC ( n = 5 ) mice five after recombination . Data represent mean ± , unpaired t - .

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

C. Blood glucose levels of control (n=5) and NICDiOE-EC (n=5) mice five weeks after recombination. Data represent mean ± SEM, unpaired t-test.


D . Plasma insulin of control ( n = 5 ) NICDiOE - EC ( n = 5 ) mice five after recombination . Data represent mean ± , Welch ' s t - .

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

D. Plasma insulin levels of control (n=5) and NICDiOE-EC (n=5) mice five weeks after recombination. Data represent mean ± SEM, Welch's t-test.


E . Blood glucose for insulin tolerance of control ( n = 7 ) NICDiOE - EC control ( n = 7 ) mice . Data represent mean ± , unpaired t - .

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

E. Blood glucose levels for insulin tolerance test of control (n=7) and NICDiOE-EC control (n=7) mice. Data represent mean ± SEM, unpaired t-test.


F . of under curve for insulin tolerance in E . Data represent mean ± , Welch ' s t - .

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

F. Quantification of area under curve for insulin tolerance test in E. Data represent mean ± SEM, Welch's t-test.


G . Blood glucose for glucose tolerance of control ( n = 6 ) or NICDiOE - EC ( n = 7 ) mice . Data represent mean ± , unpaired t - . H . of under curve for glucose tolerance in G . Data represent mean ± , unpaired t - .

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

G. Blood glucose levels for glucose tolerance test of control (n=6) or NICDiOE-EC (n=7) mice. Data represent mean ± SEM, unpaired t-test. H. Quantification of area under the curve for glucose tolerance test in G. Data represent mean ± SEM, unpaired t-test.


A . Blood glucose of control ( n = 8 ) Rbpji ∆ EC ( n = 8 ) mice five after recombination . Data represent mean ± , unpaired t - .

A. Blood glucose levels of control (n=8) and Rbpji∆EC (n=8) mice five weeks after recombination. Data represent mean ± SEM, unpaired t-test.


B . Plasma insulin of control ( n = 8 ) Rbpji ∆ EC ( n = 8 ) mice five after recombination . Data represent mean ± , unpaired t - .

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

B. Plasma insulin levels of control (n=8) and Rbpji∆EC (n=8) mice five weeks after recombination. Data represent mean ± SEM, unpaired t-test.


C . Blood glucose for insulin tolerance of control ( n = 10 ) Rbpji ∆ EC ( n = 7 ) mice . Data represent mean ± , unpaired t - .

C. Blood glucose levels for insulin tolerance test of control (n=10) and Rbpji∆EC (n=7) mice. Data represent mean ± SEM, unpaired t-test.


D . of under curve for insulin tolerance in C . Data represent mean ± , Welch ' s t - .

D. Quantification of area under curve for insulin tolerance test in C. Data represent mean ± SEM, Welch's t-test.


E . Blood glucose for glucose tolerance of control ( n = 6 ) or RbpjiΔEC ( n = 5 ) mice . Data represent mean ± , unpaired t - . F . of under curve for glucose tolerance in E . Data represent mean ± , Welch ' s t - .

E. Blood glucose levels for glucose tolerance test of control (n=6) or RbpjiΔEC (n=5) mice. Data represent mean ± SEM, unpaired t-test. F. Quantification of area under the curve for glucose tolerance test in E. Data represent mean ± SEM, Welch's t-test.


A . Uptake of FITC - insulin CAV1 expression in human umbilical venous ECs ( HUVECs ) upon Notch induction . Scale bar 20 µm .

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

A. Uptake of FITC-insulin and CAV1 expression in primary human umbilical venous ECs (HUVECs) upon Notch induction. Scale bar 20 µm.


B . Uptake of FITC - insulin CAV1 expression in ( HUVECs ) upon Notch blockage . Scale bar 20 µm .

B. Uptake of FITC-insulin and CAV1 expression in (HUVECs) upon Notch blockage. Scale bar 20 µm.


C . Representative western blot of AKT phosphorylation in gastrocnemius muscle 7 minutes after intravenous injection of PBS or 1 . 5 U / kg insulin in control ( n = 3 ) Rbpji ∆ EC ( n = 3 ) mice five after recombination . D . Densitometric of western blot in C . n = 3 , data represent mean ± , unpaired t - .

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

C. Representative western blot of AKT phosphorylation in gastrocnemius muscle 7 minutes after intravenous injection of PBS or 1.5 U/kg insulin in control (n=3) and Rbpji∆EC (n=3) mice five weeks after recombination. D. Densitometric analysis of western blot in C. n=3, data represent mean ± SEM, unpaired t-test.


E . Representative western blot of AKT phosphorylation in liver 7 minutes after intravenous injection of PBS or 1 . 5 U / kg insulin in control ( n = 3 ) Rbpji ∆ EC ( n = 3 ) mice five after recombination . F . Densitometric of western blot in E . n = 3 , data represent mean ± , unpaired t - .

O O O O O O O O O O O O O O O O O O O O O O O O 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. Representative western blot of AKT phosphorylation in liver 7 minutes after intravenous injection of PBS or 1.5 U/kg insulin in control (n=3) and Rbpji∆EC (n=3) mice five weeks after recombination. F. Densitometric analysis of western blot in E. n=3, data represent mean ± SEM, unpaired t-test.


B . AKT phosphorylation in myogenic C2C12 treated with different doses of insulin . n = 3 , data represent mean ± , unpaired t - .

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

B. AKT phosphorylation in myogenic C2C12 cells treated with different doses of insulin. n=3, data represent mean ± SEM, unpaired t-test.


C . Representative western blot of AKT phosphorylation in C2C12 upon Cav1 knockdown in mouse cardiac endothelial ( MCECs ) . D . Densitometric of western blot in C . Data represented as fold change over control . n = 3 , data represent mean ± , unpaired t - .

C. Representative western blot of AKT phosphorylation in C2C12 cells upon Cav1 knockdown in mouse cardiac endothelial cells (MCECs). D. Densitometric analysis of western blot in C. Data represented as fold change over control. n=3, data represent mean ± SEM, unpaired t-test.


E . Proximity ligation showing association of CAV1 INSR ( insulin receptor ) in MCECs upon stimulation with insulin . n = 86 - 120 from 8 microscopic high power fields per time point , data represent mean ± , unpaired t - , * * * means p < 0 . 001 .

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

E. Proximity ligation assay showing the association of CAV1 and INSR (insulin receptor) in MCECs upon stimulation with insulin. n=86-120 cells from 8 microscopic high power fields per time point, data represent mean ± SEM, unpaired t-test, *** means p<0.001.


A . Quantitative RT - PCR detection of CAV1 , CAV2 CAVIN1 in human umbilical venous endothelial ( HUVECs ) upon Notch blockade ( dnMAML ) induction ( NICD ) . n = 3 , data represent mean ± , unpaired t - .

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

A. Quantitative RT-PCR detection of CAV1, CAV2 and CAVIN1 in primary human umbilical venous endothelial cells (HUVECs) upon Notch blockade (dnMAML) and induction (NICD). n=3, data represent mean ± SEM, unpaired t-test.


B . Representative western blot of CAV1 expression in HUVECs upon Notch manipulation . C . Densitometric of western blot in B . Data represented as fold change over GFP control . n = 3 , data represent mean ± , unpaired t - .

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

B. Representative western blot of CAV1 expression in HUVECs upon Notch manipulation. C. Densitometric analysis of western blot in B. Data represented as fold change over GFP control. n=3, data represent mean ± SEM, unpaired t-test.


D . Representative western blot of CAVIN1 expression in HUVECs upon Notch induction . E . Densitometric of western blot in D . Data represented as fold change over LacZ control . n = 5 , data represent mean ± , unpaired t - .

D. Representative western blot of CAVIN1 expression in HUVECs upon Notch induction. E. Densitometric analysis of western blot in D. Data represented as fold change over LacZ control. n=5, data represent mean ± SEM, unpaired t-test.


F . Expression of CAV1 in HUVECs upon Notch manipulation HEY1 induction . n = 3 , data represent mean ± , unpaired t - .

F. Expression of CAV1 in HUVECs upon Notch manipulation and HEY1 induction. n=3, data represent mean ± SEM, unpaired t-test.


G . Expression of Cav1 , Cav2 , Cavin1 in microvascular ECs freshly isolated from skeletal muscle of RbpjiΔEC mice compared to control mice . n = 5 , data represent mean ± , unpaired t - .

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

G. Expression of Cav1, Cav2, Cavin1 in primary microvascular ECs freshly isolated from skeletal muscle of RbpjiΔEC mice compared to control mice. n=5, data represent mean ± SEM, unpaired t-test.


H . Expression of Cav1 , Cav2 , Cavin1 in microvascular ECs freshly isolated from skeletal muscle of NICDiOE - EC mice compared to control mice . n = 4 , data represent mean ± , unpaired t - .

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

H. Expression of Cav1, Cav2, Cavin1 in primary microvascular ECs freshly isolated from skeletal muscle of NICDiOE-EC mice compared to control mice. n=4, data represent mean ± SEM, unpaired t-test.


I . Expression of Cav1 , Cav2 , Cavin1 in microvascular endothelial isolated from skeletal muscle of mice kept on control diet ( CD , 10 % fat , 70 % carbohydrates ) or high - fat diet ( HFD , 60 % fat , 20 % carbohydrates ) or high - fat sugar diet ( HFS , 60 % fat , 20 % carbohydrates 42 g / l sucrose in drinking water ) . n = 4 , data represent mean ± , unpaired t - .

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

I. Expression of Cav1, Cav2, Cavin1 in microvascular endothelial cells isolated from skeletal muscle of mice kept on control diet (CD, 10% fat, 70% carbohydrates) or high-fat diet (HFD, 60% fat, 20% carbohydrates) or high-fat and sugar diet (HFS, 60% fat, 20% carbohydrates and 42 g/l sucrose in drinking water). n=4, data represent mean ± SEM, unpaired t-test.


J , K . of number of caveolae attached to apical in EC of cardiac capillaries from NICDiOE - EC mice ( 6 segments of 1000 nm from 3 hearts per genotype ) or RbpjiΔEC ( 6 segments of 1000 nm from 8 hearts per genotype ) on electron microscopy . Box plot depicts median percentiles ( 10th , 25th , 75th , 90th ) , unpaired t - , * means p < 0 . 05 .

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

J, K. Quantification of number of caveolae attached to apical membrane in EC of cardiac capillaries from NICDiOE-EC mice (6 segments of 1000 nm length from 3 hearts per genotype) or RbpjiΔEC (6 segments of 1000 nm length from 8 hearts per genotype) based on electron microscopy. Box plot depicts median and percentiles (10th, 25th, 75th, 90th), unpaired t-test, * means p<0.05.


Representative immune - histochemistry of Cav1 in heart sections from control RbpjiΔEC mice . n = 3 , scale bar 50 µm .

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

Representative immune-histochemistry of Cav1 staining in heart sections from control and RbpjiΔEC mice. n=3, scale bar 50 µm.


Representative confocal of Cav1 in heart sections from control RbpjiΔEC mice . n = 3 , scale bar 50 µm .

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

Representative confocal images of Cav1 staining in heart sections from control and RbpjiΔEC mice. n=3, scale bar 50 µm.


B . Blood glucose of control NICDiOE - EC mice during 2 - DG uptake . n = 5 , data represent mean ± , unpaired t - .

O O O B-MEASURED_VAR 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. Blood glucose levels of control and NICDiOE-EC mice during the 2-DG uptake assay. n=5, data represent mean ± SEM, unpaired t-test.


C . Plasma insulin of control NICDiOE - EC mice during 2 - DG uptake . n = 5 , data represent mean ± , unpaired t - .

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

C. Plasma insulin levels of control and NICDiOE-EC mice during the 2-DG uptake assay. n=5, data represent mean ± SEM, unpaired t-test.


D . 2 - DG uptake in skeletal muscle of control NICDiOE - EC mice . n = 5 , data represent mean ± , unpaired t - . E . 2 - DG uptake in visceral adipose tissue ( vWAT ) of control ( n = 4 ) NICDiOE - EC ( n = 5 ) mice . Data represent mean ± , unpaired t - .

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

D. 2-DG uptake levels in skeletal muscle of control and NICDiOE-EC mice. n=5, data represent mean ± SEM, unpaired t-test. E. 2-DG uptake levels in visceral white adipose tissue (vWAT) of control (n=4) and NICDiOE-EC (n=5) mice. Data represent mean ± SEM, unpaired t-test.


G . Blood glucose of control RbpjiΔEC mice during 2 - DG uptake . n = 5 , data represent mean ± , unpaired t - .

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

G. Blood glucose levels of control and RbpjiΔEC mice during the 2-DG uptake assay. n=5, data represent mean ± SEM, unpaired t-test.


H . Plasma insulin of control RbpjiΔEC mice during 2 - DG uptake . n = 5 , data represent mean ± , unpaired t - .

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

H. Plasma insulin levels of control and RbpjiΔEC mice during the 2-DG uptake assay. n=5, data represent mean ± SEM, unpaired t-test.


I . 2 - DG uptake in skeletal muscle of control RbpjiΔEC mice . n = 4 , data represent mean ± , unpaired t - . J . 2 - DG uptake in vWAT of control RbpjiΔEC mice . n = 5 , data represent mean ± , unpaired t - .

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

I. 2-DG uptake levels in skeletal muscle of control and RbpjiΔEC mice. n=4, data represent mean ± SEM, unpaired t-test. J. 2-DG uptake levels in vWAT of control and RbpjiΔEC mice. n=5, data represent mean ± SEM, unpaired t-test.


B . Blood glucose for glucose tolerance ( GTT ) of control ( n = 4 ) or RbpjiΔEC ( n = 5 ) mice kept on high - fat diet ( HFD ) . Data represent unpaired t - , mean ± . C . of under curve ( AUC ) for GTT in B . Data represent mean ± , unpaired t - .

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

B. Blood glucose levels for glucose tolerance test (GTT) of control (n=4) or RbpjiΔEC (n=5) mice kept on high-fat diet (HFD). Data represent unpaired t-test, mean ± SEM. C. Quantification of area under curve (AUC) for GTT in B. Data represent mean ± SEM, unpaired t-test.


D . Blood glucose for insulin tolerance ( ITT ) of control ( n = 4 ) or RbpjiΔEC ( n = 3 ) mice kept on HFD . Data represent unpaired t - , mean ± . E . of AUC for ITT in D . Data represent mean ± , unpaired t - .

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

D. Blood glucose levels for insulin tolerance test (ITT) of control (n=4) or RbpjiΔEC (n=3) mice kept on HFD. Data represent unpaired t-test, mean ± SEM. E. Quantification of AUC for ITT in D. Data represent mean ± SEM, unpaired t-test.


F . HbA1c of control ( n = 4 ) or RbpjiΔEC ( n = 6 ) mice kept on HFD seven after recombination . Data represent mean ± , unpaired t - .

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

F. HbA1c levels of control (n=4) or RbpjiΔEC (n=6) mice kept on HFD seven weeks after recombination. Data represent mean ± SEM, unpaired t-test.


H . Blood glucose of control RbpjiΔEC mice on HFD during 2 - DG uptake . n = 5 , data represent mean ± , unpaired t - .

H. Blood glucose levels of control and RbpjiΔEC mice on HFD during 2-DG uptake assay. n=5, data represent mean ± SEM, unpaired t-test.


I . Plasma insulin of control RbpjiΔEC mice on HFD during 2 - DG uptake . n = 5 , data represent mean ± , unpaired t - .

I. Plasma insulin levels of control and RbpjiΔEC mice on HFD during 2-DG uptake assay. n=5, data represent mean ± SEM, unpaired t-test.


J . 2 - DG uptake in skeletal muscle of control ( n = 5 ) RbpjiΔEC ( n = 4 ) mice on HFD . Data represent mean ± , unpaired t - . K . 2 - DG uptake in vWAT of control RbpjiΔEC mice . n = 4 , data represent mean ± , unpaired t - .

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

J. 2-DG uptake levels in skeletal muscle of control (n=5) and RbpjiΔEC (n=4) mice on HFD. Data represent mean ± SEM, unpaired t-test. K. 2-DG uptake levels in vWAT of control and RbpjiΔEC mice. n=4, data represent mean ± SEM, unpaired t-test.


( C - D ) - Seq data from GTEx of normalized expression of human CSAG1 ( C ) CSAG2 ( D ) in indicated tissues . Number of biological replicates are as follows : testis 361 , brain 255 , small intestine 187 , adipose 663 , muscle 803 , skin 604 , esophagus 555 , stomach 359 , liver 226 , heart 429 . blood vessel 663 , lung 578 , colon 373 , nerve 619 , pituitary 283 , blood 755 , adrenal 258 , kidney 85 , prostate 245 , salivary 162 , ovary 180 , breast 459 , pancreas 328 , vagina 156 , uterus 142 , spleen 241 , fallopian tube 9 , bladder 21 , cervix 10 . Central band indicates median , boxes define 25 75 percentiles , whiskers define 5 95 percentiles .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) RNA-Seq data from GTEx of the normalized expression of human CSAG1 (C) and CSAG2 (D) in the indicated tissues. Number of biological replicates are as follows: testis 361, brain 255, small intestine 187, white adipose 663, muscle 803, skin 604, esophagus 555, stomach 359, liver 226, heart 429. blood vessel 663, lung 578, colon 373, nerve 619, pituitary 283, blood 755, adrenal gland 258, kidney 85, prostate 245, salivary gland 162, ovary 180, breast 459, pancreas 328, vagina 156, uterus 142, spleen 241, fallopian tube 9, bladder 21, cervix 10. Central band indicates median, boxes define 25 and 75 percentiles, and whiskers define 5 and 95 percentiles.


( E - F ) Expression of CSAG1 ( E ) CSAG2 ( F ) in normal adjacent ( blue ) or tissue ( red ) from TCGA - Seq data . N . D . represents normal adjacent is not available . Data visualized by firebrowse . Number of biological replicates are as follows : ACC 41 , BLCA 307 , BRCA 524 , CESC 175 , CHOL 11 , COAD 153 , COADREAD 232 , DLBC 36 , ESCA 121 , HNSC 416 , KICH 46 , KIPAN 515 , KIRC 411 , KIRP 58 , LAML 7 , LGG 528 , LUAD 301 , LUSC 400 , MESO 31 , OV 183 , PCPG 70 , PRAD 204 , READ 79 , SARC 156 , SKCM 422 , STAD 288 , STES 409 , TGCT 126 , THCA 408 , THYM 75 , UCEC 306 , UCS 37 . Central band indicates median , boxes define 25 75 percentiles , whiskers define 5 95 percentiles .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Expression of CSAG1 (E) and CSAG2 (F) in normal adjacent (blue) or tumor tissue (red) from TCGA RNA-Seq data. N.D. represents normal adjacent is not available. Data visualized by firebrowse. Number of biological replicates are as follows: ACC 41, BLCA 307, BRCA 524, CESC 175, CHOL 11, COAD 153, COADREAD 232, DLBC 36, ESCA 121, HNSC 416, KICH 46, KIPAN 515, KIRC 411, KIRP 58, LAML 7, LGG 528, LUAD 301, LUSC 400, MESO 31, OV 183, PCPG 70, PRAD 204, READ 79, SARC 156, SKCM 422, STAD 288, STES 409, TGCT 126, THCA 408, THYM 75, UCEC 306, and UCS 37. Central band indicates median, boxes define 25 and 75 percentiles, and whiskers define 5 and 95 percentiles.


( G ) Xq28 MAGE CSAG genes are frequently co - expressed in lung squamous carcinoma tumors ( n = 184 ) . - Seq gene expression data analyzed from TCGA . Statistics shown were calculated by linear regression .

O O O O O O O O O O O O O O O O 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) Xq28 MAGE and CSAG genes are frequently co-expressed in lung squamous carcinoma tumors (n=184). RNA-Seq gene expression data analyzed from TCGA. Statistics shown were calculated by linear regression analysis.


( H - K ) Expression of CSAG1 ( H - I ) or CSAG2 ( J - K ) in from indicated cancer types correlates with poor overall survival . Data is from TCGA visualized by kmplotter .

O O O O O O O O O O O O O O O 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-K) Expression of CSAG1 (H-I) or CSAG2 (J-K) in patients from indicated cancer types correlates with poor overall survival. Data is from TCGA and visualized by kmplotter.


( A - B ) effect of stable knockdown CSAG2 in HCT116 ( A ) or A375 ( B ) were assayed for clonogenic growth on plastic for 7 days . number of colonies was quantified is shown . Data are represented as mean ± SD , n = 3 biological replicates .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) The effect of stable knockdown CSAG2 in HCT116 (A) or A375 (B) were assayed for clonogenic growth on plastic for 7 days. The number of colonies was quantified and is shown. Data are represented as the mean ± SD, n=3 biological replicates.


( C - D ) effect of stable knockdown CSAG2 in HCT116 ( C ) or A375 ( D ) were determined for anchorage - independent growth in soft agar colony formation assays for 14 days . number of colonies was quantified is shown . Data are represented as mean ± SD , n = 3 biological replicates .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) The effect of stable knockdown CSAG2 in HCT116 (C) or A375 (D) were determined for anchorage-independent growth in soft agar colony formation assays for 14 days. The number of colonies was quantified and is shown. Data are represented as the mean ± SD, n=3 biological replicates.


( E - F ) Knockdown of CSAG2 in A375 ( F ) or HCT116 ( E ) decreases xenograft growth in mice . with doxycycline - inducible shRNAs were implanted into NOD SCID gamma mice . Doxycycline ( 2 mg / mL ) was administered via drinking water . growth was monitored over time . Data are represented as mean ± SD , n = 6 mice for each group .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Knockdown of CSAG2 in A375 (F) or HCT116 (E) cells decreases xenograft tumor growth in mice. Cells with doxycycline-inducible shRNAs were implanted into NOD SCID gamma mice. Doxycycline (2 mg/mL) was administered via drinking water. Tumor growth was monitored over time. Data are represented as the mean ± SD, n=6 mice for each group.


( G - H ) Stable expression of CSAG2 in HCT116 ( G ) CSAG2 - negative H460 ( H ) increases anchorage - independent growth in soft agar colony formation assays , n = 3 biological replicates . Data are represented as mean ± SD .

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

(G-H) Stable expression of CSAG2 in HCT116 (G) and CSAG2-negative H460 (H) cells increases anchorage-independent growth in soft agar colony formation assays, n=3 biological replicates. Data are represented as the mean ± SD.


( A ) HEK293 stably expressing TAP - vector , TAP - CSAG1 , or TAP - CSAG2 were subjected to pull - down followed by SDS - PAGE LC - MS / MS . Spectral ( SC ) of identified in TAP - CSAG1 TAP - CSAG2 pulldowns , but absence in TAP - vector control , are shown .

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

(A) HEK293 cells stably expressing TAP-vector, TAP-CSAG1, or TAP-CSAG2 were subjected to pull-down followed by SDS-PAGE and LC-MS/MS. Spectral counts (SC) of the identified proteins in TAP-CSAG1 and TAP-CSAG2 pulldowns, but absence in TAP-vector control, are shown.


( B ) HEK293FT stably expressing HA - SIRT1 were transfected with Myc - CSAG1 or Myc - CSAG2 for 48 h before IP with anti - HA followed by SDS - PAGE immunoblotting for indicated .

O O O O O O O O O O O O O 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) HEK293FT cells stably expressing HA-SIRT1 were transfected with Myc-CSAG1 or Myc-CSAG2 for 48 h before IP with anti-HA followed by SDS-PAGE and immunoblotting for indicated proteins.


( C ) Recombinant GST - SIRT1 , but not GST , binds in vitro translated Myc - CSAG2 . Myc - tagged CSAG2 was in vitro translated GST - pulldown experiments were performed with recombinant GST or GST - SIRT1 followed by SDS - PAGE immunoblotting for anti - Myc .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Recombinant GST-SIRT1, but not GST, binds in vitro translated Myc-CSAG2. Myc-tagged CSAG2 was in vitro translated and GST-pulldown experiments were performed with recombinant GST or GST-SIRT1 followed by SDS-PAGE and immunoblotting for anti-Myc.


Expression of CSAG2 decreased p53 ac - K382 downregulated expression of downstream p53 target genes in H460 . were treated by either 1 μM doxorubicin ( D - E ) with 0 . 4 μM TSA for indicated times . were then harvested blotted for indicated . Quantitation of expression is shown ( E Ac - p53 K382 normalized to total p53 , whereas other were normalized to GAPDH .

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 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

Expression of CSAG2 decreased p53 ac-K382 levels and downregulated the expression of downstream p53 target genes in H460 cells. Cells were treated by either 1 μM doxorubicin (D-E) with 0.4 μM TSA for the indicated times. Cells were then harvested and blotted for the indicated proteins. Quantitation of expression levels is shown (E Ac-p53 K382 levels normalized to total p53, whereas all other proteins were normalized to GAPDH.


Expression of CSAG2 decreased p53 ac - K382 downregulated expression of downstream p53 target genes in H460 . were treated by 20 μM etoposide with 0 . 4 μM TSA for indicated times . Ac - p53 K382 normalized to total p53 , whereas other were normalized to GAPDH .

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

Expression of CSAG2 decreased p53 ac-K382 levels and downregulated the expression of downstream p53 target genes in H460 cells. Cells were treated by 20 μM etoposide with 0.4 μM TSA for the indicated times. Ac-p53 K382 levels normalized to total p53, whereas all other proteins were normalized to GAPDH.


Expression of CSAG2 decreased p53 ac - K382 downregulated expression of downstream p53 target genes in H460 . were treated by 20 μM etoposide with 0 . 4 μM TSA for indicated times . were then harvested blotted for indicated . Quantitation of expression is shown G ) as mean ± SD , n = 3 biological replicates . Ac - p53 K382 normalized to total p53 , whereas other were normalized to GAPDH .

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 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

Expression of CSAG2 decreased p53 ac-K382 levels and downregulated the expression of downstream p53 target genes in H460 cells. Cells were treated by 20 μM etoposide with 0.4 μM TSA for the indicated times. Cells were then harvested and blotted for the indicated proteins. Quantitation of expression levels is shown G) as the mean ± SD, n=3 biological replicates. Ac-p53 K382 levels normalized to total p53, whereas all other proteins were normalized to GAPDH.


Knockdown of endogenous CSAG2 in HCT116 ( H - I ) increased p53 ac - K382 . CSAG2 - knockdown stable were treated by 1 μM doxorubicin / 0 . 4 μM TSA for 6 hr . were then harvested blotted for indicated . Quantitation of ac - p53 K382 relative to total p53 is shown ( I as mean ± SD , n = 3 biological replicates .

O O 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 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

Knockdown of endogenous CSAG2 in HCT116 (H-I) cells increased p53 ac-K382 levels. CSAG2-knockdown stable cells were treated by 1 μM doxorubicin/ 0.4 μM TSA for 6 hr. Cells were then harvested and blotted for the indicated proteins. Quantitation of ac-p53 K382 levels relative to total p53 is shown (I as the mean ± SD, n=3 biological replicates.


Knockdown of endogenous CSAG2 in A375 increased p53 ac - K382 . CSAG2 - knockdown stable were treated by 1 μM doxorubicin / 0 . 4 μM TSA for 6 hr . were then harvested blotted for indicated .

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

Knockdown of endogenous CSAG2 in A375 cells increased p53 ac-K382 levels. CSAG2-knockdown stable cells were treated by 1 μM doxorubicin/ 0.4 μM TSA for 6 hr. Cells were then harvested and blotted for the indicated proteins.


Knockdown of endogenous CSAG2 in A375 increased p53 ac - K382 . CSAG2 - knockdown stable were treated by 1 μM doxorubicin / 0 . 4 μM TSA for 6 hr . were then harvested blotted for indicated . Quantitation of ac - p53 K382 relative to total p53 is shown K ) as mean ± SD , n = 3 biological replicates .

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 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

Knockdown of endogenous CSAG2 in A375 cells increased p53 ac-K382 levels. CSAG2-knockdown stable cells were treated by 1 μM doxorubicin/ 0.4 μM TSA for 6 hr. Cells were then harvested and blotted for the indicated proteins. Quantitation of ac-p53 K382 levels relative to total p53 is shown K) as the mean ± SD, n=3 biological replicates.


( L - M ) Re - expression of CSAG2 rescued p53 ac - K382 in CSAG2 knockdown stable lines . Myc - CSAG2 was transfected in HCT116 ( L ) or A375 ( M ) CSAG2 knockdown stable lines . were treated with 1 μM doxorubicin / 0 . 4 μM TSA for 6 hr then blotted for indicated . Quantitation of ac - p53 K382 relative to total p53 is shown as mean ± SD , n = 3 biological replicates .

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

(L-M) Re-expression of CSAG2 rescued p53 ac-K382 levels in CSAG2 knockdown stable cell lines. Myc-CSAG2 was transfected in HCT116 (L) or A375 (M) CSAG2 knockdown stable cell lines. Cells were treated with 1 μM doxorubicin/ 0.4 μM TSA for 6 hr and then blotted for the indicated proteins. Quantitation of ac-p53 K382 levels relative to total p53 is shown as the mean ± SD, n=3 biological replicates.


( N ) CSAG2 does not enhance anchorage - independent growth of HCT116 p53 - null . CSAG2 was stably expressed in isogenic HCT116 p53 - wild - type or p53 - null . Anchorage - independent growth in soft agar colony formation assays was determined after 14 days . number of colonies was quantified , normalized to each vector control , is shown as mean ± SD , n = 3 biological replicates .

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

(N) CSAG2 does not enhance anchorage-independent growth of HCT116 p53-null cells. CSAG2 was stably expressed in isogenic HCT116 p53-wild-type or p53-null cells. Anchorage-independent growth in soft agar colony formation assays was determined after 14 days. The number of colonies was quantified, normalized to each vector control cell line, and is shown as the mean ± SD, n=3 biological replicates.


( A ) Knockdown of SIRT1 abolished CSAG2 - mediated decrease in ac - p53 K382 . CSAG2 over - expressing HCT116 were transfected with SIRT1 siRNA for 72 hr before doxorubicin / TSA treatment for 6 hr . lysates were blotted for indicated .

O O O O O O O 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

(A) Knockdown of SIRT1 abolished CSAG2-mediated decrease in ac-p53 K382. CSAG2 over-expressing HCT116 were transfected with SIRT1 siRNA for 72 hr before doxorubicin/TSA treatment for 6 hr. Cell lysates were blotted for the indicated proteins.


( B ) Inhibition of SIRT1 enzymatic activity blocked CSAG2 - induced decrease in p53 ac - K382 . CSAG2 over - expressing HCT116 were treated with 1 μM doxorubicin / 0 . 4 μM TSA with or without 1 μM EX - 527 for 6 hr . lysates were blotted for indicated .

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

(B) Inhibition of SIRT1 enzymatic activity blocked CSAG2-induced decrease in p53 ac-K382. CSAG2 over-expressing HCT116 cells were treated with 1 μM doxorubicin/ 0.4 μM TSA with or without 1 μM EX-527 for 6 hr. Cell lysates were blotted for the indicated proteins.


Inhibition of SIRT1 enzymatic activity prevents CSAG2 - knockdown increase in p53 ac - K382 . CSAG2 - knockdown stable were treated by 1 μM doxorubicin / 0 . 4 μM TSA with or without 1 μM EX - 527 for 6 hr . were then harvested blotted for indicated ( C )

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

Inhibition of SIRT1 enzymatic activity prevents CSAG2-knockdown increase in p53 ac-K382. CSAG2-knockdown stable cells were treated by 1 μM doxorubicin/ 0.4 μM TSA with or without 1 μM EX-527 for 6 hr. Cells were then harvested and blotted for the indicated proteins (C)


Inhibition of SIRT1 enzymatic activity prevents CSAG2 - knockdown increase in p53 ac - K382 . CSAG2 - knockdown stable were treated by 1 μM doxorubicin / 0 . 4 μM TSA with or without 1 μM EX - 527 for 6 hr . were then harvested blotted for indicated quantitated ( D ; n = 3 biological replicates ) . Data shown as mean ± SD .

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

Inhibition of SIRT1 enzymatic activity prevents CSAG2-knockdown increase in p53 ac-K382. CSAG2-knockdown stable cells were treated by 1 μM doxorubicin/ 0.4 μM TSA with or without 1 μM EX-527 for 6 hr. Cells were then harvested and blotted for the indicated proteins and quantitated (D; n=3 biological replicates). Data shown as mean ± SD.


( E ) CSAG2 binds catalytically inactive SIRT1 H363Y . were transfected with indicated constructs before anti - Myc IP blotting for indicated .

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) CSAG2 binds catalytically inactive SIRT1 H363Y. Cells were transfected with the indicated constructs before anti-Myc IP and blotting for the indicated proteins.


( F ) SIRT1 H363Y blocks CSAG2 regulation of p53 ac - K382 . HCT116 were transfected with indicated constructs blotted for indicated .

(F) SIRT1 H363Y blocks CSAG2 regulation of p53 ac-K382 levels. HCT116 cells were transfected with the indicated constructs and blotted for the indicated proteins.


( G ) Inhibition of SIRT1 activity abolished CSAG2 - induced anchorage - independent growth . Soft agar colony formation assays were performed in control or CSAG2 over - expressing HCT116 with or without EX - 527 treatment . Data are represented as mean ± SD , n = 3 biological replicates .

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

(G) Inhibition of SIRT1 activity abolished CSAG2-induced anchorage-independent growth. Soft agar colony formation assays were performed in control or CSAG2 over-expressing HCT116 cells with or without EX-527 treatment. Data are represented as the mean ± SD, n=3 biological replicates.


( H ) Summary of region on CSAG2 required for SIRT1 interaction . HEK293FT stably expressing HA - SIRT1 were transfected with indicated CSAG2 constructs for 48 h before IP with anti - HA followed by SDS - PAGE immunoblotting for anti - Myc .

O O O O O O O O O O O O O O O O O O O O O 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) Summary of region on CSAG2 required for SIRT1 interaction. HEK293FT cells stably expressing HA-SIRT1 were transfected with indicated CSAG2 constructs for 48 h before IP with anti-HA followed by SDS-PAGE and immunoblotting for anti-Myc.


( I ) CSAG2 Δ37 mutant fails to interact with SIRT1 in vitro . Myc - tagged CSAG2 38 - 127 fragment was in vitro translated followed by in vitro binding with recombinant GST - SIRT1 , SDS - PAGE , immunoblotting for anti - Myc .

O O O O O O O O O O O O O O O O O O O O O O O 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) CSAG2 Δ37 mutant fails to interact with SIRT1 in vitro. Myc-tagged CSAG2 38-127 fragment was in vitro translated and followed by in vitro binding assay with recombinant GST-SIRT1, SDS-PAGE, and immunoblotting for anti-Myc.


( J ) CSAG2 Δ37 mutant does not regulate p53 ac - K382 . were transfected with indicated Myc - CSAG2 constructs for 48 hr before being treated with 1 μM doxorubicin / 0 . 4 μM TSA . lysates were blotted for indicated .

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

(J) CSAG2 Δ37 mutant does not regulate p53 ac-K382 levels. Cells were transfected with the indicated Myc-CSAG2 constructs for 48 hr before being treated with 1 μM doxorubicin/ 0.4 μM TSA. Cell lysates were blotted for the indicated proteins.


( K ) CSAG2 Δ37 fails to promote anchorage - independent growth of H460 . Soft agar colony formation assays were performed in CSAG2 - negative H460 stably expressing CSAG2 wild - type or CSAG2 Δ37 mutant ( n = 3 biological replicates ) . Data are represented as mean ± SD .

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

(K) CSAG2 Δ37 fails to promote anchorage-independent growth of H460 cells. Soft agar colony formation assays were performed in CSAG2-negative H460 cells stably expressing CSAG2 wild-type or CSAG2 Δ37 mutant (n=3 biological replicates). Data are represented as the mean ± SD.


Control or CSAG2 expressing H460 were treated with indicated concentrations of doxorubicin ( A ) for 24 hr before viability was determined by alamarBlue ( n = 6 biological replicates ) .

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

Control or CSAG2 expressing H460 cells were treated with the indicated concentrations of doxorubicin (A) for 24 hr before cell viability was determined by alamarBlue (n=6 biological replicates).


Control or CSAG2 expressing H460 were treated with indicated concentrations of H2O2 ( B ) for 24 hr before viability was determined by alamarBlue ( n = 6 biological replicates ) .

Control or CSAG2 expressing H460 cells were treated with the indicated concentrations of H2O2 (B) for 24 hr before cell viability was determined by alamarBlue (n=6 biological replicates).


shCTRL , shCSAG2 - 1 , or shCSAG2 - 2 stable HCT116 were treated with indicated concentrations of doxorubicin ( C ) for 24 hr before viability was determined by alamarBlue ( n = 6 biological replicates ) .

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

shCTRL, shCSAG2-1, or shCSAG2-2 stable HCT116 cells were treated with the indicated concentrations of doxorubicin (C) for 24 hr before cell viability was determined by alamarBlue (n=6 biological replicates).


shCTRL , shCSAG2 - 1 , or shCSAG2 - 2 stable HCT116 were treated with indicated concentrations of H2O2 ( D ) for 24 hr before viability was determined by alamarBlue ( n = 6 biological replicates ) .

shCTRL, shCSAG2-1, or shCSAG2-2 stable HCT116 cells were treated with the indicated concentrations of H2O2 (D) for 24 hr before cell viability was determined by alamarBlue (n=6 biological replicates).


( E - F ) HCT116 were transiently transfected with indicated shRNAs for 48 hr before treatment with 1 μM doxorubicin / 0 . 4 μM TSA for 6 hr . lysates were collected blotted for indicated ( E ) . Quantitation ( n = 3 biological replicates ) of normalized PARP is shown ( F ) .

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 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

(E-F) HCT116 cells were transiently transfected with the indicated shRNAs for 48 hr before treatment with 1 μM doxorubicin/ 0.4 μM TSA for 6 hr. Cell lysates were collected and blotted for the indicated proteins (E). Quantitation (n=3 biological replicates) of normalized cleaved PARP levels is shown (F).


( G ) H460 were transfected with wild type , Δ37 , or Δ63 CSAG2 for 48 hr . were then treated with indicated concentrations of H2O2 for 24 hr before viability was determined by alamarBlue ( n = 6 biological replicates ) .

O O 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

(G) H460 cells were transfected with wild type, Δ37, or Δ63 CSAG2 for 48 hr. Cells were then treated with the indicated concentrations of H2O2 for 24 hr before cell viability was determined by alamarBlue (n=6 biological replicates).


( H ) Inhibition of SIRT1 activity abolished CSAG2 induced survival under genotoxic stress . CSAG2 over - expressing H460 were treated with indicated concentrations of H2O2 with or without 1 μM EX - 527 for 24 hr before viability was determined by alamarBlue ( n = 6 biological replicates ) .

O O 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 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

(H) Inhibition of SIRT1 activity abolished CSAG2 induced cell survival under genotoxic stress. CSAG2 over-expressing H460 cells were treated with the indicated concentrations of H2O2 with or without 1 μM EX-527 for 24 hr before cell viability was determined by alamarBlue (n=6 biological replicates).


Control or Myc - CSAG2 expressing HCT116 p53 - / - were treated with indicated concentrations of H2O2 ( I ) for 24 hr before viability was determined by alamarBlue ( n = 6 biological replicates ) .

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

Control or Myc-CSAG2 expressing HCT116 p53 -/- cells were treated with the indicated concentrations of H2O2 (I) for 24 hr before cell viability was determined by alamarBlue (n=6 biological replicates).


Control or Myc - CSAG2 expressing HCT116 p53 - / - were treated with indicated concentrations of doxorubicin ( J ) for 24 hr before viability was determined by alamarBlue ( n = 6 biological replicates ) .

Control or Myc-CSAG2 expressing HCT116 p53 -/- cells were treated with the indicated concentrations of doxorubicin (J) for 24 hr before cell viability was determined by alamarBlue (n=6 biological replicates).


shCTRL , shCSAG2 - 1 , or shCSAG2 - 2 stable HCT116 p53 - / - were treated with indicated concentrations of H2O2 ( K ) for 24 hr before viability was determined by alamarBlue ( n = 6 biological replicates ) .

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

shCTRL, shCSAG2-1, or shCSAG2-2 stable HCT116 p53 -/- cells were treated with the indicated concentrations of H2O2 (K) for 24 hr before cell viability was determined by alamarBlue (n=6 biological replicates).


shCTRL , shCSAG2 - 1 , or shCSAG2 - 2 stable HCT116 p53 - / - were treated with indicated concentrations of doxorubicin ( L ) for 24 hr before viability was determined by alamarBlue ( n = 6 biological replicates ) .

shCTRL, shCSAG2-1, or shCSAG2-2 stable HCT116 p53 -/- cells were treated with the indicated concentrations of doxorubicin (L) for 24 hr before cell viability was determined by alamarBlue (n=6 biological replicates).


Control or Myc - CSAG2 expressing H1299 ( p53 mutant ) were treated with indicated concentrations of H2O2 ( M ) for 24 hr before viability was determined by alamarBlue ( n = 6 biological replicates ) .

Control or Myc-CSAG2 expressing H1299 (p53 mutant) cells were treated with the indicated concentrations of H2O2 (M) for 24 hr before cell viability was determined by alamarBlue (n=6 biological replicates).


Control or Myc - CSAG2 expressing H1299 ( p53 mutant ) were treated with indicated concentrations of doxorubicin ( N ) for 24 hr before viability was determined by alamarBlue ( n = 6 biological replicates ) .

Control or Myc-CSAG2 expressing H1299 (p53 mutant) cells were treated with the indicated concentrations of doxorubicin (N) for 24 hr before cell viability was determined by alamarBlue (n=6 biological replicates).


( A ) CSAG2 does not alter SIRT1 . HEK293FT were transfected with different amounts of Myc - CSAG2 for 48 hr before lysates were harvested immunoblotted for indicated .

O O O O O O 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) CSAG2 does not alter SIRT1 protein levels. HEK293FT cells were transfected with different amounts of Myc-CSAG2 for 48 hr before cells lysates were harvested and immunoblotted for indicated proteins.


( B ) CSAG2 does not alter SIRT1 subcellular location . U2OS were transfected with or without mcherry - CSAG2 for 48 hr before fixation , SIRT1 immunostaining , imaging . Scale bar : 10 µm .

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

(B) CSAG2 does not alter SIRT1 subcellular location. U2OS cells were transfected with or without mcherry-CSAG2 for 48 hr before fixation, SIRT1 immunostaining, and imaging. Scale bar: 10 µm.


( C ) Summary of results mapping interaction region of SIRT1 recognized by CSAG2 . HEK293FT stably expressing HA - CSAG2 were transfected with indicated Myc - SIRT1 constructs for 48 h before IP with anti - HA followed by SDS - PAGE immunoblotting for anti - Myc .

O O O O O O O O O O O O O O O O O O O O O O O O O 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) Summary of results mapping interaction region of SIRT1 recognized by CSAG2. HEK293FT cells stably expressing HA-CSAG2 were transfected with indicated Myc-SIRT1 constructs for 48 h before IP with anti-HA followed by SDS-PAGE and immunoblotting for anti-Myc.


( D ) CSAG2 directly binds SIRT1 catalytic domain in vitro . HA - tagged SIRT1 fragments were in vitro translated . In vitro binding assays with recombinant GST or GST - CSAG2 were performed followed by SDS - PAGE immunoblotting for anti - HA .

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

(D) CSAG2 directly binds SIRT1 catalytic domain in vitro. HA-tagged SIRT1 fragments were in vitro translated. In vitro binding assays with recombinant GST or GST-CSAG2 were performed followed by SDS-PAGE and immunoblotting for anti-HA.


( E ) CSAG2 enhances SIRT1 activity in vitro . In vitro SIRT1 activity towards p53 ac - K382 peptide was determined in presence of GST or GST - CSAG2 , n = 3 biological replicates . Nicotinamide ( 1 mM ) was added to reaction were indicated to block SIRT1 activity .

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

(E) CSAG2 enhances SIRT1 activity in vitro. In vitro SIRT1 activity towards p53 ac-K382 peptide was determined in the presence of GST or GST-CSAG2, n=3 biological replicates. Nicotinamide (1 mM) was added to the reaction were indicated to block SIRT1 activity.


( F ) CSAG2 Δ37 mutant that does not bind SIRT1 does not enhance SIRT1 activity in vitro . In vitro SIRT1 activity towards p53 ac - K382 peptide was determined in presence of GST , GST - CSAG2 or GST - CSAG2 38 - 127 , n = 3 biological replicates .

(F) CSAG2 Δ37 mutant that does not bind SIRT1 does not enhance SIRT1 activity in vitro. In vitro SIRT1 activity towards p53 ac-K382 peptide was determined in the presence of GST, GST-CSAG2 or GST-CSAG2 38-127, n=3 biological replicates.


( G ) CSAG2 enhances SIRT1 deacetylation of p53 ac - K382 peptide through increasing kcat , but not Km . not affecting binding ability with its substrate p53 . SIRT1 activity assays were performed as described above , except varying amounts of p53 ac - K382 fluorometric peptide were used in presence of saturating NAD + ( 3 mM ) , n = 3 biological replicates .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) CSAG2 enhances SIRT1 deacetylation of p53 ac-K382 peptide through increasing kcat, but not Km. not affecting the binding ability with its substrate p53. SIRT1 activity assays were performed as described above, except varying amounts of p53 ac-K382 fluorometric peptide were used in the presence of saturating NAD+ (3 mM), n=3 biological replicates.


( H - I ) U2OS were transfected with Myc - Vector or Myc - CSAG2 for 48 hr before being treated with or without 0 . 4 μM TSA for 6 hr . lysates were blotted for indicated ( H ) . Quantitation of expression of acetylated Histone relative to total Histone is shown as mean ± SD , n = 3 biological replicates . circles indicate Myc - Vector black circles indicate Myc - CSAG2 .

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

(H-I) U2OS cells were transfected with Myc-Vector or Myc-CSAG2 for 48 hr before being treated with or without 0.4 μM TSA for 6 hr. Cell lysates were blotted for the indicated proteins (H). Quantitation of expression levels of acetylated Histone relative to total Histone is shown as the mean ± SD, n=3 biological replicates. White circles indicate Myc-Vector and black circles indicate Myc-CSAG2.


( J ) HeLa , U2OS , or HCT116 were transfected with Myc - Vector or Myc - CSAG2 for 48 hr . lysates were blotted for indicated . Quantitation of expression of acetylated Histone relative to total Histone is shown as mean ± SD , n = 3 biological replicates . circles indicate Myc - Vector black circles indicate Myc - CSAG2 .

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

(J) HeLa, U2OS, or HCT116 cells were transfected with Myc-Vector or Myc-CSAG2 for 48 hr. Cell lysates were blotted for the indicated proteins. Quantitation of expression levels of acetylated Histone relative to total Histone is shown as the mean ± SD, n=3 biological replicates. White circles indicate Myc-Vector and black circles indicate Myc-CSAG2.


( K ) Knockdown of endogenous CSAG2 in A375 increased ac - H4K16 . lysates were blotted for indicated . Quantitation of expression of ac - H4K16 relative to total H4 is shown as mean ± SD , n = 3 biological replicates .

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

(K) Knockdown of endogenous CSAG2 in A375 cells increased ac-H4K16 levels. Cell lysates were blotted for the indicated proteins. Quantitation of expression levels of ac-H4K16 relative to total H4 is shown as the mean ± SD, n=3 biological replicates.


A . Four representative of RHOB immunostaining in human non - small - lung cancers . Figures correspond to of analyzed .

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

A. Four representative images of RHOB immunostaining in human non-small-cell lung cancers. Figures correspond to the percentage of tumor analyzed.


B . Characteristics of 96 with an EGFR - activating mutation who were treated with EGFR - TKI . Each column corresponds to one . Data include general characteristics ( stage at diagnosis , , sex , tobacco usage , position of EGFR mutation , TKI type ) , progression - free survival ( PFS ; lower graph ) of RHOB as determined by immunohistochemistry ( null : 0 , weak : + , moderate : + + high : + + + ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Characteristics of 96 patients with an EGFR-activating mutation who were treated with EGFR-TKI. Each column corresponds to one patient. Data include general characteristics (stage at diagnosis, age, sex, tobacco usage, position of EGFR mutation, TKI type), progression-free survival (PFS; lower graph) and intensity of RHOB staining as determined by immunohistochemistry (null: 0, weak: +, moderate: ++ and high: +++).


C . Representative scans of with low or high RHOB - expressing lung tumors , before after erlotinib treatment . Red arrows indicate lung tumors .

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

C. Representative scans of patients with low or high RHOB-expressing lung tumors, before and after erlotinib treatment. Red arrows indicate lung tumors.


D . Progression - free survival of erlotinib - treated with EGFR - mutated lung tumors , according to RHOB expression , assessed by immunohistochemistry ( low RHOB group = negative + weak ; high RHOB group = moderate + high ) .

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

D. Progression-free survival of erlotinib-treated patients with EGFR-mutated lung tumors, according to RHOB expression, assessed by immunohistochemistry (low RHOB group = negative + weak staining; high RHOB group = moderate + high staining).


E . Progression - free survival of who received EGFR - TKi as first - therapy ( n = 63 ) . F . Progression - free survival of who received EGFR - TKi as 2nd - ( n = 28 ) , 3rd - ( n = 3 ) or 4th - ( n = 2 ) therapy .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Progression-free survival of patients who received EGFR-TKi as first-line therapy (n=63). F. Progression-free survival of patients who received EGFR-TKi as 2nd-line (n=28), 3rd-line (n=3) or 4th-line (n=2) therapy.


G . RHOB immunostaining score evolution in EGFR - mutated lung tumors before treatment after EGFR - TKI relapse .

G. RHOB immunostaining score evolution in EGFR-mutated lung tumors before treatment and after EGFR-TKI relapse.


A . Representative H & amp ; amp ; E of whole lungs from EGFRL858R / Rhob - / - , EGFRL858R / Rhob + / - EGFRL858R / Rhob + / + mice treated or not with erlotinib ( 12 . 5 mg / kg / day ) for four days . Scale bar : 5 mm . B . of / lung . n = 7 for each group except for EGFRL858R / Rhob + / - placebo ( n = 6 ) .

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

A. Representative H&amp;E staining of whole lungs from EGFRL858R/Rhob-/-, EGFRL858R/Rhob+/- and EGFRL858R/Rhob+/+ mice treated or not with erlotinib (12.5 mg/kg/day) for four days. Scale bar: 5 mm. B. Quantification of the tumor/lung ratio. n=7 for each group except for EGFRL858R/Rhob+/- placebo (n=6).


C . Representative Ki67 immunostaining of EGFRL858R / Rhob - / - , EGFRL858R / Rhob + / - EGFRL858R / Rhob + / + mice treated or not with erlotinib ( 12 . 5 mg / kg / day ) for four days ( scale bar : 50 µm ) , corresponding ( D ) . Three independent zones / mice lung were used for . n = 21 ( 7 mice ) for each group except for EGFRL858R / Rhob + / - placebo ( n = 18 ; 6 mice ) .

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

C. Representative Ki67 immunostaining of EGFRL858R/Rhob-/-, EGFRL858R/Rhob+/- and EGFRL858R/Rhob+/+ mice treated or not with erlotinib (12.5 mg/kg/day) for four days (scale bar: 50 µm), and the corresponding quantification (D). Three independent zones/mice lung were used for quantification. n=21 (7 mice) for each group except for EGFRL858R/Rhob+/- placebo (n=18; 6 mice).


E . Immunostaining of caspase - 3 in lung tumors from EGFRL858R / Rhob + / + or EGFRL858R / Rhob - / - mice treated for 24 h with erlotinib at 12 . 5 mg / kg . Scale bar : 50 µm . ( * * : p < 0 . 001 vs . placebo ; * * * : p < 0 . 0001 vs . placebo ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Immunostaining of cleaved caspase-3 in lung tumors from EGFRL858R/Rhob+/+ or EGFRL858R/Rhob-/- mice treated for 24 h with erlotinib at 12.5 mg/kg. Scale bar: 50 µm. (**: p<0.001 vs. placebo; ***: p<0.0001 vs. placebo).


HCC4006 were ( A ) transfected with two siRNA against RHOB ( siB1 , siB2 ) then treated with increasing doses of erlotinib . surviving fraction was determined by an MTS after 72 h compared to untreated .

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

HCC4006 cells were (A) transfected with two siRNA against RHOB (siB1, siB2) then treated with increasing doses of erlotinib. The surviving cell fraction was determined by an MTS assay after 72 h and compared to untreated cells.


HCC4006 were ( A ) transfected with two siRNA against RHOB ( siB1 , siB2 ) . RHOB overexpression was monitored by western blotting . ( * * * : p < 0 . 0001 vs . control ) . Data are representative of at least three independent experiments .

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

HCC4006 cells were (A) transfected with two siRNA against RHOB (siB1, siB2). RHOB overexpression was monitored by western blotting. (***: p<0.0001 vs. control cells). Data are representative of at least three independent experiments.


HCC4006 were ( B ) transduced with control ( AdCont ) or RHOB - overexpressing adenoviruses ( AdRHOB ) then treated with increasing doses of erlotinib . surviving fraction was determined by an MTS after 72 h compared to untreated .

O 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

HCC4006 cells were (B) transduced with control (AdCont) or RHOB-overexpressing adenoviruses (AdRHOB) then treated with increasing doses of erlotinib. The surviving cell fraction was determined by an MTS assay after 72 h and compared to untreated cells.


HCC4006 were ( B ) transduced with control ( AdCont ) or RHOB - overexpressing adenoviruses ( AdRHOB ) . RHOB overexpression was monitored by western blotting . ( * * * : p < 0 . 0001 vs . control ) . Data are representative of at least three independent experiments .

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

HCC4006 cells were (B) transduced with control (AdCont) or RHOB-overexpressing adenoviruses (AdRHOB). RHOB overexpression was monitored by western blotting. (***: p<0.0001 vs. control cells). Data are representative of at least three independent experiments.


HCC4006 were ( C ) both transfected with siRNA transduced by adenoviruses ; then treated with increasing doses of erlotinib . surviving fraction was determined by an MTS after 72 h compared to untreated .

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

HCC4006 cells were (C) both transfected with siRNA and transduced by adenoviruses; then treated with increasing doses of erlotinib. The surviving cell fraction was determined by an MTS assay after 72 h and compared to untreated cells.


HCC4006 were ( C ) both transfected with siRNA transduced by adenoviruses ; then treated with increasing doses of erlotinib . RHOB overexpression was monitored by western blotting . ( * * * : p < 0 . 0001 vs . control ) . Data are representative of at least three independent experiments .

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

HCC4006 cells were (C) both transfected with siRNA and transduced by adenoviruses; then treated with increasing doses of erlotinib. RHOB overexpression was monitored by western blotting. (***: p<0.0001 vs. control cells). Data are representative of at least three independent experiments.


Erlotinib IC50 values were quantified in RHOB - overexpressing or RHOB - depleted ( D ) HCC827 , ( E ) HCC2935 ( F ) H3255 , as determined by an MTS after 72 h treatment . RHOB overexpression or inhibition was monitored by western blotting for each condition .

Erlotinib IC50 values were quantified in RHOB-overexpressing or RHOB-depleted (D) HCC827 cells, (E) HCC2935 cells and (F) H3255 cells, as determined by an MTS assay after 72 h treatment. RHOB overexpression or inhibition was monitored by western blotting for each condition.


G . HCC4006 were transduced with control ( AdCont ) or RHOB - overexpressing adenoviruses ( AdRHOB ) at an increasing multiplicity of infection ( MOI ) . RHOB overexpression was monitored by western blotting . ( * * * : p < 0 . 0001 vs . control ) . Data are representative of at least three independent experiments .

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

G. HCC4006 cells were transduced with control (AdCont) or RHOB-overexpressing adenoviruses (AdRHOB) at an increasing multiplicity of infection (MOI). RHOB overexpression was monitored by western blotting. (***: p<0.0001 vs. control cells). Data are representative of at least three independent experiments.


G . HCC4006 were transduced with control ( AdCont ) or RHOB - overexpressing adenoviruses ( AdRHOB ) at an increasing multiplicity of infection ( MOI ) , then erlotinib IC50 values were determined after 72 h by an MTS a correlation was 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 B-CONTROLLED_VAR O O O O O O O O O O O O O O O

G. HCC4006 cells were transduced with control (AdCont) or RHOB-overexpressing adenoviruses (AdRHOB) at an increasing multiplicity of infection (MOI), then erlotinib IC50 values were determined after 72 h by an MTS assay and a correlation analysis was performed.


A . HCC4006 , HCC827 , HCC2935 H3255 were transduced with control ( AdCont ) or RHOB - overexpressing ( AdRHOB ) adenoviruses treated for four hours with erlotinib at concentrations corresponding to respective IC50 values determined for each control . phosphorylation status of AKT , ERK1 / 2 EGFR was assessed by western blotting normalized according to total . RHOB overexpression was also monitored by western blotting .

O O O O 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

A. HCC4006, HCC827, HCC2935 and H3255 cells were transduced with control (AdCont) or RHOB-overexpressing (AdRHOB) adenoviruses and treated for four hours with erlotinib at concentrations corresponding to the respective IC50 values determined for each control cell line. The phosphorylation status of AKT, ERK1/2 and EGFR was assessed by western blotting and normalized according to total protein levels. RHOB overexpression was also monitored by western blotting.


B . Representative immunostaining of phospho - AKT ( Ser473 ) , phospho ERK1 / 2 their total amounts in lung tumors from EGFRL858R / Rhob - / - or EGFRL858R / Rhob + / + mice treated or not with erlotinib ( 12 . 5 mg / kg / day ) for four days . remaining hyperplastic areas were selected in erlotinib - treated mice to efficiently characterize effect of erlotinib on ERK AKT pathways in both Rhob genotypes . Scale bar : 100 µm .

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

B. Representative immunostaining of phospho-AKT (Ser473), phospho ERK1/2 and their total protein amounts in lung tumors from EGFRL858R/Rhob-/- or EGFRL858R/Rhob+/+ mice treated or not with erlotinib (12.5 mg/kg/day) for four days. The remaining hyperplastic areas were selected in erlotinib-treated mice to efficiently characterize the effect of erlotinib on ERK and AKT pathways in both Rhob genotypes. Scale bar: 100 µm.


C . HCC4006 were transfected with a plasmid coding for a constitutively active AKT mutant ( AKTmyr , myristoylated ) or an empty vector ( ø ) , treated for 72 h with increasing concentrations of erlotinib . surviving fraction was determined by an MTS . Data are representative of at least three independent experiments .

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

C. HCC4006 cells were transfected with a plasmid coding for a constitutively active AKT mutant (AKTmyr, myristoylated) or an empty vector (ø), and treated for 72 h with increasing concentrations of erlotinib. The surviving cell fraction was determined by an MTS assay. Data are representative of at least three independent experiments.


C . HCC4006 were transfected with a plasmid coding for a constitutively active AKT mutant ( AKTmyr , myristoylated ) or an empty vector ( ø ) , treated for 72 h with increasing concentrations of erlotinib . AKT overexpression phosphorylation at Ser473 were assessed by western blotting . Data are representative of at least three independent experiments .

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

C. HCC4006 cells were transfected with a plasmid coding for a constitutively active AKT mutant (AKTmyr, myristoylated) or an empty vector (ø), and treated for 72 h with increasing concentrations of erlotinib. AKT overexpression and phosphorylation at Ser473 were assessed by western blotting. Data are representative of at least three independent experiments.


A . HCC4006 were transduced with control ( AdCont ) or RHOB - overexpressing ( AdRHOB ) adenoviruses treated for four hours with erlotinib ( 100 nM ) , G594 ( 100 nM ) , or a combination of both drugs . phosphorylation status of GSK3β ( Ser9 ) , ERK1 / 2 EGFR ( Tyr1173 ) was assessed by western blotting normalized according to total . RHOB overexpression was also monitored by western blotting .

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 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

A. HCC4006 cells were transduced with control (AdCont) or RHOB-overexpressing (AdRHOB) adenoviruses and treated for four hours with erlotinib (100 nM), G594 (100 nM), or a combination of both drugs. The phosphorylation status of GSK3β (Ser9), ERK1/2 and EGFR (Tyr1173) was assessed by western blotting and normalized according to the total protein levels. RHOB overexpression was also monitored by western blotting.


B . HCC4006 were transduced with control ( AdCont ) or RHOB - overexpressing ( AdRHOB ) adenoviruses treated for 72 h with erlotinib alone ( black red curves ) or in combination with AKT inhibitor G594 at 100 nM ( green blue curves ) . surviving fraction was determined by an MTS .

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

B. HCC4006 cells were transduced with control (AdCont) or RHOB-overexpressing (AdRHOB) adenoviruses and treated for 72 h with erlotinib alone (black and red curves) or in combination with the AKT inhibitor G594 at 100 nM (green and blue curves). The surviving cell fraction was determined by an MTS assay.


C . HCC4006 were transduced with control ( AdCont ) or RHOB - overexpressing adenoviruses ( AdRHOB ) treated for 72 h with increasing concentrations of erlotinib in absence or presence of increasing doses of G594 . surviving fraction was determined by an MTS , erlotinib IC50 values were determined for each condition . ( * * : p < 0 . 001 vs . AdCont ; * * * : p < 0 . 0001 vs . AdCont ) . HCC4006 were transduced with control ( AdCont ) or RHOB - overexpressing ( AdRHOB ) adenoviruses treated for 48 h with erlotinib ( 100 nM ) , G594 ( 100 nM ) , or a combination of both drugs .

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

C. HCC4006 cells were transduced with control (AdCont) or RHOB-overexpressing adenoviruses (AdRHOB) and treated for 72 h with increasing concentrations of erlotinib in the absence or presence of increasing doses of G594. The surviving cell fraction was determined by an MTS assay, and erlotinib IC50 values were determined for each condition. (**: p<0.001 vs. AdCont cells; ***: p<0.0001 vs. AdCont cells). HCC4006 cells were transduced with control (AdCont) or RHOB-overexpressing (AdRHOB) adenoviruses and treated for 48 h with erlotinib (100 nM), G594 (100 nM), or a combination of both drugs.


HCC4006 were transduced with control ( AdCont ) or RHOB - overexpressing ( AdRHOB ) adenoviruses treated for 48 h with erlotinib ( 100 nM ) , G594 ( 100 nM ) , or a combination of both drugs . Apoptosis was then determined by of subG1 population ( D ) .

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 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

HCC4006 cells were transduced with control (AdCont) or RHOB-overexpressing (AdRHOB) adenoviruses and treated for 48 h with erlotinib (100 nM), G594 (100 nM), or a combination of both drugs. Apoptosis was then determined by quantification of the subG1 cell population (D).


HCC4006 were transduced with control ( AdCont ) or RHOB - overexpressing ( AdRHOB ) adenoviruses treated for 48 h with erlotinib ( 100 nM ) , G594 ( 100 nM ) , or a combination of both drugs . Apoptosis was then determined by detection of PARP caspase - 3 ( E ) .

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 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

HCC4006 cells were transduced with control (AdCont) or RHOB-overexpressing (AdRHOB) adenoviruses and treated for 48 h with erlotinib (100 nM), G594 (100 nM), or a combination of both drugs. Apoptosis was then determined by detection of cleaved PARP and caspase-3 (E).


A . Representative H & amp ; amp ; E of lung tumors from EGFRL858R / Rhob - / - , EGFRL858R / Rhob + / - EGFRL858R / Rhob + / + mice treated or not during four days with erlotinib ( 12 . 5 mg / kg / day ) , with AKT inhibitor G594 ( 25 mg / kg / day ) , or in combination with both drugs . Scale bar : 500 µm . B . of / lung of mice treated or not with drugs or with a combination of both .

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

A. Representative H&amp;E staining of lung tumors from EGFRL858R/Rhob-/-, EGFRL858R/Rhob+/- and EGFRL858R/Rhob+/+ mice treated or not during four days with erlotinib (12.5 mg/kg/day), with the AKT inhibitor G594 (25 mg/kg/day), or in combination with both drugs. Scale bar: 500 µm. B. Quantification of the tumor/lung ratio of mice treated or not with the individual drugs or with a combination of both.


C . Representative Ki67 immunostaining of lung tumors from EGFRL858R / Rhob - / - , EGFRL858R / Rhob + / - EGFRL858R / Rhob + / + mice treated or not with erlotinib alone ( 12 . 5 mg / kg / day ) or in combination with AKT inhibitor G594 ( 25 mg / kg / day ) for four days ( scale bar : 50 µm ) , corresponding ( D ) .

O O O O O O O O O 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 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

C. Representative Ki67 immunostaining of lung tumors from EGFRL858R/Rhob-/-, EGFRL858R/Rhob+/- and EGFRL858R/Rhob+/+ mice treated or not with erlotinib alone (12.5 mg/kg/day) or in combination with the AKT inhibitor G594 (25 mg/kg/day) for four days (scale bar: 50 µm), and the corresponding quantification (D).