f3Four - 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 and photographed . ( d , e ) Total liver mass ( d ) were measured from indicated mice . ( 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 . ( 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 ) and quantified ( l ) . ( 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 .

B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O 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-PANEL_START 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

f3Four-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 and photographed. (d,e) Total liver mass (d) were measured from indicated mice.(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.(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) and quantified (l).(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.


f4Four - 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 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 .

B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

f4Four-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 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.


f5Four - 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 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 .

B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

f5Four-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 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.


f6Four - 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 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 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 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 ) .

B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

f6Four-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 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 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 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).


f7 ( 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 ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

f7(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).


Figure 1 ( A ) Western blotting of AXL and OPCML protein levels in AXL - expressing , OPCML - null SKOV3 and PEA1 cell lines transduced with OPCML " O " or control Empty vector " E " ( B ) Mammalian 2 - Hybrid assay between OPCML and AXL Data in ( A ) to ( M ) are representative of at least three experiments with graphs depicting means ± SEM s ; * P < 0 . 05 , * * * P < 0 . 01 , and * * * * P < 0 . 0001 by Student " s t tests ( C ) Western blotting of the OPCML - GST pull down . Input : 1 / 20 of SKOV3 - Empty whole cell lysate ( D ) Western blotting of the anti - AXL immunoprecipitation . Input : 1 / 100 of SKOV3 - OPCML whole cell lysate . IP : Immunoprecipitated protein , IB : Immunoblotted protein ( E ) Immunostaining of OPCML ( red ) and AXL ( green ) in SKOV3 - OPCML and PEA1 - OPCML cells . Scale bar = 10μm Data i M , ( E ) was performed once due to the limited numbers of primary cells ; ( G ) FRET efficiency for AXL - OPCML interaction in SKOV3 - Empty " E " and SKOV3 - OPCML " O " , using AXL - AXL FRET as a positive control . Cells were labeled with Anti - Axl rabbit ( conjugated to donor probe ) and Anti - OPCML mouse ( conjugated to acceptor probe ) , or Anti - Axl mouse ( conjugated to acceptor probe ) and Anti - Axl rabbit ( conjugated to donor probe ) Data in ( A ) to ( M ) are representative of at least three experiments with graphs depicting means ± SE s ; * P < 0 . 05 , * * * P < 0 . 01 , and * * * * P < 0 . 0001 by Student " s t tests ( H ) PLA of AXL - OPCML ( red ) in SKOV3 and PEA1 cells transduced with empty vector " E " or OPCML " O " . Scale bar = 50μmPrimary ovarian tumour cells were characterised for AXL and OPCML levels by ( I ) Western blottin PAX8 was used to identify ovarian cancer cellsPrimary ovarian tumour cells were characterised for AXL and OPCML levels b ( J ) Immunostaining . PAX8 was used to identify ovarian cancer cells ( K ) Quantitation of AXL - OPCML PLA in primary cells Data in ( A ) to ( M ) are representative of at least three experiments with graphs depicting means ± SEM s ; * P < 0 . 05 , * * * P < 0 . 01 , and * * * * P < 0 . 0001 by Student " s t tests ( L ) Western blotting of membrane fractionation of SKOV3 - Empty cells : total cell lysate " T " , liquid - disordered soluble fraction " S " , detergent resistant membrane fraction " R " . Caveolin - 1 is used as a marker of the R fraction and Calnexin is used as a marker for the S fraction , E : SKOV3 - Empty , O : SKOV3 - OPCML ( M ) Densitometry of AXL band intensities in ( J ) in S and R , normalized to AXL intensity in input . Data in ( A ) to ( M ) are representative of at least three experiments with graphs depicting means ± SE s ; * P < 0 . 05 , * * * P < 0 . 01 , and * * * * P < 0 . 0001 by 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START 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-PANEL_START O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1 (A) Western blotting of AXL and OPCML protein levels in AXL-expressing, OPCML-null SKOV3 and PEA1 cell lines transduced with OPCML "O" or control Empty vector "E" (B) Mammalian 2-Hybrid assay between OPCML and AXL Data in (A) to (M) are representative of at least three experiments with graphs depicting means ± SEM s; *P < 0.05, ***P < 0.01, and ****P < 0.0001 by Student"s t tests(C) Western blotting of the OPCML-GST pull down. Input: 1/20 of SKOV3-Empty whole cell lysate(D) Western blotting of the anti-AXL immunoprecipitation. Input: 1/100 of SKOV3-OPCML whole cell lysate. IP: Immunoprecipitated protein, IB: Immunoblotted protein(E) Immunostaining of OPCML (red) and AXL (green) in SKOV3-OPCML and PEA1-OPCML cells. Scale bar = 10μm Data i M, (E) was performed once due to the limited numbers of primary cells;(G) FRET efficiency for AXL-OPCML interaction in SKOV3-Empty "E" and SKOV3-OPCML "O", using AXL-AXL FRET as a positive control. Cells were labeled with Anti-Axl rabbit (conjugated to donor probe) and Anti-OPCML mouse (conjugated to acceptor probe), or Anti-Axl mouse (conjugated to acceptor probe) and Anti-Axl rabbit (conjugated to donor probe) Data in (A) to (M) are representative of at least three experiments with graphs depicting means ± SE s; *P < 0.05, ***P < 0.01, and ****P < 0.0001 by Student"s t tests(H) PLA of AXL-OPCML (red) in SKOV3 and PEA1 cells transduced with empty vector "E" or OPCML "O". Scale bar = 50μmPrimary ovarian tumour cells were characterised for AXL and OPCML levels by (I) Western blottin PAX8 was used to identify ovarian cancer cellsPrimary ovarian tumour cells were characterised for AXL and OPCML levels b (J) Immunostaining. PAX8 was used to identify ovarian cancer cells(K) Quantitation of AXL-OPCML PLA in primary cells Data in (A) to (M) are representative of at least three experiments with graphs depicting means ± SEM s; *P < 0.05, ***P < 0.01, and ****P < 0.0001 by Student"s t tests(L) Western blotting of membrane fractionation of SKOV3-Empty cells: total cell lysate "T", liquid-disordered soluble fraction "S", detergent resistant membrane fraction "R". Caveolin-1 is used as a marker of the R fraction and Calnexin is used as a marker for the S fraction, E: SKOV3-Empty, O: SKOV3-OPCML(M) Densitometry of AXL band intensities in (J) in S and R, normalized to AXL intensity in input. Data in (A) to (M) are representative of at least three experiments with graphs depicting means ± SE s; *P < 0.05, ***P < 0.01, and ****P < 0.0001 by Student"s t tests


Figure 2 SKOV3 - OPCML cells stimulated with Gas6 over a 12 hour time course and ( A ) stained with DAPI ( cyan ) , Anti - OPCML ( green ) and Anti - AXL ( red ) antibodies ( scale bar = 10μm ) ( C , D ) OPCML - AXL interaction ( red ) was visualised by PLA ( scale bar = 50μm ) and quantified , n = 3 Data in ( A ) to ( O ) are representative of at least three experiments with graphs depicting means ± SE s ; * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , and * * * * P < 0 . 0001 by Student " s t testsd ; ( C , D ) OPCML - AXL interaction ( red ) was visualised by PLA ( scale bar = 50μm ) and quantified , n = 3 ( E ) Quantitation OPCML - AXL PLA interaction in primary ovarian tumour cells , n = 1 Data i M , ( E ) was performed once due to the limited numbers of primary cells ; * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , and * * * * P < 0 . 0001 by Student " s t tests ( F ) Co - immunoprecipitation with anti - AXL antibody in cells stimulated with Gas6 as indicated . Input : 1 / 50 of whole cell lysate from SKOV3 - OPCML cells . IP : Immunoprecipitated protein , IB : Immunoblotted protein ( G ) Co - immunoprecipitation with anti - pAXL antibody in SKOV3 - OPCML cells stimulated with Gas6 ( H ) OPCML - GST pull down assay in SKOV3 - Empty cells stimulated with Gas6 ( I ) Membrane fractionation in SKOV3 - Empty " E " and SKOV3 - OPCML " O " cells into the detergent resistant " R " membrane fraction and the detergent soluble " S " fraction upon Gas6 stimulation ( K ) FRAP recovery curves in insoluble lipid domain or soluble plasma membrane fraction in SKOV3 - Empty " E " and SKOV3 - OPCML " O " cells treated with Gas6 Data in ( A ) to ( O ) are representative of at least three experiments with graphs depicting means ± SEM s ; * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , and * * * * P < 0 . 0001 by Student " s t tests ( L ) Diffusion Co - efficient in insoluble lipid domain or soluble plasma membrane fraction in SKOV3 - Empty " E " and SKOV3 - OPCML " O " cells treated with Gas6 Data in ( A ) to ( O ) are representative of at least three experiments with graphs depicting means ± SEM s ; * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , and * * * * P < 0 . 0001 by Student " s t tests ( M ) percentage of AXL localised in insoluble lipid domain or soluble plasma membrane fraction in SKOV3 - Empty " E " and SKOV3 - OPCML " O " cells treated with Gas6 Data in ( A ) to ( O ) are representative of at least three experiments with graphs depicting means ± SE s ; * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , and * * * * P < 0 . 0001 by Student " s t tests ( N ) FRET Efficiency in SKOV3 - OPCML " O " cells stimulated with Gas6 , and labeled with Anti - Axl ( donor probe ) and Anti - OPCML ( acceptor probe ) Data in ( A ) to ( O ) are representative of at least three experiments with graphs depicting means ± SEM s ; * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , and * * * * P < 0 . 0001 by Student " s t tests ( O ) Western blotting of the " S " and " R " membrane fractions in SKOV3 - Empty " E " and OCPML " O " cells stimulated with Gas6 for 3 hours , for Gas6 , AXL and pAXL . Data in ( A ) to ( O ) are representative of at least three experiments with graphs depicting means ±

O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2 SKOV3-OPCML cells stimulated with Gas6 over a 12 hour time course and (A) stained with DAPI (cyan), Anti-OPCML (green) and Anti-AXL (red) antibodies (scale bar = 10μm) (C,D) OPCML-AXL interaction (red) was visualised by PLA (scale bar = 50μm) and quantified, n = 3 Data in (A) to (O) are representative of at least three experiments with graphs depicting means ± SE s; *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001 by Student"s t testsd; (C,D) OPCML-AXL interaction (red) was visualised by PLA (scale bar = 50μm) and quantified, n = 3(E) Quantitation OPCML-AXL PLA interaction in primary ovarian tumour cells, n=1 Data i M, (E) was performed once due to the limited numbers of primary cells; *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001 by Student"s t tests(F) Co-immunoprecipitation with anti-AXL antibody in cells stimulated with Gas6 as indicated. Input: 1/50 of whole cell lysate from SKOV3-OPCML cells. IP: Immunoprecipitated protein, IB: Immunoblotted protein(G) Co-immunoprecipitation with anti-pAXL antibody in SKOV3-OPCML cells stimulated with Gas6(H) OPCML-GST pull down assay in SKOV3-Empty cells stimulated with Gas6(I) Membrane fractionation in SKOV3-Empty "E" and SKOV3-OPCML "O" cells into the detergent resistant "R" membrane fraction and the detergent soluble "S" fraction upon Gas6 stimulation(K) FRAP recovery curves in insoluble lipid domain or soluble plasma membrane fraction in SKOV3-Empty "E" and SKOV3-OPCML "O" cells treated with Gas6 Data in (A) to (O) are representative of at least three experiments with graphs depicting means ± SEM s; *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001 by Student"s t tests(L) Diffusion Co-efficient in insoluble lipid domain or soluble plasma membrane fraction in SKOV3-Empty "E" and SKOV3-OPCML "O" cells treated with Gas6 Data in (A) to (O) are representative of at least three experiments with graphs depicting means ± SEM s; *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001 by Student"s t tests(M) percentage of AXL localised in insoluble lipid domain or soluble plasma membrane fraction in SKOV3-Empty "E" and SKOV3-OPCML "O" cells treated with Gas6 Data in (A) to (O) are representative of at least three experiments with graphs depicting means ± SE s; *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001 by Student"s t tests(N) FRET Efficiency in SKOV3-OPCML "O" cells stimulated with Gas6, and labeled with Anti-Axl (donor probe) and Anti-OPCML (acceptor probe) Data in (A) to (O) are representative of at least three experiments with graphs depicting means ± SEM s; *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001 by Student"s t tests(O) Western blotting of the "S" and "R" membrane fractions in SKOV3-Empty "E" and OCPML "O" cells stimulated with Gas6 for 3 hours, for Gas6, AXL and pAXL. Data in (A) to (O) are representative of at least three experiments with graphs depicting means ± SEM


Figure 5 ( A ) Filipin staining of SKOV3 - OPCML cells treated for 30 minutes with 5mM MBCD and allowed to recover for 0 , 30 minutes , and 12 hours in serum free ( SF ) medium ( B ) Western blotting of SKOV3 - OPCML cells treated with MBCD and separated into the detergent resistant " R " membrane fraction and the detergent soluble " S " fraction upon Gas6 stimulation ( C ) Ratio of AXL band intensity in " R " or pAXL band intensity in " S " relative to total AXL " T " from SKOV3 - OPCML " O " in ( B ) ( D ) MBCD treatment of SKOV3 - EMPTY and SKOV3 - OPCML cell lines was followed by the analysis of Gas6 signalling kinetics by western blot . GAPDH was used as loading control ( E , F ) Signalling kinetics are presented for pAXL and pERK in SKOV3 - EMPTY " E " and SKOV3 - OCPML " O " , treated with MBCD and no treatment control , normalised to GAPDH , from ( D ) ( G ) PLA assay of AXL - OPCML interaction ( red ) upon Gas6 stimulation in both MBCD treated and non - treated SKOV3 - OPCML cells . Scale bar =

O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5 (A) Filipin staining of SKOV3-OPCML cells treated for 30 minutes with 5mM MBCD and allowed to recover for 0, 30 minutes, and 12 hours in serum free (SF) medium(B) Western blotting of SKOV3-OPCML cells treated with MBCD and separated into the detergent resistant "R" membrane fraction and the detergent soluble "S" fraction upon Gas6 stimulation(C) Ratio of AXL band intensity in "R" or pAXL band intensity in "S" relative to total AXL "T" from SKOV3-OPCML "O" in (B)(D) MBCD treatment of SKOV3-EMPTY and SKOV3-OPCML cell lines was followed by the analysis of Gas6 signalling kinetics by western blot. GAPDH was used as loading control(E,F) Signalling kinetics are presented for pAXL and pERK in SKOV3-EMPTY "E" and SKOV3-OCPML "O", treated with MBCD and no treatment control, normalised to GAPDH, from (D)(G) PLA assay of AXL-OPCML interaction (red) upon Gas6 stimulation in both MBCD treated and non-treated SKOV3-OPCML cells. Scale bar = 50μm


Figure 7 ( B ) Western Blotting of SKOV3 - Empty " E " and SKOV3 - OPCML " O " cells pre incubated with either no drug ( DMSO only control ) , 1 . 09 μM R428 , or 2 . 84 μM R428 for an hour and stimulated with Gas6 for the indicated times . GAPDH was used as loading control ( C ) Immunofluorescence staining of pERK ( red ) and OPCML ( magenta ) in SKOV3 - Empty " E " and SKOV3 - OPCML " O " cells treated with R428 and stimulated with Gas6 for the indicted time points . Scale bars =

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 7(B) Western Blotting of SKOV3-Empty "E" and SKOV3-OPCML "O" cells pre incubated with either no drug (DMSO only control), 1.09 μM R428, or 2.84 μM R428 for an hour and stimulated with Gas6 for the indicated times. GAPDH was used as loading control(C) Immunofluorescence staining of pERK (red) and OPCML (magenta) in SKOV3-Empty "E" and SKOV3-OPCML "O" cells treated with R428 and stimulated with Gas6 for the indicted time points. Scale bars = 50μm


Figure 1Bone marrow cells were polarized in the presence of 40 ng / ml GM - CSF and treated with 0 . 2 mM of the indicated compounds for 7 days . Gr1 - CD11b + cells were sorted and lysed for microarray . The hierarchical clustering was based on the different expression genes between BSA ( Con ) and oleate group . Signature genes involved in lipid metabolism , dendritic cell maturation , macrophage maturation , tumor associated macrophages ( TAMs ) phenotype , MHCII complex and innate immune response are listed and validated via flow cytometry or catalytic activity assay . Data are expressed as mean ± SD from two to four independent experiments . Unpaired Student ' s two - tailed t tests were performed to compare the expression level of indicated proteins in control and oleate groups . * p < 0 . 05 ; * * p ≤ 0 .

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Figure 1Bone marrow cells were polarized in the presence of 40 ng/ml GM-CSF and treated with 0.2 mM of the indicated compounds for 7 days. Gr1-CD11b+ cells were sorted and lysed for microarray. The hierarchical clustering was based on the different expression genes between BSA (Con) and oleate group.Signature genes involved in lipid metabolism, dendritic cell maturation, macrophage maturation, tumor associated macrophages (TAMs) phenotype, MHCII complex and innate immune response are listed and validated via flow cytometry or catalytic activity assay. Data are expressed as mean±SD from two to four independent experiments. Unpaired Student's two-tailed t tests were performed to compare the expression level of indicated proteins in control and oleate groups. *p<0.05; **p≤0.01


Figure 2Gr1 - CD11b + cells , polarized by the indicated treatment , were purified for mitochondrial respiration detection . The oxygen consumption rate ( OCR ) of these cells was monitored after the addition of oligomycin ( OA ) ( 1 µM ) , Carbonyl cyanide - 4 - ( trifluoromethoxy ) phenylhydrazone ( FCCP , 1 µM ) , and the electron transport inhibitor rotenone and antimycin A ( R / AA ) ( 0 . 5 µM ) at indicated time points . The basal OCR , basal extracellular acidification rate ( ECAR ) , spare respiratory capacity , proton leak , ATP production and maximal respiration based on the OCR value were quantified . Forty µM etomoxir were added starting from day 0 of bone marrow polarization , followed by mitochondrial respiration assay by using the same amount of polarized Gr1 - CD11b + myeloid cells on day 7 . The mitochondrial respiration was monitored and analyzed via XFe96 Analyzer . Exemplary plots of the proportion of CD206 + cells , the mean fluorescence intensity ( MFI ) of CD38 and CD73 on polarized myeloid cells was determined via flow cytometry . T cell proliferation assay was performed via co - culture with purified CD4 + T cells in the ratios of M ( myeloid cells ) : T ( T cells ) = 1 : 30 . NO production from the co - culture supernatant was quantified by Griess reaction .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O 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-PANEL_START O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O

Figure 2Gr1-CD11b+ cells, polarized by the indicated treatment, were purified for mitochondrial respiration detection. The oxygen consumption rate (OCR) of these cells was monitored after the addition of oligomycin (OA) (1 µM), Carbonyl cyanide-4-(trifluoromethoxy) phenylhydrazone (FCCP, 1 µM), and the electron transport inhibitor rotenone and antimycin A (R/AA) (0.5 µM) at indicated time points. The basal OCR, basal extracellular acidification rate (ECAR), spare respiratory capacity, proton leak, ATP production and maximal respiration based on the OCR value were quantified.Forty µM etomoxir were added starting from day 0 of bone marrow polarization, followed by mitochondrial respiration assay by using the same amount of polarized Gr1-CD11b+ myeloid cells on day 7. The mitochondrial respiration was monitored and analyzed via XFe96 Analyzer.Exemplary plots of the proportion of CD206+ cells, the mean fluorescence intensity (MFI) of CD38 and CD73 on polarized myeloid cells was determined via flow cytometry.T cell proliferation assay was performed via co-culture with purified CD4+ T cells in the ratios of M (myeloid cells): T (T cells) = 1:30.NO production from the co-culture supernatant was quantified by Griess reaction.


Figure 3The oxygen consumption rate ( OCR ) of 1 × 105 purified Gr1 - CD11b + cells was monitored as described in Figure 2 . The basal OCR , basal ECAR ( extracellular acidification rate ) , spare respiratory capacity , proton leak , ATP production and maximal respiration based on the OCR value were quantified . The polarization state was evaluated via the expression of CD206 . T cell proliferation assay was performed employing co - culture with purified CD4 + T cell in the variant ratios . The lipid droplets accumulation was determined by BODIPY staining . The percentage of divided cells and proliferation index were calculated . Nitric oxide ( NO ) concentration in the co - culture supernatant was quantified by Griess reaction .

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Figure 3The oxygen consumption rate (OCR) of 1×105 purified Gr1-CD11b+ cells was monitored as described in Figure 2. The basal OCR, basal ECAR (extracellular acidification rate), spare respiratory capacity, proton leak, ATP production and maximal respiration based on the OCR value were quantified.The polarization state was evaluated via the expression of CD206.T cell proliferation assay was performed employing co-culture with purified CD4+ T cell in the variant ratios.The lipid droplets accumulation was determined by BODIPY staining. The percentage of divided cells and proliferation index were calculated.Nitric oxide (NO) concentration in the co-culture supernatant was quantified by Griess reaction.


Figure 4Bone marrow - derived myeloid cells were treated with BSA - control ( Con ) or oleate in the presence or absence of 10 nM rapamycin ( Rapa ) . The polarization of myeloid cells was detected via flow cytometry as indicated by CD11b and CD206 expression . Purified Gr1 - CD11b + myeloid cells were co - cultured with CD4 + T cells for proliferation assay . The supernatant was collected for nitric oxide ( NO ) detection . The oxygen consumption rate ( OCR ) of 1 × 105 differentiated myeloid cells were monitored after the addition of oligomycin ( OA ) ( 1 µM ) , the uncoupler carbonyl cyanide - 4 - ( trifluoromethoxy ) phenylhydrazone ( FCCP , 1 µM ) , and the electron transport inhibitor rotenone and antimycin A ( R / AA , 5 µM ) at indicated time points . The basal OCR , basal extracellular acidification rate ( ECAR ) , spare respiratory capacity , ATP production and maximal respiration based on the OCR value were quantified . Mean fluorescence intensity ( MFI ) of intracellular phosphor - mTOR ( pS2448 ) in polarized myeloid cells as determined by flow cytometryMean fluorescence intensity ( MFI ) of intracellular phosphor - mTOR ( pS2448 ) in polarized myeloid cells lysed for western blot .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O

Figure 4Bone marrow-derived myeloid cells were treated with BSA-control (Con) or oleate in the presence or absence of 10 nM rapamycin (Rapa). The polarization of myeloid cells was detected via flow cytometry as indicated by CD11b and CD206 expression.Purified Gr1-CD11b+ myeloid cells were co-cultured with CD4+ T cells for proliferation assay. The supernatant was collected for nitric oxide (NO) detection.The oxygen consumption rate (OCR) of 1×105 differentiated myeloid cells were monitored after the addition of oligomycin (OA) (1 µM), the uncoupler carbonyl cyanide-4-(trifluoromethoxy) phenylhydrazone (FCCP, 1 µM), and the electron transport inhibitor rotenone and antimycin A (R/AA, 5 µM) at indicated time points. The basal OCR, basal extracellular acidification rate (ECAR), spare respiratory capacity, ATP production and maximal respiration based on the OCR value were quantified.Mean fluorescence intensity (MFI) of intracellular phosphor-mTOR (pS2448) in polarized myeloid cells as determined by flow cytometryMean fluorescence intensity (MFI) of intracellular phosphor-mTOR (pS2448) in polarized myeloid cells lysed for western blot.


Figure 5Tumor infiltrating immune cells were isolated from the either CT26 or MCA205 tumor bearing model and analyzed for the expression of BODIPY in CD206 + ( black ) and CD206 - ( grey ) cells . Balb / c mice were inoculated with 5 × 105 CT26 tumor cells subcutaneously . Since day 6 , vehicle or DGAT inhibitors ( iDGAT ) were administrated via intra - tumoral injection once daily . At day 13 , tumor infiltrated immune cells were isolated and stained for CD206 and CD11b . 5 × 105 MCA205 were injected subcutaneously into C57BL / 6 mice . PBS , liposome control or iDGAT - liposome was injected into the peritoneum every other day starting at day 7 . Tumor size and body weight were measured every 7 days and the mice were sacrificed on day 31 for analysis . Immune cells from spleen , tumor - draining lymph nodes ( TDLN ) or tumor were isolated and stained for CD11b and with BODIPY for lipid droplets quantification in myeloid cells . Tumor infiltrating CD8 + T cells ( red ) were stained together with nuclei ( DAPI / blue ) after sacrificing the tumor bearing mice .

O B-PANEL_START O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5Tumor infiltrating immune cells were isolated from the either CT26 or MCA205 tumor bearing model and analyzed for the expression of BODIPY in CD206+ (black) and CD206- (grey) cells.Balb/c mice were inoculated with 5×105 CT26 tumor cells subcutaneously. Since day 6, vehicle or DGAT inhibitors (iDGAT) were administrated via intra-tumoral injection once daily.At day 13, tumor infiltrated immune cells were isolated and stained for CD206 and CD11b.5×105 MCA205 were injected subcutaneously into C57BL/6 mice. PBS, liposome control or iDGAT-liposome was injected into the peritoneum every other day starting at day 7. Tumor size and body weight were measured every 7 days and the mice were sacrificed on day 31 for analysis. Immune cells from spleen, tumor-draining lymph nodes (TDLN) or tumor were isolated and stained for CD11b and with BODIPY for lipid droplets quantification in myeloid cells.Tumor infiltrating CD8+ T cells (red) were stained together with nuclei (DAPI/blue) after sacrificing the tumor bearing mice.


Figure 61 × 106 CD14 + myeloid cells from healthy donors were polarized by 1 ng / ml GM - CSF in the presence of BSA - control ( C ) , 0 . 2 mM oleate ( O ) or 0 . 2 mM stearate ( S ) over a 6 - day period . The expression of CD206 , CD204 and CD38 was analyzed via flow cytometry . The level of lipid droplets was determined via BODIPY staining . The oxygen consumption rate ( OCR ) of differentiated myeloid cells was monitored as described in Materials and Methods . ( D ) The purified CFSE - labeled autologous CD4 + T cells were co - cultured with polarized myeloid cells with indicated treatment for T cell proliferation assay . Tumor tissue ( tumor ) as well as corresponding non - tumorous adjacent tissue ( control ) were collected from colorectal cancer patients and prepared for histopathology . The expression of CD68 ( red ) , CD206 ( green ) and ADRP ( blue ) indicate the lipid droplets in tumor infiltrating myeloid cells . Nuclei ( white ) were stained using DAPI . The absolute number of positive cells was quantified in 5 high - power fields ( hpf ) ( scale bar = 20 µm ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 61×106 CD14+ myeloid cells from healthy donors were polarized by 1 ng/ml GM-CSF in the presence of BSA-control (C), 0.2 mM oleate (O) or 0.2 mM stearate (S) over a 6-day period. The expression of CD206, CD204 and CD38 was analyzed via flow cytometry.The level of lipid droplets was determined via BODIPY staining.The oxygen consumption rate (OCR) of differentiated myeloid cells was monitored as described in Materials and Methods.(D) The purified CFSE-labeled autologous CD4+ T cells were co-cultured with polarized myeloid cells with indicated treatment for T cell proliferation assay.Tumor tissue (tumor) as well as corresponding non-tumorous adjacent tissue (control) were collected from colorectal cancer patients and prepared for histopathology. The expression of CD68 (red), CD206 (green) and ADRP (blue) indicate the lipid droplets in tumor infiltrating myeloid cells. Nuclei (white) were stained using DAPI. The absolute number of positive cells was quantified in 5 high-power fields (hpf) (scale bar = 20 µm).


Figure 1A ) Deletion of the CID domain dramatically reduces the interaction of Nrd1 with Sen1 . Coimmunoprecipitation ( CoIP ) experiments using TAP - tagged Nrd1 ( either wt or ∆ CID ) as the bait . Representative gel of one out of two independent experiments . C ) Deletion of the NIM decreases substantially the association of Nrd1 with Sen1 . CoIP experiments using Nrd1 - TAP as the bait in a SEN1 or sen1 ∆ NIM background . Representative gel of one out of two independent experiments . D ) CoIP experiments using HA - tagged Sen1 , either wt or ∆ NIM , as the bait . Representative gel of one out of two independent experiments . Protein extracts were treated with RNaseA prior to immunoprecipitation . In these experiment , Sen1 could not be detected in the input extracts .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1A) Deletion of the CID domain dramatically reduces the interaction of Nrd1 with Sen1. Coimmunoprecipitation (CoIP) experiments using TAP-tagged Nrd1 (either wt or ∆CID) as the bait. Representative gel of one out of two independent experiments.C) Deletion of the NIM decreases substantially the association of Nrd1 with Sen1. CoIP experiments using Nrd1-TAP as the bait in a SEN1 or sen1∆NIM background. Representative gel of one out of two independent experiments.D) CoIP experiments using HA-tagged Sen1, either wt or ∆NIM, as the bait. Representative gel of one out of two independent experiments. Protein extracts were treated with RNaseA prior to immunoprecipitation. In these experiment, Sen1 could not be detected in the input extracts.


Figure 3A ) Deletion of the N - terminal of Sen1 is lethal . A ∆ sen1 strain ( YDL2767 ) covered by an URA3 - containing plasmid ( pFL38 ) expressing wt Sen1 was transformed with a TRP1 - plasmid ( pFL39 ) carrying either the wt or the mutant versions of SEN1 indicated in the scheme on the left . After over - night growth in non - selective medium , cells were plated on minimal medium ( CSM ) containing 5 - fluorootic acid ( 5 - FOA ) to select those that have lost the URA3 plasmid . The absence of growth in 5 - FOA implies that the SEN1 version expressed in the TRP1 - plasmid does not support viability . B ) Deletion of the N - terminal domain of Sen1 provokes dramatic defects in transcription termination in vivo . Northern blot analyses of three well - characterized NNS - targets , the CUT NEL025c and the snoRNAs SNR13 and SNR33 , in a Sen1 - AID ( auxin - induced degron ) strain carrying an empty vector or a plasmid expressing either the wt or the indicated versions of SEN1 . Sen1 - AID was depleted for 2h by the addition of 500 μM indole - 3 - acetic acid ( IAA ) to monitor the capacity of the plasmid - borne versions of SEN1 to induce transcription termination . The expected RNA species resulting from inefficient termination ( RT for readthrough species ) are indicated . assays were performed in a ∆ rrp6 background to detect the primary products of NNS - dependent termination and the U4 RNA is used as a loading control . C ) Overexpression of sen1 ∆ Nter restores viability . Growth test of strains harboring the indicated version of SEN1 at the endogenous locus under the control of the GAL1 promoter ( pGAL ) in the presence of galactose . D ) Overexpression of SEN1 partially suppresses the termination defects associated with deletion of the N - terminal domain . Northern blot analysis of a typical NNS - target in strains expressing the indicated SEN1 versions from pGAL in the presence of galactose . assays were performed in a ∆ rrp6 background to detect the primary products of NNS - dependent termination and the U4 RNA is used as a loading control .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3A) Deletion of the N-terminal of Sen1 is lethal. A ∆sen1 strain (YDL2767) covered by an URA3-containing plasmid (pFL38) expressing wt Sen1 was transformed with a TRP1-plasmid (pFL39) carrying either the wt or the mutant versions of SEN1 indicated in the scheme on the left. After over-night growth in non-selective medium, cells were plated on minimal medium (CSM) containing 5-fluorootic acid (5-FOA) to select those that have lost the URA3 plasmid. The absence of growth in 5-FOA implies that the SEN1 version expressed in the TRP1-plasmid does not support viability.B) Deletion of the N-terminal domain of Sen1 provokes dramatic defects in transcription termination in vivo. Northern blot analyses of three well-characterized NNS-targets, the CUT NEL025c and the snoRNAs SNR13 and SNR33, in a Sen1-AID (auxin-induced degron) strain carrying an empty vector or a plasmid expressing either the wt or the indicated versions of SEN1. Sen1-AID was depleted for 2h by the addition of 500 μM indole-3-acetic acid (IAA) to monitor the capacity of the plasmid-borne versions of SEN1 to induce transcription termination. The expected RNA species resulting from inefficient termination (RT for readthrough species) are indicated. assays were performed in a ∆rrp6 background to detect the primary products of NNS-dependent termination and the U4 RNA is used as a loading control.C) Overexpression of sen1∆Nter restores viability. Growth test of strains harboring the indicated version of SEN1 at the endogenous locus under the control of the GAL1 promoter (pGAL) in the presence of galactose.D) Overexpression of SEN1 partially suppresses the termination defects associated with deletion of the N-terminal domain. Northern blot analysis of a typical NNS-target in strains expressing the indicated SEN1 versions from pGAL in the presence of galactose. assays were performed in a ∆rrp6 background to detect the primary products of NNS-dependent termination and the U4 RNA is used as a loading control.


Figure 4RNAPII CRAC were performed in the presence of the indicated versions of SEN1 expressed from its own promoter in a centromeric plasmid , upon depletion of the chromosomally - encoded version of Sen1 in a Sen1 - AID strain . A ) Metagene analysis of the RNAPII distribution relative to the annotated polyadenylation site ( PAS ) of mRNAs . Values on the y axis correspond to the median coverage . B ) Box - plot representation of the readthrough ( RT ) index as a readout of the termination efficiency at each mRNA . The RT index was calculated as the average signal over a window of 200 nt downstream of the PAS , divided by the average signal from the TSS to the PAS of each mRNA . Boxes include all the values between the 25th and the 75th percentiles and the horizontal line indicates the median . The upper error bars indicate the distance between the largest value smaller than or equal to the upper limit of the box + 1 . 5 * IQR ( where IQR is the interquartile distance or distance between the first and the third quartile ) . The lower error bars indicate the distance between the lowest value greater than or equal to the lower limit of the box - 1 . 5 * IQR . For each mRNA ( n = 5752 ) , the values correspond to the average of two independent biological replicates . Data populations were compared using a t - test ( bilateral distributions , unpaired data ) and p - values ≥ 0 . 05 are indicated as ns ( not significant ) . RNAPII CRAC were performed in the presence of the indicated versions of SEN1 expressed from its own promoter in a centromeric plasmid , upon depletion of the chromosomally - encoded version of Sen1 in a Sen1 - AID strain . C - D ) IGV screenshots of two well - characterized protein - coding genes displaying substantial defects in premature termination in some mutants . The values correspond to the counts per million of counts ( CPM ) multiplied by 10 . RNAPII CRAC were performed in the presence of the indicated versions of SEN1 expressed from its own promoter in a centromeric plasmid , upon depletion of the chromosomally - encoded version of Sen1 in a Sen1 - AID strain . E ) Metagene analyses of transcribing RNAPII at CUTs performed as for mRNAs but using the annotated transcription termination site ( TTS ) as the reference point . F ) Analysis of termination defects in the different mutants at CUTs . Box - plots performed as in ( B ) . The RT index is calculated as the average signal over a window of 400 nt downstream of the TTS , divided by the average signal over a window of 100 nt downstream from the TSS ( CUT100 ) . For each CUT ( n = 329 ) , the values correspond to the average of two independent biological replicates . P - values < 0 . 001 are indicated by three asterisks . RNAPII CRAC were performed in the presence of the indicated versions of SEN1 expressed from its own promoter in a centromeric plasmid , upon depletion of the chromosomally - encoded version of Sen1 in a Sen1 - AID strain . G - H ) IGV screenshots of examples of CUTs displaying substantial termination defects in the mutants . RNAPII CRAC were performed in the presence of the indicated versions of SEN1 expressed from its own promoter in a centromeric plasmid , upon depletion of the chromosomally - encoded version of Sen1 in a Sen1 - AID strain . Analysis of RNAPII occupancy around snoRNAs performed as for CUTs . Box - plots performed as in ( B ) . The RT index is calculated as the average signal over a window of 300 nt downstream of the TTS , divided by the average signal over the mature snoRNA . RNAPII CRAC were performed in the presence of the indicated versions of SEN1 expressed from its own promoter in a centromeric plasmid , upon depletion of the chromosomally - encoded version of Sen1 in a Sen1 - AID strain . Analysis of RNAPII occupancy around snoRNAs performed as for CUTs .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4RNAPII CRAC were performed in the presence of the indicated versions of SEN1 expressed from its own promoter in a centromeric plasmid, upon depletion of the chromosomally-encoded version of Sen1 in a Sen1-AID strain. A) Metagene analysis of the RNAPII distribution relative to the annotated polyadenylation site (PAS) of mRNAs. Values on the y axis correspond to the median coverage. B) Box-plot representation of the readthrough (RT) index as a readout of the termination efficiency at each mRNA. The RT index was calculated as the average signal over a window of 200 nt downstream of the PAS, divided by the average signal from the TSS to the PAS of each mRNA. Boxes include all the values between the 25th and the 75th percentiles and the horizontal line indicates the median. The upper error bars indicate the distance between the largest value smaller than or equal to the upper limit of the box + 1.5 *IQR (where IQR is the interquartile distance or distance between the first and the third quartile). The lower error bars indicate the distance between the lowest value greater than or equal to the lower limit of the box - 1.5 *IQR. For each mRNA (n=5752), the values correspond to the average of two independent biological replicates. Data populations were compared using a t-test (bilateral distributions, unpaired data) and p-values≥ 0.05 are indicated as ns (not significant).RNAPII CRAC were performed in the presence of the indicated versions of SEN1 expressed from its own promoter in a centromeric plasmid, upon depletion of the chromosomally-encoded version of Sen1 in a Sen1-AID strain. C-D) IGV screenshots of two well-characterized protein-coding genes displaying substantial defects in premature termination in some mutants. The values correspond to the counts per million of counts (CPM) multiplied by 10.RNAPII CRAC were performed in the presence of the indicated versions of SEN1 expressed from its own promoter in a centromeric plasmid, upon depletion of the chromosomally-encoded version of Sen1 in a Sen1-AID strain. E) Metagene analyses of transcribing RNAPII at CUTs performed as for mRNAs but using the annotated transcription termination site (TTS) as the reference point. F) Analysis of termination defects in the different mutants at CUTs. Box-plots performed as in (B). The RT index is calculated as the average signal over a window of 400 nt downstream of the TTS, divided by the average signal over a window of 100 nt downstream from the TSS (CUT100). For each CUT (n=329), the values correspond to the average of two independent biological replicates. P-values < 0.001 are indicated by three asterisks. RNAPII CRAC were performed in the presence of the indicated versions of SEN1 expressed from its own promoter in a centromeric plasmid, upon depletion of the chromosomally-encoded version of Sen1 in a Sen1-AID strain. G-H) IGV screenshots of examples of CUTs displaying substantial termination defects in the mutants.RNAPII CRAC were performed in the presence of the indicated versions of SEN1 expressed from its own promoter in a centromeric plasmid, upon depletion of the chromosomally-encoded version of Sen1 in a Sen1-AID strain. Analysis of RNAPII occupancy around snoRNAs performed as for CUTs. Box-plots performed as in (B). The RT index is calculated as the average signal over a window of 300 nt downstream of the TTS, divided by the average signal over the mature snoRNA.RNAPII CRAC were performed in the presence of the indicated versions of SEN1 expressed from its own promoter in a centromeric plasmid, upon depletion of the chromosomally-encoded version of Sen1 in a Sen1-AID strain. Analysis of RNAPII occupancy around snoRNAs performed as for CUTs.


Figure 5A ) Deletion of Sen1 N - terminal domain does not prevent the interaction of Sen1 with RNAPII . CoIP experiments using Rbp3 - FLAG as the bait . Assays were performed in a Sen1 - AID strain harboring a plasmid expressing either SEN1 or sen1 ∆ Nter upon depletion of Sen1 - AID in the presence of IAA for 2h . An asterisk denotes a major proteolytic Sen1 fragment detected in the extracts of roughly the size of sen1 ∆ Nter . Nrd1 is detected as a positive control . Representative gel of one out of two independent experiments . Protein extracts were treated with RNaseA before immunoprecipitation . B ) Deletion of the Sen1 N - terminal domain reduces the interaction of Sen1 with the S5P - CTD . CoIP experiments using TAP - Sen1 as the bait . Sen1 proteins were expressed from pGAL in the presence of galactose . Nab3 is detected as a positive control . Representative gel of one out of three independent experiments . Protein extracts were treated with RNaseA before immunoprecipitation . C ) Replacing the Nter of Sen1 by the CID of Nrd1 restores viability . Growth test performed in the same conditions as in figure 3A but in the presence of a TRP1 - plasmid carrying the SEN1 versions indicated in the scheme on the left . The growth of the strain expressing the Nrd1 CID - sen1 ∆ Nter chimera in 5 - FOA implies that this gene can support viability . D ) Substituting the Nter of Sen1 by Nrd1 CID but not Pcf11 CID partially suppresses the termination defects detected in the sen1 ∆ Nter mutant . Northern blot assays performed in a Sen1 - AID strain carrying an empty vector or a plasmid expressing the indicated versions of SEN1 upon depletion of the endogenous Sen1 protein as in figure 3B . Experiments performed in a ∆ rrp6 background . Representative gel of one out of two independent experiments . The U4 RNA is used as a loading control . E ) Sen1 Nter interacts with the C - terminal domain ( Cter ) of Sen1 both in the presence and in the absence of the NIM in vitro . Pull - down experiments using either a wt or a ∆ NIM version of recombinant Sen1 Cter immobilized on glutathione - sepharose beads and a TAP - tagged version of Sen1 Nter expressed in yeast . Representative gel of one out of three independent experiments .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5A) Deletion of Sen1 N-terminal domain does not prevent the interaction of Sen1 with RNAPII. CoIP experiments using Rbp3-FLAG as the bait. Assays were performed in a Sen1-AID strain harboring a plasmid expressing either SEN1 or sen1∆Nter upon depletion of Sen1-AID in the presence of IAA for 2h. An asterisk denotes a major proteolytic Sen1 fragment detected in the extracts of roughly the size of sen1∆Nter. Nrd1 is detected as a positive control. Representative gel of one out of two independent experiments. Protein extracts were treated with RNaseA before immunoprecipitation.B) Deletion of the Sen1 N-terminal domain reduces the interaction of Sen1 with the S5P-CTD. CoIP experiments using TAP-Sen1 as the bait. Sen1 proteins were expressed from pGAL in the presence of galactose. Nab3 is detected as a positive control. Representative gel of one out of three independent experiments. Protein extracts were treated with RNaseA before immunoprecipitation.C) Replacing the Nter of Sen1 by the CID of Nrd1 restores viability. Growth test performed in the same conditions as in figure 3A but in the presence of a TRP1-plasmid carrying the SEN1 versions indicated in the scheme on the left. The growth of the strain expressing the Nrd1 CID-sen1∆Nter chimera in 5-FOA implies that this gene can support viability.D) Substituting the Nter of Sen1 by Nrd1 CID but not Pcf11 CID partially suppresses the termination defects detected in the sen1∆Nter mutant. Northern blot assays performed in a Sen1-AID strain carrying an empty vector or a plasmid expressing the indicated versions of SEN1 upon depletion of the endogenous Sen1 protein as in figure 3B. Experiments performed in a ∆rrp6 background. Representative gel of one out of two independent experiments.The U4 RNA is used as a loading control.E) Sen1 Nter interacts with the C-terminal domain (Cter) of Sen1 both in the presence and in the absence of the NIM in vitro. Pull-down experiments using either a wt or a ∆NIM version of recombinant Sen1 Cter immobilized on glutathione-sepharose beads and a TAP-tagged version of Sen1 Nter expressed in yeast. Representative gel of one out of three independent experiments.


Figure 6A - C ) Comparison of the distribution of Sen1 and RNAPII ( Rpb1 ) at mRNAs and CUTs . Values on the y axis correspond to the median coverage . Note that the scale of Sen1 and Rpb1 datasets have been modified to be able to visualize both in the same plot . D - F ) IGV screenshots of two mRNAs and one CUT exemplifying the trend observed in the metagene plots . G ) Metagene analysis of the distribution of the different Sen1 variants at mRNAs using either the TSS or the PAS as reference point . Values correspond to the median of Sen1 data normalized by the RNAPII signal obtained in the corresponding background . H - I ) Screenshots of two well - characterized protein - coding genes that are regulated by NNS - dependent premature termination . Values correspond to unnormalized Sen1 occupancy . J ) Profile of Sen1 binding at CUTs calculated as for mRNAs . K - L ) Examples of CUTs exhibiting differences in Sen1 occupancy upon deletion of the NIM or the Nter . Plots correspond to unnormalized data . M - O ) Analysis of the distribution of the different Sen1 versions around snoRNAs performed as for CUTs . P ) Comparison of the total levels of the different Sen1 variants at mRNAs , CUTs and the termination region of snoRNAs ( i . e . a window of 200 nt downstream of the 3 ' end of the mature snoRNA , snoRNA - ter ) . Boxes in box - plots include all the values between the 25th and the 75th percentiles and the horizontal line indicates the median . The upper error bars indicate the distance between the largest value smaller than or equal to the upper limit of the box + 1 . 5 * IQR . The lower error bars indicate the distance between the lowest value greater than or equal to the lower limit of the box - 1 . 5 * IQR . Datasets for the different Sen1 versions were compared using a t - test ( bilateral distributions , unpaired data ) . p - values ≥ 0 . 05 are indicated as ns ( not significant ) , one asterisk is used when 0 . 05 > p - value ≥ 0 . 01 , two asterisks when 0 . 01 > p - value ≥ 0 . 001 and three asterisks for p - value < 0 . 001 .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O 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-PANEL_START O O O O O O O O O O O O B-PANEL_START O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6A-C) Comparison of the distribution of Sen1 and RNAPII (Rpb1) at mRNAs and CUTs. Values on the y axis correspond to the median coverage. Note that the scale of Sen1 and Rpb1 datasets have been modified to be able to visualize both in the same plot.D-F) IGV screenshots of two mRNAs and one CUT exemplifying the trend observed in the metagene plots.G) Metagene analysis of the distribution of the different Sen1 variants at mRNAs using either the TSS or the PAS as reference point. Values correspond to the median of Sen1 data normalized by the RNAPII signal obtained in the corresponding background.H-I) Screenshots of two well-characterized protein-coding genes that are regulated by NNS-dependent premature termination. Values correspond to unnormalized Sen1 occupancy.J) Profile of Sen1 binding at CUTs calculated as for mRNAs.K-L) Examples of CUTs exhibiting differences in Sen1 occupancy upon deletion of the NIM or the Nter. Plots correspond to unnormalized data.M-O) Analysis of the distribution of the different Sen1 versions around snoRNAs performed as for CUTs.P) Comparison of the total levels of the different Sen1 variants at mRNAs, CUTs and the termination region of snoRNAs (i.e. a window of 200 nt downstream of the 3' end of the mature snoRNA, snoRNA-ter). Boxes in box-plots include all the values between the 25th and the 75th percentiles and the horizontal line indicates the median. The upper error bars indicate the distance between the largest value smaller than or equal to the upper limit of the box + 1.5 *IQR. The lower error bars indicate the distance between the lowest value greater than or equal to the lower limit of the box - 1.5 *IQR. Datasets for the different Sen1 versions were compared using a t-test (bilateral distributions, unpaired data). p-values≥ 0.05 are indicated as ns (not significant), one asterisk is used when 0.05>p-value≥0.01, two asterisks when 0.01>p-value≥0.001 and three asterisks for p-value <0.001.


Figure 7A ) Decreasing the elongation rate alleviates the termination defects associated with deletion of Sen1 Nter . Northern blot assays performed in a Sen1 - AID , ∆ rrp6 strain carrying a plasmid expressing the indicated versions of SEN1 upon depletion of the endogenous Sen1 protein as in former experiments . Where indicated , cells were treated with 50 mg / L of 6 - azauracile ( 6AU ) for 2h . Representative gel of one out of two independent biological replicates . The U4 RNA is used as a loading control .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 7A) Decreasing the elongation rate alleviates the termination defects associated with deletion of Sen1 Nter. Northern blot assays performed in a Sen1-AID, ∆rrp6 strain carrying a plasmid expressing the indicated versions of SEN1 upon depletion of the endogenous Sen1 protein as in former experiments. Where indicated, cells were treated with 50 mg/L of 6-azauracile (6AU) for 2h. Representative gel of one out of two independent biological replicates.The U4 RNA is used as a loading control.


Figure 1Flow cytometry analysis of PD - L1 expression in ( A ) KPC and ( B ) Pan02 cells following treatment with gemcitabine and RT . Gemcitabine was diluted in DMSO ( vehicle ) and DMSO was used for the ' untreated ' control group ( mean ± SD , n = 3 , One - way ANOVA , Bonferroni test ) . Western blot analysis of indicated proteins in both KPC ( C ) and Pan02 ( D ) cells following radiation and chemotherapy ± AG490 , a JAK / Stat kinase inhibitor . Actin represents loading control . Flow cytometry analysis of PD - L1 after gemcitabine and RT ( as described above ) in KPC ( E ) and Pan02 ( F ) following Stat1 downregulation by siRNA ( mean ± SD , n = 3 , Student ' s t - test ) . MFI , mean fluorescence index ;

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1Flow cytometry analysis of PD-L1 expression in (A) KPC and (B) Pan02 cells following treatment with gemcitabine and RT. Gemcitabine was diluted in DMSO (vehicle) and DMSO was used for the 'untreated' control group (mean ± SD, n=3, One-way ANOVA, Bonferroni test).Western blot analysis of indicated proteins in both KPC (C) and Pan02 (D) cells following radiation and chemotherapy ± AG490, a JAK/Stat kinase inhibitor. Actin represents loading control.Flow cytometry analysis of PD-L1 after gemcitabine and RT (as described above) in KPC (E) and Pan02 (F) following Stat1 downregulation by siRNA (mean ± SD, n=3, Student's t-test). MFI, mean fluorescence index;


Figure 2 ( A ) Tumor growth delay was measured in the different groups , as indicated ( n = 6 mice per group ) . Anti - PD - L1 was given at days 0 3 , 6 and 9 ( black arrows ) . The average time ( days ) for tumors to reach a volume of 400 mm3 from day 0 is shown ( means ± SD , n = 1 , One - way ANOVA , Bonferroni test ) . No weight loss was observed in the in vivo experiment ( B ) Quantitative data on the percentage of gated cells are shown ( n = 5 per group ; means ± SD , n = 1 , One - way ANOVA , Bonferroni test ) .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2(A) Tumor growth delay was measured in the different groups, as indicated (n=6 mice per group). Anti-PD-L1 was given at days 0 3, 6 and 9 (black arrows). The average time (days) for tumors to reach a volume of 400 mm3 from day 0 is shown (means ± SD, n=1, One-way ANOVA, Bonferroni test). No weight loss was observed in the in vivo experiment(B) Quantitative data on the percentage of gated cells are shown (n=5 per group; means ± SD, n=1, One-way ANOVA, Bonferroni test).


Figure 3 ( A ) Tumor growth delay was measured by treating tumor with RT ( 12 Gy or 5x3 Gy daily ; initiated at day 0 ) and anti - PD - L1 alone or combination of RT ( n = 6 mice per group ) . Anti - PD - L1 was given at days 0 , 3 , 6 and 9 ( black arrows ) . The radiosensitizing effect of anti - PD - L1 after 12 Gy and 5x3 Gy was assessed as shown by the capped lines ( means ± SD , n = 1 , One - way ANOVA , Bonferroni test ) . No weight loss was observed in the in vivo experiment . ( B ) Quantitative data on the percentage of gated cells are shown ( n = 5 per group ; means ± SD , n = 1 , One - way ANOVA , Bonferroni test ) .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3(A) Tumor growth delay was measured by treating tumor with RT (12 Gy or 5x3 Gy daily; initiated at day 0) and anti-PD-L1 alone or combination of RT (n=6 mice per group). Anti-PD-L1 was given at days 0, 3, 6 and 9 (black arrows). The radiosensitizing effect of anti-PD-L1 after 12 Gy and 5x3 Gy was assessed as shown by the capped lines (means ± SD, n=1, One-way ANOVA, Bonferroni test). No weight loss was observed in the in vivo experiment.(B) Quantitative data on the percentage of gated cells are shown (n=5 per group; means ± SD, n=1, One-way ANOVA, Bonferroni test).


Figure 4 ( A ) KPC tumor growth delay after treatment with either 12 Gy ( day 0 ) , anti - PD - L1 ( days 0 , 3 , 6 and 9 ; black arrows ) , anti - CD8 alone ( days 0 3 , 6 and 9 ) or their combinations , as indicated . ( B ) KPC tumor growth delay after treatment with either 5x3 Gy ( day 0 - 4 ) , anti - PD - L1 alone ( days 0 , 3 , 6 and 9 ; black arrows ) , anti - CD8 alone ( days 0 , 3 , 6 and 9 ) or their combinations , as indicated . ( C ) KPC tumor growth delay after either simultaneous ( day 0 , 3 , 6 and 9 ; black arrows - upper row ) or sequential ( day 6 , 9 , 13 and 16 ; black arrows - lower row ) addition of anti - PD - L1 to RT ( day 0 ) . The average time ( days ) for tumors to reach a volume of 400 mm3 from day 0 for Figure 4A - C is also shown ( means ± SD , n = 1 , One - way ANOVA , Bonferroni test ) . No weight loss was observed in the in vivo experiments .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4(A) KPC tumor growth delay after treatment with either 12 Gy (day 0), anti-PD-L1 (days 0, 3, 6 and 9; black arrows), anti-CD8 alone (days 0 3, 6 and 9) or their combinations, as indicated.(B) KPC tumor growth delay after treatment with either 5x3 Gy (day 0-4), anti-PD-L1 alone (days 0, 3, 6 and 9; black arrows), anti-CD8 alone (days 0, 3, 6 and 9) or their combinations, as indicated.(C) KPC tumor growth delay after either simultaneous (day 0, 3, 6 and 9; black arrows-upper row) or sequential (day 6, 9, 13 and 16; black arrows-lower row) addition of anti-PD-L1 to RT (day 0). The average time (days) for tumors to reach a volume of 400 mm3 from day 0 for Figure 4A-C is also shown (means ± SD, n=1, One-way ANOVA, Bonferroni test). No weight loss was observed in the in vivo experiments.


Figure 5 ( A ) Mice were treated with either gemcitabine ( day 0 , 3 ) , anti - PD - L1 ( day 4 , 7 , 10 and 13 ; black arrows ) , 12 Gy ( day 4 ) alone or in combinations , as indicated ( n = 8 mice per group ) . The average time ( days ) for tumors to reach a volume of 400 mm3 from day 0 is shown ( means ± SD , n = 1 , One - way ANOVA , Bonferroni test ) No weight loss was observed in the in vivo experiment . RT plus anti - PD - L1 ( B ) reverses T - cell exhaustion . Bar graphs show quantitation of T cell activation markers in tumor samples based on flow cytometry analysis , as indicated ( n = 5 per group ; means ± SD , n = 1 , One - way ANOVA , Bonferroni test ) . RT plus anti - PD - L1 ( C ) increases CD8 : Treg ratio . Bar graphs show ratio of CD8 : Treg ratio in tumor samples based on flow cytometry analysis , as indicated ( n = 5 per group ; means ± SD , n = 1 , One - way ANOVA , Bonferroni test ) .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5(A) Mice were treated with either gemcitabine (day 0, 3), anti-PD-L1 (day 4, 7, 10 and 13; black arrows), 12 Gy (day 4) alone or in combinations, as indicated (n=8 mice per group). The average time (days) for tumors to reach a volume of 400 mm3 from day 0 is shown (means ± SD, n=1, One-way ANOVA, Bonferroni test) No weight loss was observed in the in vivo experiment.RT plus anti-PD-L1 (B) reverses T-cell exhaustion . Bar graphs show quantitation of T cell activation markers in tumor samples based on flow cytometry analysis, as indicated (n=5 per group; means ± SD, n=1, One-way ANOVA, Bonferroni test).RT plus anti-PD-L1 (C) increases CD8:Treg ratio. Bar graphs show ratio of CD8:Treg ratio in tumor samples based on flow cytometry analysis, as indicated (n=5 per group; means ± SD, n=1, One-way ANOVA, Bonferroni test).


Figure 6 ( A ) Representative images of livers from the different treatment groups , as indicated . The corresponding liver weights are shown below ( n = 6 mice per group ) . ( B ) Flow cytometry analysis of CD45 + CD8 + , CD11b + Gr1 + and CD4 + CD25 + FOXP3 + cell populations in the livers of the mice from the different treatment groups , as indicated ( n = 5 per group ; ( means ± SD , n = 1 , One - way ANOVA , Bonferroni test ) .

O O B-PANEL_START O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6(A) Representative images of livers from the different treatment groups, as indicated. The corresponding liver weights are shown below (n=6 mice per group).(B) Flow cytometry analysis of CD45+CD8+, CD11b+Gr1+ and CD4+CD25+FOXP3+ cell populations in the livers of the mice from the different treatment groups, as indicated (n=5 per group; (means ± SD, n=1, One-way ANOVA, Bonferroni test).


Figure 1A . The survival of age - and sex - matched HDAC6 + / + mice and HDAC6 - / - mice was monitored for 9 days after intravenous VSV - Indiana infection ( 2 × 108 pfu / mouse ; n = 12 per group ; log - rank test ) . B . Determination of the viral load in organs by standard plaque assay . HDAC6 + / + and HDAC6 - / - mice were intravenously infected with VSV - Indiana , and the brain and spleen were collected at 5 dpi ( 2 × 108 pfu / mouse ; n = 6 per group ; Mann - Whitney test ) . C . Determination of viral loads in organs by Q - PCR of VSV viral transcripts . The brain and spleen were collected at 5 dpi after HDAC6 + / + and HDAC6 - / - mice were intravenously infected with VSV - Indiana ( 2 × 108 pfu / mouse ; n = 9 per group ; Mann - Whitney test ) . D . The viral load in the serum was assessed by standard plaque assay . HDAC6 + / + and HDAC6 - / - mice were intravenously infected with VSV - GFP . Serum was collected at the indicated time points and analyzed ( 4 × 108 pfu / mouse ; n = 3 per group ; Student ' s t - test ) . E . ELISA of IFN - β ( left ) and IL - 6 ( right ) in the serum of mice described in ( d ) ( Student ' s t - test ) . F . ELISA of serum IFN - β ( left ) and IL - 6 ( right ) after poly ( I : C ) injection . HDAC6 + / + and HDAC6 - / - mice were intravenously injected with poly ( I : C ) . Serum was collected at the indicated time points and analyzed ( 200 μg / mouse ; n = 3 per group ; Student ' s t - test ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1A. The survival of age- and sex-matched HDAC6+/+ mice and HDAC6-/- mice was monitored for 9 days after intravenous VSV-Indiana infection (2 × 108 pfu/mouse; n = 12 per group; log-rank test).B. Determination of the viral load in organs by standard plaque assay. HDAC6+/+ and HDAC6-/- mice were intravenously infected with VSV-Indiana, and the brain and spleen were collected at 5 dpi (2 × 108 pfu/mouse; n = 6 per group; Mann-Whitney test).C. Determination of viral loads in organs by Q-PCR of VSV viral transcripts. The brain and spleen were collected at 5 dpi after HDAC6+/+and HDAC6-/- mice were intravenously infected with VSV-Indiana (2 × 108 pfu/mouse; n = 9 per group; Mann-Whitney test).D. The viral load in the serum was assessed by standard plaque assay. HDAC6+/+ and HDAC6-/- mice were intravenously infected with VSV-GFP. Serum was collected at the indicated time points and analyzed (4 × 108 pfu/mouse; n = 3 per group; Student's t-test).E. ELISA of IFN-β (left) and IL-6 (right) in the serum of mice described in (d) (Student's t-test).F. ELISA of serum IFN-β (left) and IL-6 (right) after poly(I:C) injection. HDAC6+/+ and HDAC6-/- mice were intravenously injected with poly(I:C). Serum was collected at the indicated time points and analyzed (200 μg/mouse; n = 3 per group; Student's t-test).


Figure 2A , B . virus replication at 12 and 24 hpi , in HDAC6 + / + and HDAC6 - / - BMDMs in response to VSV - GFP ( MOI = 10 ) infection ( A ) and PR8 - GFP ( MOI = 5 ) infection ( B ) . C . Virus replication in HDAC6 + / + and HDAC6 - / - BMDMs in response to HSV - GFP ( MOI = 2 ) infection . D . Virus replication in HDAC6 + / + and HDAC6 - / - PBMCs in response to VSV - GFP ( MOI = 10 ) infection . E . ELISA of IFN - β ( upper ) , IL - 6 ( lower ) levels in the supernatant of ( A ) and ( B ) , and in HDAC6 + / + and HDAC6 - / - BMDMs treated with poly ( I : C ) ( 20 µg / ml ) or transfected with 5 ' ppp - dsRNA ( 1 µg / ml ) . F . Immunoblot analysis of the phosphorylated and inactive forms of IRF3 , IKBα , TBK1 , RIG - I , MAVS , HDAC6 , and β - actin at the indicated times ( 0 , 2 , 4 , 8 , and 16 h ) in HDAC6 + / + and HDAC6 - / - BMDMs . BMDMs were stimulated with PR8 - GFP ( MOI = 3 ) . G . Induction of mRNA for type I IFN , IL - 6 , and other IFN - related antiviral genes in HDAC6 + / + and HDAC6 - / - BMDMs in response to a RIG - I agonist stimulation at 6 h . HDAC6 + / + and HDAC6 - / - BMDMs were stimulated with 5 ' ppp - dsRNA ( 0 . 5 µg / ml ) for 6 h .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O 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-PANEL_START O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2A, B. virus replication at 12 and 24 hpi, in HDAC6+/+ and HDAC6-/- BMDMs in response to VSV-GFP (MOI = 10) infection (A) and PR8-GFP (MOI = 5) infection (B).C. Virus replication in HDAC6+/+ and HDAC6-/- BMDMs in response to HSV-GFP (MOI = 2) infection.D. Virus replication in HDAC6+/+ and HDAC6-/- PBMCs in response to VSV-GFP (MOI = 10) infection.E. ELISA of IFN-β (upper), IL-6 (lower) levels in the supernatant of (A) and (B), and in HDAC6+/+ and HDAC6-/- BMDMs treated with poly(I:C) (20 µg/ml) or transfected with 5'ppp-dsRNA (1 µg/ml).F. Immunoblot analysis of the phosphorylated and inactive forms of IRF3, IKBα, TBK1, RIG-I, MAVS, HDAC6, and β-actin at the indicated times (0, 2, 4, 8, and 16 h) in HDAC6+/+ and HDAC6-/- BMDMs. BMDMs were stimulated with PR8-GFP (MOI = 3).G. Induction of mRNA for type I IFN, IL-6, and other IFN-related antiviral genes in HDAC6+/+ and HDAC6-/- BMDMs in response to a RIG-I agonist stimulation at 6 h. HDAC6+/+ and HDAC6-/- BMDMs were stimulated with 5'ppp-dsRNA (0.5 µg/ml) for 6 h.


Figure 3A , B . Fluorescence microscopy at 24 hpi showing green fluorescence absorbance at 12 and 24 hpi , and virus replication at 12 and 24 hpi , in control and HDAC6 knockdown RAW264 . 7 cells in response to VSV - GFP ( MOI = 1 ) infection ( A ) and PR8 - GFP ( MOI = 1 ) infection ( B ) . bar , 100µmC . ELISA of IFN - β ( upper ) , IL - 6 ( lower ) levels in the supernatant of ( A ) and ( B ) , and in control and HDAC6 knockdown RAW264 . 7 cells treated with poly ( I : C ) ( 20 µg / ml ) or transfected with 5 ' ppp - dsRNA ( 1 µg / ml ) . D , E . Fluorescence microscopy at 24 hpi showing green fluorescence absorbance at 12 and 24 hpi , and virus replication at 12 and 24 hpi , in HDAC6 + / + and HDAC6 - / - MEFs in response to VSV - GFP ( MOI = 1 ) infection ( D ) and PR8 - GFP ( MOI = 1 ) infection ( E ) . bar , 50µmF . ELISA of IFN - β ( upper ) , IL - 6 ( lower ) levels in the supernatant of ( D ) and ( E ) , and in HDAC6 + / + and HDAC6 - / - MEFs transfected with poly ( I : C ) ( 1 µg / ml ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3A, B. Fluorescence microscopy at 24 hpi showing green fluorescence absorbance at 12 and 24 hpi, and virus replication at 12 and 24 hpi, in control and HDAC6 knockdown RAW264.7 cells in response to VSV-GFP (MOI = 1) infection (A) and PR8-GFP (MOI = 1) infection (B). bar, 100µmC. ELISA of IFN-β (upper), IL-6 (lower) levels in the supernatant of (A) and (B), and in control and HDAC6 knockdown RAW264.7 cells treated with poly(I:C) (20 µg/ml) or transfected with 5'ppp-dsRNA (1 µg/ml).D, E. Fluorescence microscopy at 24 hpi showing green fluorescence absorbance at 12 and 24 hpi, and virus replication at 12 and 24 hpi, in HDAC6+/+ and HDAC6-/- MEFs in response to VSV-GFP (MOI = 1) infection (D) and PR8-GFP (MOI = 1) infection (E). bar, 50µmF. ELISA of IFN-β (upper), IL-6 (lower) levels in the supernatant of (D) and (E), and in HDAC6+/+ and HDAC6-/- MEFs transfected with poly(I:C) (1 µg/ml).


Figure 4A , B . Fluorescence microscopy at 24 hpi showing green fluorescence absorbance at 12 and 24 hpi , and virus replication at 12 and 24 hpi , in vector , HDAC6 , or HDAC6 - CDM - overexpressing stable RAW264 . 7 cells in response to VSV - GFP ( MOI = 1 ) infection ( A ) and PR8 - GFP ( MOI = 1 ) infection ( B ) . bar , 100µm . C . ELISA of IFN - b ( upper ) and IL - 6 ( lower ) levels in the supernatant of ( A ) , ( B ) , and in vector , HDAC6 , or HDAC6 - CDM - overexpressing stable RAW264 . 7 cells treated with poly ( I : C ) ( 20 μg / ml ) or transfected with 5 ' ppp - dsRNA ( 1 µg / ml ) . D , E . Luciferase assay in 293T cells transfected with an IFN - β luciferase promoter and TK - Renilla together with mutant HDAC6 or HDAC6 - CDM ( 100 , 200 , 400 , or 800 ng ) , followed by PR8 - GFP ( MOI = 2 ) infection ( D ) or by poly ( I : C ) ( E ) ( 1 μg / ml ) transfection for another 12 h . F , G . Luciferase assay in 293T cells transfected with RIG - I ( F ) , MDA - 5 ( G ) , an IFN - β luciferase promoter , and TK - Renilla together with mutant HDAC6 or HDAC6 - CDM ( 100 , 200 , 400 , or 800 ng ) . 24 h later , IFN - β activity was measured by luciferase reporter assay .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4A, B. Fluorescence microscopy at 24 hpi showing green fluorescence absorbance at 12 and 24 hpi, and virus replication at 12 and 24 hpi, in vector, HDAC6, or HDAC6-CDM-overexpressing stable RAW264.7 cells in response to VSV-GFP (MOI = 1) infection (A) and PR8-GFP (MOI = 1) infection (B). bar, 100µm.C. ELISA of IFN-b (upper) and IL-6 (lower) levels in the supernatant of (A), (B), and in vector, HDAC6, or HDAC6-CDM-overexpressing stable RAW264.7 cells treated with poly(I:C) (20 μg/ml) or transfected with 5'ppp-dsRNA (1 µg/ml).D, E. Luciferase assay in 293T cells transfected with an IFN-β luciferase promoter and TK-Renilla together with mutant HDAC6 or HDAC6-CDM (100, 200, 400, or 800 ng), followed by PR8-GFP (MOI = 2) infection (D) or by poly(I:C) (E) (1 μg/ml) transfection for another 12 h.F, G. Luciferase assay in 293T cells transfected with RIG-I (F), MDA-5 (G), an IFN-β luciferase promoter, and TK-Renilla together with mutant HDAC6 or HDAC6-CDM (100, 200, 400, or 800 ng). 24 h later, IFN-β activity was measured by luciferase reporter assay.