Prom1 and radixin expression were silenced in WT ( Prom1 + / + ) and KO ( Prom1 - / - ) hepatocytes by infection with an adenovirus harboring sh - control ( sh - Con ) , sh - radixin ( sh - Rdx ) or sh - Prom1 for 24 hours . Hepatocytes were further serum - starved for 18 hours and stimulated with glucagon ( 10 nM ) for 10 minutes . ( B ) The nuclear localization of p - CREB after glucagon stimulation was determined by immunofluorescence staining . Blue ; DAPI , Red ; p - CREB . The percentage of cells with nuclear p - CREB staining among more than 300 cells in each group was also statistically analyzed ( Figure EV3G ) . Scale bar , 20 μm .

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

Prom1 and radixin expression were silenced in WT (Prom1+/+) and KO (Prom1-/-) hepatocytes by infection with an adenovirus harboring sh-control (sh-Con), sh-radixin (sh-Rdx) or sh-Prom1 for 24 hours. Hepatocytes were further serum-starved for 18 hours and stimulated with glucagon (10 nM) for 10 minutes. (B) The nuclear localization of p-CREB after glucagon stimulation was determined by immunofluorescence staining. Blue; DAPI, Red; p-CREB. The percentage of cells with nuclear p-CREB staining among more than 300 cells in each group was also statistically analyzed (Figure EV3G). Scale bar, 20 μm.


Prom1 and radixin expression were silenced in WT ( Prom1 + / + ) and KO ( Prom1 - / - ) hepatocytes by infection with an adenovirus harboring sh - control ( sh - Con ) , sh - radixin ( sh - Rdx ) or sh - Prom1 for 24 hours . Hepatocytes were further serum - starved for 18 hours and stimulated with glucagon ( 10 nM ) for 10 minutes . ( C ) Levels of p - PKA substrates , p - CREB , CREB , p - IP3R , IP3R , Prom1 , radixin and GAPDH after glucagon stimulation ( 10 minutes ) were determined by immunoblotting .

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

Prom1 and radixin expression were silenced in WT (Prom1+/+) and KO (Prom1-/-) hepatocytes by infection with an adenovirus harboring sh-control (sh-Con), sh-radixin (sh-Rdx) or sh-Prom1 for 24 hours. Hepatocytes were further serum-starved for 18 hours and stimulated with glucagon (10 nM) for 10 minutes. (C) Levels of p-PKA substrates, p-CREB, CREB, p-IP3R, IP3R, Prom1, radixin and GAPDH after glucagon stimulation (10 minutes) were determined by immunoblotting.


D . Immunofluorescence staining of Prom1 in mouse liver tranduced with adenoviral particles . Confocal microscope images of WT mouse liver infected with adenovirus harboring sh - Con or sh - Rdx linked with IRES - GFP . Scale bar , 20 μm .

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

D. Immunofluorescence staining of Prom1 in mouse liver tranduced with adenoviral particles. Confocal microscope images of WT mouse liver infected with adenovirus harboring sh-Con or sh-Rdx linked with IRES-GFP. Scale bar, 20 μm.


12 - week - old male wild type mice were infected with an adenovirus harboring sh - control ( sh - Con ) or sh - radixin ( sh - Rdx ) for 3 days , fasted for 4 hours and intraperitoneally injected with glucagon . ( E ) Blood glucose levels ( mg / dL ) were measured 0 , 15 , 30 , 60 and 120 minutes after glucagon administration ( 200 µg / kg body weight , n = 10 mice / group ) .

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

12-week-old male wild type mice were infected with an adenovirus harboring sh-control (sh-Con) or sh-radixin (sh-Rdx) for 3 days, fasted for 4 hours and intraperitoneally injected with glucagon. (E) Blood glucose levels (mg/dL) were measured 0, 15, 30, 60 and 120 minutes after glucagon administration (200 µg/kg body weight, n = 10 mice/ group).


12 - week - old male wild type mice were infected with an adenovirus harboring sh - control ( sh - Con ) or sh - radixin ( sh - Rdx ) for 3 days , fasted for 4 hours and intraperitoneally injected with glucagon . ( F ) Levels of p - PKA , p - CREB , CREB , radixin and GAPDH in the liver after a 10 - minutes glucagon stimulation ( 2 mg / kg body weight ) were determined by immunoblotting .

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

12-week-old male wild type mice were infected with an adenovirus harboring sh-control (sh-Con) or sh-radixin (sh-Rdx) for 3 days, fasted for 4 hours and intraperitoneally injected with glucagon. (F) Levels of p-PKA, p-CREB, CREB, radixin and GAPDH in the liver after a 10-minutes glucagon stimulation (2 mg/kg body weight) were determined by immunoblotting.


12 - week - old male wild type mice were infected with an adenovirus harboring sh - control ( sh - Con ) or sh - radixin ( sh - Rdx ) for 3 days , fasted for 4 hours and intraperitoneally injected with glucagon . ( G ) Glucose disposal rates in sh - Con or sh - Rdx ( radixin ) mice were measured using glucose , insulin and pyruvate tolerance tests ( n = 10 mice / group ) .

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

12-week-old male wild type mice were infected with an adenovirus harboring sh-control (sh-Con) or sh-radixin (sh-Rdx) for 3 days, fasted for 4 hours and intraperitoneally injected with glucagon. (G) Glucose disposal rates in sh-Con or sh-Rdx (radixin) mice were measured using glucose, insulin and pyruvate tolerance tests (n = 10 mice/group).


Primary hepatocytes were obtained from 12 - week - old male WT ( Prom1 + / + ) and KO ( Prom1 - / - ) mice or KO ( Prom1 - / - ) hepatocytes infected with an adenovirus harboring LacZ ( Ad - LacZ ) or Prom1 ( Ad - Prom1 ) for 24 hours . The molecular interaction between Prom1 and radixin ( A ) in these hepatocytes was determined by a proximity ligation assay using anti - Prom1 and anti - radixin antibodies ( A )

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

Primary hepatocytes were obtained from 12-week-old male WT (Prom1+/+) and KO (Prom1-/-) mice or KO (Prom1-/-) hepatocytes infected with an adenovirus harboring LacZ (Ad-LacZ) or Prom1 (Ad-Prom1) for 24 hours. The molecular interaction between Prom1 and radixin (A) in these hepatocytes was determined by a proximity ligation assay using anti-Prom1 and anti-radixin antibodies (A)


Primary hepatocytes were obtained from 12 - week - old male WT ( Prom1 + / + ) and KO ( Prom1 - / - ) mice or KO ( Prom1 - / - ) hepatocytes infected with an adenovirus harboring LacZ ( Ad - LacZ ) or Prom1 ( Ad - Prom1 ) for 24 hours . The molecular interaction between Prom1 and PKA regulatory subunits ( B ) in these hepatocytes was determined by a proximity ligation assay using anti - Prom1 and anti - PKA regulatory subunits ( B ) , respectively . Scale bar , 20 μm .

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

Primary hepatocytes were obtained from 12-week-old male WT (Prom1+/+) and KO (Prom1-/-) mice or KO (Prom1-/-) hepatocytes infected with an adenovirus harboring LacZ (Ad-LacZ) or Prom1 (Ad-Prom1) for 24 hours. The molecular interaction between Prom1 and PKA regulatory subunits (B) in these hepatocytes was determined by a proximity ligation assay using anti-Prom1 and anti-PKA regulatory subunits (B), respectively. Scale bar, 20 μm.


C . Human liver specimens were analyzed by double immunostaining for PROM1 ( red ) and radixin ( green ) . The lower panels represent the magnification of the square frame in the upper panels . Scale bar , 20 μm .

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

C. Human liver specimens were analyzed by double immunostaining for PROM1 (red) and radixin (green). The lower panels represent the magnification of the square frame in the upper panels. Scale bar, 20 μm.


D . The molecular interaction between Prom1 , radixin ( RDX ) and actin in WT mouse primary hepatocytes was determined by co - immunoprecipitation with an anti - Prom1 antibody or isotype control IgG . WCL ; whole cell lysate .

D. The molecular interaction between Prom1, radixin (RDX) and actin in WT mouse primary hepatocytes was determined by co-immunoprecipitation with an anti-Prom1 antibody or isotype control IgG. WCL; whole cell lysate.


HEK293 cells were transiently transfected with the combination of Prom1 - FLAG and Myc - radixin plasmids for 24 hours . The molecular interaction between Prom1 - FLAG and Myc - radixin was determined by co - immunoprecipitation using isotype control IgG , anti - Myc ( E ) antibodies .

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

HEK293 cells were transiently transfected with the combination of Prom1-FLAG and Myc-radixin plasmids for 24 hours. The molecular interaction between Prom1-FLAG and Myc-radixin was determined by co-immunoprecipitation using isotype control IgG, anti-Myc (E) antibodies.


HEK293 cells were transiently transfected with the combination of Prom1 - FLAG and Myc - radixin plasmids for 24 hours . The molecular interaction between Prom1 - FLAG and Myc - radixin was determined by co - immunoprecipitation using isotype control IgG , anti - FLAG ( F ) antibodies .

HEK293 cells were transiently transfected with the combination of Prom1-FLAG and Myc-radixin plasmids for 24 hours. The molecular interaction between Prom1-FLAG and Myc-radixin was determined by co-immunoprecipitation using isotype control IgG, anti-FLAG (F) antibodies.


G . The cellular localization of radixin and actin in WT ( Prom1 + / + ) and KO ( Prom1 - / - ) hepatocytes was determined by immunofluorescence staining for radixin and phalloidin staining . Scale bar , 20 μm .

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

G. The cellular localization of radixin and actin in WT (Prom1+/+) and KO (Prom1-/-) hepatocytes was determined by immunofluorescence staining for radixin and phalloidin staining. Scale bar, 20 μm.


H . Detergent - resistant lipid rafts were isolated from WT ( Prom1 + / + ) and KO ( Prom1 - / - ) mouse liver samples . Levels of the Prom1 , radixin ( RDX ) , flotillin and transferrin receptor ( TfR ) proteins were determined in each fraction after sucrose gradient ultracentrifugation using immunoblotting .

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

H. Detergent-resistant lipid rafts were isolated from WT (Prom1+/+) and KO (Prom1-/-) mouse liver samples. Levels of the Prom1, radixin (RDX), flotillin and transferrin receptor (TfR) proteins were determined in each fraction after sucrose gradient ultracentrifugation using immunoblotting.


I . Detergent - resistant lipid rafts were isolated from WT ( Prom1 + / + ) and KO ( Prom1 - / - ) mouse primary hepatocytes after cholesterol depletion by methyl - β - cyclodextrin ( mβCD , 30 mM ) for 30 minutes at 4 oC . Levels of the Prom1 , radixin , flotillin and transferrin receptor proteins were determined in each fraction after sucrose gradient ultracentrifugation using immunoblotting .

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

I. Detergent-resistant lipid rafts were isolated from WT (Prom1+/+) and KO (Prom1-/-) mouse primary hepatocytes after cholesterol depletion by methyl-β-cyclodextrin (mβCD, 30 mM) for 30 minutes at 4 oC. Levels of the Prom1, radixin, flotillin and transferrin receptor proteins were determined in each fraction after sucrose gradient ultracentrifugation using immunoblotting.


The molecular interactions between PROM1 and radixin mutants were determined by co - immunoprecipitation with an anti - FLAG antibody . HEK293 cells were co - transfected with plasmids expressing Myc - radixin and various deletion mutants of PROM1 - FLAG ( A ) for 24 hours .

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

The molecular interactions between PROM1 and radixin mutants were determined by co-immunoprecipitation with an anti-FLAG antibody. HEK293 cells were co-transfected with plasmids expressing Myc-radixin and various deletion mutants of PROM1-FLAG (A) for 24 hours.


The molecular interactions between PROM1 and radixin mutants were determined by co - immunoprecipitation with an anti - FLAG antibody . HEK293 cells were co - transfected with plasmids expressing PROM1 - FLAG and various deletion mutants of Myc - radixin ( B ) for 24 hours .

The molecular interactions between PROM1 and radixin mutants were determined by co-immunoprecipitation with an anti-FLAG antibody. HEK293 cells were co-transfected with plasmids expressing PROM1-FLAG and various deletion mutants of Myc-radixin (B) for 24 hours.


C . The molecular interaction between PROM1 - FLAG and Myc - radixin in the presence of increasing amounts of Myc - FERM was determined by co - immunoprecipitation with an anti - FLAG antibody .

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

C. The molecular interaction between PROM1-FLAG and Myc-radixin in the presence of increasing amounts of Myc-FERM was determined by co-immunoprecipitation with an anti-FLAG antibody.


WT primary hepatocytes were infected with an adenovirus harboring FERM - Myc ( Ad - FERM - Myc ) or LacZ ( Ad - LacZ ) for 48 hours . The cellular localization of radixin was determined by immunofluorescence staining . Scale bar , 20 μm .

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

WT primary hepatocytes were infected with an adenovirus harboring FERM-Myc (Ad-FERM-Myc) or LacZ (Ad-LacZ) for 48 hours. The cellular localization of radixin was determined by immunofluorescence staining. Scale bar, 20 μm.


WT primary hepatocytes were infected with an adenovirus harboring FERM - Myc ( Ad - FERM - Myc ) or LacZ ( Ad - LacZ ) for 48 hours . the nuclear localization of p - CREB after glucagon stimulation for 10 minutes ( E ) was determined by immunofluorescence staining . Scale bar , 20 μm .

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

WT primary hepatocytes were infected with an adenovirus harboring FERM-Myc (Ad-FERM-Myc) or LacZ (Ad-LacZ) for 48 hours. the nuclear localization of p-CREB after glucagon stimulation for 10 minutes (E) was determined by immunofluorescence staining. Scale bar, 20 μm.


WT primary hepatocytes were infected with an adenovirus harboring FERM - Myc ( Ad - FERM - Myc ) or LacZ ( Ad - LacZ ) for 48 hours . ( F ) Ad - FERM - Myc - or Ad - LacZ - expressing WT hepatocytes were serum - starved for 16 hours and stimulated with glucagon ( 10 nM ) for 10 minutes . Levels of p - PKA substrates , p - CREB , CREB , FERM - Myc and GAPDH were determined by immunoblotting .

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

WT primary hepatocytes were infected with an adenovirus harboring FERM-Myc (Ad-FERM-Myc) or LacZ (Ad-LacZ) for 48 hours. (F) Ad-FERM-Myc- or Ad-LacZ-expressing WT hepatocytes were serum-starved for 16 hours and stimulated with glucagon (10 nM) for 10 minutes. Levels of p-PKA substrates, p-CREB, CREB, FERM-Myc and GAPDH were determined by immunoblotting.


Twelve - week - old male wild type mice were infected with an adenovirus harboring GFP or FERM for 3 days , fasted for 4 hours and intraperitoneally injected with glucagon . ( G ) Blood glucose levels ( mg / dL ) were measured 0 , 15 , 30 , 60 and 120 minutes after glucagon stimulation ( 0 . 2 mg / kg body weight , n = 10 mice / group ) .

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

Twelve-week-old male wild type mice were infected with an adenovirus harboring GFP or FERM for 3 days, fasted for 4 hours and intraperitoneally injected with glucagon. (G) Blood glucose levels (mg/dL) were measured 0, 15, 30, 60 and 120 minutes after glucagon stimulation (0.2 mg/kg body weight, n = 10 mice/ group).


Twelve - week - old male wild type mice were infected with an adenovirus harboring GFP or FERM for 3 days , fasted for 4 hours and intraperitoneally injected with glucagon . ( H ) Levels of p - CREB , CREB , p - PKA substrates , and FERM in the liver 10 minutes after glucagon stimulation ( 2 mg / kg body weight ) were determined by immunoblotting ( n = 3 mice / group ) .

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

Twelve-week-old male wild type mice were infected with an adenovirus harboring GFP or FERM for 3 days, fasted for 4 hours and intraperitoneally injected with glucagon. (H) Levels of p-CREB, CREB, p-PKA substrates, and FERM in the liver 10 minutes after glucagon stimulation (2 mg/kg body weight) were determined by immunoblotting (n = 3 mice/ group).


Twelve - week - old male wild type mice were infected with an adenovirus harboring GFP or FERM for 3 days , fasted for 4 hours and intraperitoneally injected with glucagon . ( I ) Glucose disposal rates in GFP - or FERM - overexpressing mice were measured using glucose , pyruvate and insulin tolerance tests ( n = 10 mice / group ) .

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

Twelve-week-old male wild type mice were infected with an adenovirus harboring GFP or FERM for 3 days, fasted for 4 hours and intraperitoneally injected with glucagon. (I) Glucose disposal rates in GFP- or FERM-overexpressing mice were measured using glucose, pyruvate and insulin tolerance tests (n = 10 mice/group).


( A ) Wild type primary hepatocytes were infected with an adenovirus harboring sh - control ( sh - Con ) , sh - radixin ( sh - Rdx ) , GFP ( Ad - GFP ) , and / or sh - resistant wildtype radixin ( Ad - RdxR ) or AKAP - dead LPTD mutant of radixin ( Ad - LPTDR ) for 24 hours .

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

(A) Wild type primary hepatocytes were infected with an adenovirus harboring sh-control (sh-Con), sh-radixin (sh-Rdx), GFP (Ad-GFP), and/or sh-resistant wildtype radixin (Ad-RdxR) or AKAP-dead LPTD mutant of radixin (Ad-LPTDR) for 24 hours.


( B ) Increasing amount of Ad - RdxR or Ad - LPTDR were infected to WT primary hepatocytes for 24 hours . Hepatocytes were further serum - starved for 18 hours and stimulated with glucagon ( 10 nM ) for 10 minutes . Levels of p - PKA substrates , p - CREB , CREB , radixin , Myc - radixin and GAPDH were determined by immunoblotting .

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

(B) Increasing amount of Ad-RdxR or Ad-LPTDR were infected to WT primary hepatocytes for 24 hours. Hepatocytes were further serum-starved for 18 hours and stimulated with glucagon (10 nM) for 10 minutes. Levels of p-PKA substrates, p-CREB, CREB, radixin, Myc-radixin and GAPDH were determined by immunoblotting.


Four - week - old male WT ( Prom1 + / + ) and KO ( Prom1 - / - ) mice were fed a high - fat diet for 8 weeks ( Diet - Induced Obesity ; DIO ) , and fasted for 4 hours before experiments . ( C ) DIO WT ( Prom1 + / + ) and KO ( Prom1 - / - ) mice were intraperitoneally injected with glucagon . Blood glucose levels ( mg / dL ) were measured 0 , 15 , 30 , 60 and 120 minutes after glucagon ( 100 µg / kg body weight ) stimulation ( n = 10 mice / group ) .

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

Four-week-old male WT (Prom1+/+) and KO (Prom1-/-) mice were fed a high-fat diet for 8 weeks (Diet-Induced Obesity; DIO), and fasted for 4 hours before experiments. (C) DIO WT (Prom1+/+) and KO (Prom1-/-) mice were intraperitoneally injected with glucagon. Blood glucose levels (mg/dL) were measured 0, 15, 30, 60 and 120 minutes after glucagon (100 µg/kg body weight) stimulation (n = 10 mice/ group).


Four - week - old male WT ( Prom1 + / + ) and KO ( Prom1 - / - ) mice were fed a high - fat diet for 8 weeks ( Diet - Induced Obesity ; DIO ) , and fasted for 4 hours before experiments . ( D ) Levels of p - PKA substrates , p - CREB , CREB , Prom1 and GAPDH were determined by immunoblotting 10 minutes after glucagon ( 2 mg / kg body weight ) stimulation ( n = 3 mice / group ) .

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

Four-week-old male WT (Prom1+/+) and KO (Prom1-/-) mice were fed a high-fat diet for 8 weeks (Diet-Induced Obesity; DIO), and fasted for 4 hours before experiments. (D) Levels of p-PKA substrates, p-CREB, CREB, Prom1 and GAPDH were determined by immunoblotting 10 minutes after glucagon (2 mg/kg body weight) stimulation (n = 3 mice/group).


Four - week - old male WT ( Prom1 + / + ) and KO ( Prom1 - / - ) mice were fed a high - fat diet for 8 weeks ( Diet - Induced Obesity ; DIO ) , and fasted for 4 hours before experiments . ( E ) Glucose disposal rates in DIO WT ( Prom1 + / + ) and KO ( Prom1 - / - ) mice were measured using glucose and insulin tolerance tests ( n = 10 mice / group ) .

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

Four-week-old male WT (Prom1+/+) and KO (Prom1-/-) mice were fed a high-fat diet for 8 weeks (Diet-Induced Obesity; DIO), and fasted for 4 hours before experiments. (E) Glucose disposal rates in DIO WT (Prom1+/+) and KO (Prom1-/-) mice were measured using glucose and insulin tolerance tests (n = 10 mice/group).


Four - week - old male WT ( Prom1 + / + ) and KO ( Prom1 - / - ) mice were fed a high - fat diet for 8 weeks ( Diet - Induced Obesity ; DIO ) , and fasted for 4 hours before experiments . ( F ) DIO KO ( Prom1 - / - ) mice were infected with an adenovirus harboring LacZ or Prom1 for 24 hours , fasted for 4 hours and intraperitoneally injected with glucagon ( 100 µg / kg body weight ) . Blood glucose levels ( mg / dL ) were measured 0 , 15 , 30 , 60 and 120 minutes after glucagon stimulation ( GST ) . Glucose disposal rates were measured using the pyruvate tolerance test ( PTT ) ( n = 10 mice / group ) .

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

Four-week-old male WT (Prom1+/+) and KO (Prom1-/-) mice were fed a high-fat diet for 8 weeks (Diet-Induced Obesity; DIO), and fasted for 4 hours before experiments. (F) DIO KO (Prom1-/-) mice were infected with an adenovirus harboring LacZ or Prom1 for 24 hours, fasted for 4 hours and intraperitoneally injected with glucagon (100 µg/kg body weight). Blood glucose levels (mg/dL) were measured 0, 15, 30, 60 and 120 minutes after glucagon stimulation (GST). Glucose disposal rates were measured using the pyruvate tolerance test (PTT) (n = 10 mice/group).


( D and E ) Growth comparisons of bir1 ∆ - ad2 strains that contain ( 18 strains ) or do not contain ( 28 strains ) confirmed suppressor mutations as measured by area of colony growth after serial dilution . The mean ( red line ) is shown . 10 - fold serial dilutions were made on YPAD plates containing either 0 . 1 % DMSO ( D ) or 10µg / mL of benomyl ( E ) .

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

(D and E) Growth comparisons of bir1∆-ad2 strains that contain (18 strains) or do not contain (28 strains) confirmed suppressor mutations as measured by area of colony growth after serial dilution. The mean (red line) is shown. 10-fold serial dilutions were made on YPAD plates containing either 0.1% DMSO (D) or 10µg/mL of benomyl (E).


( A ) Serial dilutions on YPAD media at the permissive ( 25 ˚ C ) or restrictive ( 35 ˚ C ) temperatures for the ipl1 - 321 mutation . The suppressor mutations from the Dam1c ( blue ) were the only mutants that rescued viability at the restrictive temperature .

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

(A) Serial dilutions on YPAD media at the permissive (25˚C) or restrictive (35˚C) temperatures for the ipl1-321 mutation. The suppressor mutations from the Dam1c (blue) were the only mutants that rescued viability at the restrictive temperature.


( B ) Top : representative images showing Bub3 - mNeonGreen localization to kinetochores ( Nuf2 - mRuby3 ) , indicating spindle assembly checkpoint activation in wild - type yeast . Scale bar is 2µm . Bottom : quantification of the proportion of prometaphase cells with Bub3 - mNeonGreen foci . Only the mutations in Dam1 complex ( blue ) show a significant increase in SAC activity . Data are from at least 5 independent experiments . Means and standard deviations are shown .

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

(B) Top: representative images showing Bub3-mNeonGreen localization to kinetochores (Nuf2-mRuby3), indicating spindle assembly checkpoint activation in wild-type yeast. Scale bar is 2µm. Bottom: quantification of the proportion of prometaphase cells with Bub3-mNeonGreen foci. Only the mutations in Dam1 complex (blue) show a significant increase in SAC activity. Data are from at least 5 independent experiments. Means and standard deviations are shown.


( B ) Serial dilutions of strains engineered with the indicated mutations were tested for rescue of BIR1 deletion . 10 - fold serial dilutions on the indicated media are shown . dam1 ( S20D ) rescues loss of CPC activity to a similar extent as a suppressor mutant in Duo1 .

(B) Serial dilutions of strains engineered with the indicated mutations were tested for rescue of BIR1 deletion. 10-fold serial dilutions on the indicated media are shown. dam1(S20D) rescues loss of CPC activity to a similar extent as a suppressor mutant in Duo1.


( B ) Left : representative images showing the localization of Dad3 - mNeonGreen ( Dam1c member ) and Nuf2 - mRuby3 ( kinetochore marker ) in prometaphase cells . Scale bar is 2µm . Right : quantification of the intensity of Dad3 - mNeonGreen at kinetochores / spindles in prometaphase cells . Each individual point on the graph represents a single measurement of Dad3 - mNeonGreen intensity ; up to 10 measurements per replicate . Data are from 5 independent experiments .

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

(B) Left: representative images showing the localization of Dad3-mNeonGreen (Dam1c member) and Nuf2-mRuby3 (kinetochore marker) in prometaphase cells. Scale bar is 2µm. Right: quantification of the intensity of Dad3-mNeonGreen at kinetochores/spindles in prometaphase cells. Each individual point on the graph represents a single measurement of Dad3-mNeonGreen intensity; up to 10 measurements per replicate. Data are from 5 independent experiments.


( C ) Left : representative images showing the localization of Dad3 - mNeonGreen ( Dam1c member ) and Nuf2 - mRuby3 ( kinetochore marker ) in anaphase cells . Scale bar is 2µm . Right : quantification of the intensity of Dad3 - mNeonGreen at the spindle in anaphase cells . The intensity was measured in the middle of the spindle using a perpendicular line - scan . Each individual point on the graph represents a single measurement of Dad3 - mNeonGreen intensity ; up to 10 measurements per replicate . Data are from 5 independent experiments .

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

(C) Left: representative images showing the localization of Dad3-mNeonGreen (Dam1c member) and Nuf2-mRuby3 (kinetochore marker) in anaphase cells. Scale bar is 2µm. Right: quantification of the intensity of Dad3-mNeonGreen at the spindle in anaphase cells. The intensity was measured in the middle of the spindle using a perpendicular line-scan. Each individual point on the graph represents a single measurement of Dad3-mNeonGreen intensity; up to 10 measurements per replicate. Data are from 5 independent experiments.


( D ) Quantification of the intensity of Dad3 - mNeonGreen at kinetochores in anaphase cells . Kinetochore position was based on Nuf2 - mRuby3 localization . The differences in localization at the anaphase kinetochores are much weaker than those measured along the spindle ( Fig 5C ) . Each individual point on the graph represents a single measurement of Dad3 - mNeonGreen intensity ; up to 10 measurements per replicate . Data are from 5 independent experiments . The mean ( red line ) is shown .

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

(D) Quantification of the intensity of Dad3-mNeonGreen at kinetochores in anaphase cells. Kinetochore position was based on Nuf2-mRuby3 localization. The differences in localization at the anaphase kinetochores are much weaker than those measured along the spindle (Fig 5C). Each individual point on the graph represents a single measurement of Dad3-mNeonGreen intensity; up to 10 measurements per replicate. Data are from 5 independent experiments. The mean (red line) is shown.


( A ) Serial dilutions of strains engineered with the indicated mutations were tested for rescue of BIR1 deletion . 10 - fold serial dilutions on the indicated media are shown . The met30 - 6 and cdc4 - 1 alleles are temperature sensitive . Two independent clones of each of these alleles are shown . The serial dilutions were performed at the permissive temperature ( 20 ˚ C ) .

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

(A) Serial dilutions of strains engineered with the indicated mutations were tested for rescue of BIR1 deletion. 10-fold serial dilutions on the indicated media are shown. The met30-6 and cdc4-1 alleles are temperature sensitive. Two independent clones of each of these alleles are shown. The serial dilutions were performed at the permissive temperature (20˚C).


( B ) The amount of mNeonGreen - Sli15 ( CPC member ) located at kinetochores ( Nuf2 - mCherry ) was measured in prometaphase . Sgo1 was depleted by placing it under a galactose - inducible promoter and switching to glucose - containing media ( YPAD ) . Representative images are on the left . Scale bar is 2µm . Averages from 3 independent experiments are shown .

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

(B) The amount of mNeonGreen-Sli15 (CPC member) located at kinetochores (Nuf2-mCherry) was measured in prometaphase. Sgo1 was depleted by placing it under a galactose-inducible promoter and switching to glucose-containing media (YPAD). Representative images are on the left. Scale bar is 2µm. Averages from 3 independent experiments are shown.


( C ) Measurements of Aurora B intensity at the inner centromere of prometaphase kinetochores . Inner centromeres were identified as the regions directly between two centromeres ( ACA staining ) . Images from an example WT cell are shown to the right . Scale bar is 5µm long . Each data point is an individual chromosome . More than 15 cells for each cell line were measured across two independent experiments . The mean ( red line ) is shown .

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

(C) Measurements of Aurora B intensity at the inner centromere of prometaphase kinetochores. Inner centromeres were identified as the regions directly between two centromeres (ACA staining). Images from an example WT cell are shown to the right. Scale bar is 5µm long. Each data point is an individual chromosome. More than 15 cells for each cell line were measured across two independent experiments. The mean (red line) is shown.


A CAFs isolation from human pancreatic tumour resections ( left panel ) and in vitro primo - culture ( right panel ) .

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

A CAFs isolation from human pancreatic tumour resections (left panel) and in vitro primo-culture (right panel).


B Isolated CAFs and PaSC characterization in vitro by immunofluorescence using anti - vimentin ( left panel ) or anti - α - SMA ( right panel ) antibody ( one representative field of n = 3 ) .

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

B Isolated CAFs and PaSC characterization in vitro by immunofluorescence using anti-vimentin (left panel) or anti-α-SMA (right panel) antibody (one representative field of n = 3).


D , E Panc - 1 cell viability was assessed by MTT . Results ( mean ± SD ) are presented for each treatment ( CM from PaSC , or from CAF , or from CAF ± SOM230 , SOM230 ) as a percentage of the respective gemcitabine - untreated cells ( = 100 % ) ( n = 4 ; from left to right : * * P = 0 . 007 , # # P = 0 . 006 in D ; * P = 0 . 032 , # P = 0 . 041 , # P = 0 . 047 in E ) .

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

D, E Panc-1 cell viability was assessed by MTT. Results (mean ± SD) are presented for each treatment (CM from PaSC, or from CAF, or from CAF ± SOM230, SOM230) as a percentage of the respective gemcitabine-untreated cells (= 100%) (n = 4; from left to right: **P = 0.007, ##P = 0.006 in D; *P = 0.032, #P = 0.041, #P = 0.047 in E).


F Apoptosis induced by gemcitabine was evaluated by Western blot using an anti - cleaved caspase - 3 or anti - PARP antibody ( representative of n = 3 ) .

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

F Apoptosis induced by gemcitabine was evaluated by Western blot using an anti-cleaved caspase-3 or anti-PARP antibody (representative of n = 3).


G Panc - 1 was analysed by flow cytometry . Percentages ( mean ± SD ) of annexin V - positive cells are indicated ( n = 3 ; from left to right : * * P = 0 . 005 , # # P = 0 . 004 , * * P = 0 . 006 ) .

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

G Panc-1 was analysed by flow cytometry. Percentages (mean ± SD) of annexin V-positive cells are indicated (n = 3; from left to right: **P = 0.005, ##P = 0.004, **P = 0.006).


Immunoblotting of protein extracts from PaSCs or CAFs treated ( + ) or not with SOM230 ( 10 − 7 M ) for 48 h , using the anti - puromycin antibody ( representative of n = 3 ) .

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

Immunoblotting of protein extracts from PaSCs or CAFs treated (+) or not with SOM230 (10−7 M) for 48 h, using the anti-puromycin antibody (representative of n = 3).


Polysomes profiles of CAFs treated or not with SOM230 for 48 h ( representative of n = 3 ) .

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

Polysomes profiles of CAFs treated or not with SOM230 for 48 h (representative of n = 3).


Immunoblotting using an anti - P - Akt , anti - P - S6 or anti - β - actin ( loading control ) antibody of protein extracts from PaSCs or CAFs treated ( + ) with SOM230 for 30 min ( representative of n = 3 ) .

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

Immunoblotting using an anti-P-Akt, anti-P-S6 or anti-β-actin (loading control) antibody of protein extracts from PaSCs or CAFs treated (+) with SOM230 for 30 min (representative of n = 3).


Immunoblotting of protein extracts from CAFs treated ( + ) or not with SOM230 ( 10 − 7 M ) for the indicated times , using anti - P - Akt , anti - P - S6 or anti - 4E - BP1 antibody ( representative of n = 3 ) .

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

Immunoblotting of protein extracts from CAFs treated (+) or not with SOM230 (10−7 M) for the indicated times, using anti-P-Akt, anti-P-S6 or anti-4E-BP1 antibody (representative of n = 3).


Protein concentration in untreated or SOM230 - treated CAF - CM or extracts normalized per 1 × 106 cells . Results ( mean ± SD ) are presented as a percentage of the untreated CAFs ( = 100 % ) ( n = 3 ; from left to right : # # P = 0 . 008 , # # P = 0 . 007 ) .

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

Protein concentration in untreated or SOM230-treated CAF-CM or extracts normalized per 1 × 106 cells. Results (mean ± SD) are presented as a percentage of the untreated CAFs (= 100%) (n = 3; from left to right: ##P = 0.008, ##P = 0.007).


Immunoblotting using an anti - 4E - BP1 or anti - GAPDH ( loading control ) antibody of protein extracts from siCTR - or si4E - BP1 - transfected CAFs ( representative of n = 3 ) .

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

Immunoblotting using an anti-4E-BP1 or anti-GAPDH (loading control) antibody of protein extracts from siCTR- or si4E-BP1-transfected CAFs (representative of n = 3).


Immunoblotting of equal amounts of protein from siCTR - or si4E - BP1 - transfected CAFs treated ( + ) or not with SOM230 for 48 h , using the anti - puromycin antibody ( representative of n = 3 ) .

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

Immunoblotting of equal amounts of protein from siCTR- or si4E-BP1-transfected CAFs treated (+) or not with SOM230 for 48 h, using the anti-puromycin antibody (representative of n = 3).


Panc - 1 cell viability was assessed by MTT . Panc - 1 cells were incubated with gemcitabine in the presence of CM from untreated or SOM230 - treated CAFs transfected with the siCTR or si4E - BP - 1 . Results ( mean ± SD ) are presented as a percentage of the untreated CAFs ( = 100 % ) ( n = 3 ; * * P = 0 . 003 , § § P = 0 . 002 ) .

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

Panc-1 cell viability was assessed by MTT. Panc-1 cells were incubated with gemcitabine in the presence of CM from untreated or SOM230-treated CAFs transfected with the siCTR or si4E-BP-1. Results (mean ± SD) are presented as a percentage of the untreated CAFs (= 100%) (n = 3; **P = 0.003, §§P = 0.002).


Caspase - 3 and PARP cleavage induced by gemcitabine in Panc - 1 was evaluated by Western blot using the respective antibodies ( representative of n = 3 ) . Arrow indicates cleaved forms of PARP .

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

Caspase-3 and PARP cleavage induced by gemcitabine in Panc-1 was evaluated by Western blot using the respective antibodies (representative of n = 3). Arrow indicates cleaved forms of PARP.


A Expression of the somatostatin receptor subtype 1 ( sst1 ) analysed by RT - qPCR in the human pancreatic endocrine cell line BON and in CAFs isolated from 11 different patients ( mean ± SD ) .

A Expression of the somatostatin receptor subtype 1 (sst1) analysed by RT-qPCR in the human pancreatic endocrine cell line BON and in CAFs isolated from 11 different patients (mean ± SD).


B Immunoblotting of protein extracts from PaSCs and CAFs , using anti - sst1 , anti - α - SMA , anti - P - S6 or anti - GAPDH ( loading control ) antibody ( representative of n = 3 ) .

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

B Immunoblotting of protein extracts from PaSCs and CAFs, using anti-sst1, anti-α-SMA, anti-P-S6 or anti-GAPDH (loading control) antibody (representative of n = 3).


C Immunoblotting of protein extracts from BON , CAFs and indicated pancreatic cancer cell lines , using anti - sst1 or anti - GAPDH ( loading control ) antibody ( representative of n = 3 ) .

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

C Immunoblotting of protein extracts from BON, CAFs and indicated pancreatic cancer cell lines, using anti-sst1 or anti-GAPDH (loading control) antibody (representative of n = 3).


D Immunofluorescence confocal microscopy analyses of α - SMA and sst1 in CAFs . Co - localization of α - SMA and sst1 is shown ( merge ) ( one representative field of n = 3 ) ( scale bar = 100 μm ) .

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

D Immunofluorescence confocal microscopy analyses of α-SMA and sst1 in CAFs. Co-localization of α-SMA and sst1 is shown (merge) (one representative field of n = 3) (scale bar = 100 μm).


E - G Representative immunofluorescence confocal microscopy analyses of sst1 co - localization with cytokeratin - 19 ( n = 42 PDAC samples ) ( E ) , α - SMA ( n = 42 PDAC samples ) ( F ) or with P - 4E - BP1 ( n = 25 PDAC samples ) ( G ) in serial slides ( scale bar = 100 μm ) . Dashed white lines represent the limits between stroma and tumour epithelial glands .

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

E-G Representative immunofluorescence confocal microscopy analyses of sst1 co-localization with cytokeratin-19 (n = 42 PDAC samples) (E), α-SMA (n = 42 PDAC samples) (F) or with P-4E-BP1 (n = 25 PDAC samples) (G) in serial slides (scale bar = 100 μm). Dashed white lines represent the limits between stroma and tumour epithelial glands.


H , I Immunoblotting of protein extracts from siCTR - or sisst1 - transfected CAFs treated ( + ) or not with SOM230 for 48 h ( sst1 , puromycin and GAPDH as loading control , H ) or for 30 min ( P - Akt , P - S6 , 4E - BP1 and GAPDH as loading control , I ) ( representative of n = 3 ) .

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

H, I Immunoblotting of protein extracts from siCTR- or sisst1-transfected CAFs treated (+) or not with SOM230 for 48 h (sst1, puromycin and GAPDH as loading control, H) or for 30 min (P-Akt, P-S6, 4E-BP1 and GAPDH as loading control, I) (representative of n = 3).


J Panc - 1 cell viability was assessed by MTT . Panc - 1 cells were incubated with gemcitabine in the presence of CM from untreated or SOM230 - treated CAFs transfected with siCTR or si - sst1 . Results ( mean ± SD ) are presented as the percentage of the untreated cells ( = 100 % ) ( n = 3 ; * * P = 0 . 003 , § § P = 0 . 002 ) .

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

J Panc-1 cell viability was assessed by MTT. Panc-1 cells were incubated with gemcitabine in the presence of CM from untreated or SOM230-treated CAFs transfected with siCTR or si-sst1. Results (mean ± SD) are presented as the percentage of the untreated cells (= 100%) (n = 3; **P = 0.003, §§P = 0.002).


A - D Immunoblotting using an anti - P - Akt , anti - P - S6 , anti - 4EB - P1 or anti - GAPDH ( loading control ) antibody of protein extracts from CAFs pre - treated ( + ) or not overnight with PTX ( 100 ng / ml ) ( A ) , gallein ( 10 μM ) ( B ) , NSC87877 ( 10 μM ) ( C ) , Src inhibitor ( 5 μM ) ( D ) , treated ( + ) or not with SOM230 ( 10 − 7 M ) for 30 min ( representative of n = 3 ) .

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

A-D Immunoblotting using an anti-P-Akt, anti-P-S6, anti-4EB-P1 or anti-GAPDH (loading control) antibody of protein extracts from CAFs pre-treated (+) or not overnight with PTX (100 ng/ml) (A), gallein (10 μM) (B), NSC87877 (10 μM) (C), Src inhibitor (5 μM) (D), treated (+) or not with SOM230 (10−7 M) for 30 min (representative of n = 3).


E , F Immunoblotting using anti - P - Akt , anti - P - S6 , anti - 4EB - P1 or anti - GAPDH ( loading control ) antibody of protein extracts from CAF siCTR - or siSHP2 - ( E ) or siPTPε - ( F ) transfected CAF treated or not with SOM230 for 30 min ( representative of n = 3 ) .

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

E, F Immunoblotting using anti-P-Akt, anti-P-S6, anti-4EB-P1 or anti-GAPDH (loading control) antibody of protein extracts from CAF siCTR- or siSHP2- (E) or siPTPε- (F) transfected CAF treated or not with SOM230 for 30 min (representative of n = 3).


G , H Immunoblotting using an anti - P - Akt , anti - P - S6 , anti - 4EB - P1 or anti - GAPDH ( loading control ) antibody of protein extracts from CAFs treated with the indicated molecules ( PDGF receptor inhibitor , 5 - 10 μM ; Jak1 / 2 inhibitor ruxolitinib , 5 or 10 μM ; EGFR inhibitor , 150 or 300 nM and recombinant PDGF , 5 μg / ml ) ( representative of n = 3 ) .

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

G, H Immunoblotting using an anti-P-Akt, anti-P-S6, anti-4EB-P1 or anti-GAPDH (loading control) antibody of protein extracts from CAFs treated with the indicated molecules (PDGF receptor inhibitor, 5-10 μM; Jak1/2 inhibitor ruxolitinib, 5 or 10 μM; EGFR inhibitor, 150 or 300 nM and recombinant PDGF, 5 μg/ml) (representative of n = 3).


A - C MIA PaCa - 2 - GLuc cells were injected with or without CAFs into the pancreas of nude mice . Mice were treated with each indicated treatment ( SOM230 - LAR at day 7 and gemcitabine at days 7 , 10 , 14 and 17 ) , and the plasmatic luciferase activity was measured ( mean ± SD ) at days 7 , 14 and 21 ( n = 5 ) ( P = 0 . 003 for gemcitabine + SOM230LAR versus untreated ) ( A ) . Tumours were excised , photographed ( scale bars are 1 cm ) ( B ) , weighted ( P = 0 . 0008 ) ( C ) and paraffin - embedded for immunohistofluorescence analyses using an anti - cleaved caspase - 3 antibody ( E ) .

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

A-C MIA PaCa-2-GLuc cells were injected with or without CAFs into the pancreas of nude mice. Mice were treated with each indicated treatment (SOM230-LAR at day 7 and gemcitabine at days 7, 10, 14 and 17), and the plasmatic luciferase activity was measured (mean ± SD) at days 7, 14 and 21 (n = 5) (P = 0.003 for gemcitabine + SOM230LAR versus untreated) (A). Tumours were excised, photographed (scale bars are 1 cm) (B), weighted (P = 0.0008) (C) and paraffin-embedded for immunohistofluorescence analyses using an anti-cleaved caspase-3 antibody (E).


D Human tumours were subcutaneously xenografted in nude mice and tumour volumes ( mean ± SD ) measured ( at day 37 , P = 0 . 0009 for gemcitabine + SOM230LAR versus untreated and P = 0 . 021 for gemcitabine + SOM230LAR versus gemcitabine ) . Mice were treated with the indicated treatments ( SOM230 - LAR at days 3 and 31 , and gemcitabine at day 3 and twice a week thereafter ) .

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

D Human tumours were subcutaneously xenografted in nude mice and tumour volumes (mean ± SD) measured (at day 37, P = 0.0009 for gemcitabine + SOM230LAR versus untreated and P = 0.021 for gemcitabine + SOM230LAR versus gemcitabine). Mice were treated with the indicated treatments (SOM230-LAR at days 3 and 31, and gemcitabine at day 3 and twice a week thereafter).


E , F Tumours excised from mice as in ( A - C ) were paraffin - embedded for immunohistofluorescence analyses using an anti - cleaved caspase - 3 antibody ( scale bar = 100 μm ) ( E ) . Cleaved caspase - 3 quantification was performed by counting the mean number of positive cells per field in five independent tumours ( mean ± SD ) ( F ) ( * P = 0 . 036 , * * P = 0 . 009 ) .

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

E, F Tumours excised from mice as in (A-C) were paraffin-embedded for immunohistofluorescence analyses using an anti-cleaved caspase-3 antibody (scale bar = 100 μm) (E). Cleaved caspase-3 quantification was performed by counting the mean number of positive cells per field in five independent tumours (mean ± SD) (F) (*P = 0.036, **P = 0.009).


G , H Tumours excised from mice as in ( D ) were paraffin - embedded for immunohistofluorescence analyses using an anti - cleaved caspase - 3 antibody ( scale bar = 100 μm ) ( G ) . Cleaved caspase - 3 quantification was performed by counting the mean number of positive cells per field in five independent tumours ( mean ± SD ) ( H ) ( * P = 0 . 027 ) .

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

G, H Tumours excised from mice as in (D) were paraffin-embedded for immunohistofluorescence analyses using an anti-cleaved caspase-3 antibody (scale bar = 100 μm) (G). Cleaved caspase-3 quantification was performed by counting the mean number of positive cells per field in five independent tumours (mean ± SD) (H) (*P = 0.027).


A Membrane antibody array assay using CM from SOM230 - treated or not CAFs ( representative of n = 3 ) . Controls are circled in blue dashed line and IL - 6 in red square .

A Membrane antibody array assay using CM from SOM230-treated or not CAFs (representative of n = 3). Controls are circled in blue dashed line and IL-6 in red square.


B , C Anti - IL - 6 ELISA assay ( mean ± SD ) using CM or protein extracts from SOM230 - treated or not CAF ( from left to right : § § § P = 0 . 0002 , § § § P = 0 . 0002 ) ( B ) , or CM from siCTR - or si4E - BP1 - transfected CAFs treated or not with SOM230 ( from left to right : * * * P = 0 . 0003 , $ $ P = 0 . 004 ) ( C ) ( n = 3 ) .

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

B, C Anti-IL-6 ELISA assay (mean ± SD) using CM or protein extracts from SOM230-treated or not CAF (from left to right: §§§P = 0.0002, §§§P = 0.0002) (B), or CM from siCTR- or si4E-BP1-transfected CAFs treated or not with SOM230 (from left to right: ***P = 0.0003, $$P = 0.004) (C) (n = 3).


D Panc - 1 cell viability was assessed by MTT . Results ( mean ± SD ) are presented for each treatment as the percentage of the respective untreated cells ( = 100 % ) ( n = 3 ; from left to right : * * P = 0 . 004 , # # P = 0 . 001 , § P = 0 . 023 , § P = 0 . 019 ) .

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

D Panc-1 cell viability was assessed by MTT. Results (mean ± SD) are presented for each treatment as the percentage of the respective untreated cells (= 100%) (n = 3; from left to right: **P = 0.004, ##P = 0.001, §P = 0.023, §P = 0.019).


E , F Immunoblotting using an anti - cleaved caspase - 3 , anti - PARP or anti - GAPDH ( loading control ) antibody of protein extracts from Panc - 1 cells incubated with CAF - CM pre - incubated or not with an anti - IL - 6 neutralizing antibody ( E ) , or with human recombinant IL - 6 ( representative of n = 3 ) ( F ) .

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

E, F Immunoblotting using an anti-cleaved caspase-3, anti-PARP or anti-GAPDH (loading control) antibody of protein extracts from Panc-1 cells incubated with CAF-CM pre-incubated or not with an anti-IL-6 neutralizing antibody (E), or with human recombinant IL-6 (representative of n = 3) (F).


G , H Immunoblotting using an anti - survivin or anti - GAPDH ( loading control ) antibody of protein extracts from Panc - 1 cells stimulated or not with CAF - CM pre - incubated or not with anti - IL - 6 neutralizing antibody ( G ) , or with human recombinant IL - 6 ( representative of n = 3 ) ( H ) .

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

G, H Immunoblotting using an anti-survivin or anti-GAPDH (loading control) antibody of protein extracts from Panc-1 cells stimulated or not with CAF-CM pre-incubated or not with anti-IL-6 neutralizing antibody (G), or with human recombinant IL-6 (representative of n = 3) (H).


I Immunoblotting using an anti - P - Akt , anti - P - S6 or anti - β - actin ( loading control ) antibody of protein extracts from SOM230 - treated or SOM230 - untreated CAF incubated or not with IL - 6 neutralizing antibody ( representative of n = 3 ) .

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

I Immunoblotting using an anti-P-Akt, anti-P-S6 or anti-β-actin (loading control) antibody of protein extracts from SOM230-treated or SOM230-untreated CAF incubated or not with IL-6 neutralizing antibody (representative of n = 3).


A - C Immunohistofluorescence using an anti - α - SMA or anti - IL - 6 antibody in paraffin - embedded sections from MIA PaCa - 2 - Gluc and CAF cells orthotopic co - xenografted tumours from Fig5A ( A ) or from human tumour subcutaneously xenografted from Fig5D ( B ) in nude mice treated or not with gemcitabine + SOM230 ( representative of five different tumours ) . Representative co - localization of α - SMA with IL - 6 in human PDAC samples ( C ) ( n = 15 ) . Scale bar = 100 μm .

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

A-C Immunohistofluorescence using an anti-α-SMA or anti-IL-6 antibody in paraffin-embedded sections from MIA PaCa-2-Gluc and CAF cells orthotopic co-xenografted tumours from Fig5A (A) or from human tumour subcutaneously xenografted from Fig5D (B) in nude mice treated or not with gemcitabine + SOM230 (representative of five different tumours). Representative co-localization of α-SMA with IL-6 in human PDAC samples (C) (n = 15). Scale bar = 100 μm.


D , E Plasma was collected from MIA PaCa - 2GLuc and CAFs ( * P = 0 . 034 , * * P = 0 . 007 ) ( D ) , or human pancreatic tumour ( * P = 0 . 028 , * * P = 0 . 004 ) ( E ) , xenografts in nude mice and human IL - 6 plasmatic concentrations ( mean ± SD ) were quantified by ELISA ( n = 5 mice / group ) . * treated versus untreated mice .

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

D, E Plasma was collected from MIA PaCa-2GLuc and CAFs (*P = 0.034, **P = 0.007) (D), or human pancreatic tumour (*P = 0.028, **P = 0.004) (E), xenografts in nude mice and human IL-6 plasmatic concentrations (mean ± SD) were quantified by ELISA (n = 5 mice / group). * treated versus untreated mice.


C - D : Confocal images ( C ) and quantification ( D ) of immunofluorescence staining of STAT5b , actin filaments ( phalloidin ) and nuclei ( DAPI ) in cardiomyocytes of cryosections from the hearts of mice transduced with either control - or ERBB4ECD - AAV . Panel D depicts quantification of colocalization of STAT5b - and DAPI - specific signals . One dot in the boxplot corresponds to the median value in 20 - 50 images of stained sections from one heart ( control AAV : n = 4 , ERBB4ECD - AAV : n = 5 , biological replicates ) . Two - tailed unpaired T - test was used for statistics . Scale bar 10µm . White arrows point to cardiomyocyte nuclei . Data information : For all boxplots the central band represents the median , the box the interquartile range and whiskers the whole range of values .

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

C-D: Confocal images (C) and quantification (D) of immunofluorescence staining of STAT5b, actin filaments (phalloidin) and nuclei (DAPI) in cardiomyocytes of cryosections from the hearts of mice transduced with either control- or ERBB4ECD-AAV. Panel D depicts quantification of colocalization of STAT5b- and DAPI-specific signals. One dot in the boxplot corresponds to the median value in 20-50 images of stained sections from one heart (control AAV: n = 4, ERBB4ECD-AAV: n=5, biological replicates). Two-tailed unpaired T-test was used for statistics. Scale bar 10µm. White arrows point to cardiomyocyte nuclei. Data information: For all boxplots the central band represents the median, the box the interquartile range and whiskers the whole range of values.


E : Real - time RT - PCR analysis of expression of the indicated STAT5b target genes in the heart of mice transduced with either control - or ERBB4ECD - AAV . One dot corresponds to analysis of one heart ( n = 10 ; biological replicates combined from two replicate experiments ) . Two - tailed unpaired T - test was used for statistics . Data information : For all boxplots the central band represents the median , the box the interquartile range and whiskers the whole range of values .

E: Real-time RT-PCR analysis of expression of the indicated STAT5b target genes in the heart of mice transduced with either control- or ERBB4ECD-AAV. One dot corresponds to analysis of one heart (n = 10; biological replicates combined from two replicate experiments). Two-tailed unpaired T-test was used for statistics. Data information: For all boxplots the central band represents the median, the box the interquartile range and whiskers the whole range of values.


F - G : Confocal images ( F ) and quantification ( G ) of immunofluorescence staining of myosin heavy chain in primary mouse cardiomyocytes . The cells were treated with either control or Stat5b - targeting shRNAs and stimulated with NRG - 1 for two days . Myosin immunoreactivity was used to quantify cell area . One dot in the boxplot corresponds to one cell ( Control no NRG - 1 : n = 83 , control with NRG1 : n = 73 , Stat5b # 1 with NRG - 1 : n = 80 , Stat5b # 2 with NRG1 : n = 75 ; technical replicates combined from four biological replicate experiments ) . Non - parametric Kruskal - Wallis ANOVA was used . Post hoc analyses were conducted with the Mann Whitney U - test and the resulting P - values corrected with the method of Benjamini , Krieger and Yekutieli . Scale bar 20µm . Data information : For all boxplots the central band represents the median , the box the interquartile range and whiskers the whole range of values .

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

F-G: Confocal images (F) and quantification (G) of immunofluorescence staining of myosin heavy chain in primary mouse cardiomyocytes. The cells were treated with either control or Stat5b-targeting shRNAs and stimulated with NRG-1 for two days. Myosin immunoreactivity was used to quantify cell area. One dot in the boxplot corresponds to one cell (Control no NRG-1: n = 83, control with NRG1: n=73, Stat5b#1 with NRG-1: n=80, Stat5b#2 with NRG1: n=75; technical replicates combined from four biological replicate experiments). Non-parametric Kruskal-Wallis ANOVA was used. Post hoc analyses were conducted with the Mann Whitney U-test and the resulting P-values corrected with the method of Benjamini, Krieger and Yekutieli. Scale bar 20µm. Data information: For all boxplots the central band represents the median, the box the interquartile range and whiskers the whole range of values.


H - I : Confocal images ( H ) and quantification ( I ) of immunofluorescence staining of STAT5b and nuclei ( DAPI ) in mouse cardiomyocytes . The cells were treated with either control or Erbb4 - targeting shRNAs . Panel I depicts quantification of colocalization of STAT5b - and DAPI - specific signals ( left ) and the cell perimeter ( right ) . One dot in the boxplots corresponds to one cell ( Control : n = 24 , Erbb4 # 1 : n = 26 , Erbb4 # 2 : n = 35 ; technical replicates combined from two biological replicate experiments ) . Non - parametric Kruskal - Wallis ANOVA and Dunn ' s multicomparison test ( colocalization analysis ) and Brown - Forsythe one - way ANOVA and Dunnett ' s multicomparison test ( cardiomyocyte size analysis ) with multiple test correction were used for statistics . Scale bar 20µm . Data information : For all boxplots the central band represents the median , the box the interquartile range and whiskers the whole range of values .

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

H-I: Confocal images (H) and quantification (I) of immunofluorescence staining of STAT5b and nuclei (DAPI) in mouse cardiomyocytes. The cells were treated with either control or Erbb4-targeting shRNAs. Panel I depicts quantification of colocalization of STAT5b- and DAPI-specific signals (left) and the cell perimeter (right). One dot in the boxplots corresponds to one cell (Control: n = 24, Erbb4#1: n=26, Erbb4#2: n=35; technical replicates combined from two biological replicate experiments). Non-parametric Kruskal-Wallis ANOVA and Dunn's multicomparison test (colocalization analysis) and Brown-Forsythe one-way ANOVA and Dunnett's multicomparison test (cardiomyocyte size analysis) with multiple test correction were used for statistics. Scale bar 20µm. Data information: For all boxplots the central band represents the median, the box the interquartile range and whiskers the whole range of values.


J : Real - time RT - PCR analysis of expression of the indicated STAT5b target genes in mouse cardiomyocytes . The cells were treated with either control , Stat5b - or Erbb4 - targeting shRNAs and stimulated with NRG - 1 for 30 minutes . One dot corresponds to the mean of technical repeats in one experiment and the whiskers the standard deviation ( for each group n = 3 ; biological replicate experiments ) . One - way ANOVA and the Dunnett ' s multicomparison test with multiple test correction was used for statistics . * , P ≤ 0 . 05 ; * * P < 0 . 01 ; * * * , P < 0 . 001 against control shRNA ( Igf - 1 panel : Stat5b # 1 P = 0 . 0038 , Stat5b # 2 P = 0 . 0153 , Erbb4 # 1 P = 0 . 0188 , Erbb4 # 2 P = 0 . 0288 ; Myc panel : Stat5b # 1 P = 0 . 0093 , Stat5b # 2 P = 0 . 0119 , Erbb4 # 1 P = 0 . 0197 , Erbb4 # 2 P = 0 . 0512 ; Cdkn1a panel : Stat5b # 1 P = 0 . 0091 , Stat5b # 2 P = 0 . 0107 , Erbb4 # 1 P = 0 . 0235 , Erbb4 # 2 P = 0 . 048 ) . Data information : For all boxplots the central band represents the median , the box the interquartile range and whiskers the whole range of values .

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

J: Real-time RT-PCR analysis of expression of the indicated STAT5b target genes in mouse cardiomyocytes. The cells were treated with either control, Stat5b- or Erbb4-targeting shRNAs and stimulated with NRG-1 for 30 minutes. One dot corresponds to the mean of technical repeats in one experiment and the whiskers the standard deviation (for each group n = 3; biological replicate experiments). One-way ANOVA and the Dunnett's multicomparison test with multiple test correction was used for statistics. *, P ≤ 0.05; ** P < 0.01; ***, P < 0.001 against control shRNA (Igf-1 panel: Stat5b#1 P = 0.0038, Stat5b#2 P = 0.0153, Erbb4#1 P = 0.0188, Erbb4#2 P = 0.0288; Myc panel: Stat5b#1 P = 0.0093, Stat5b#2 P = 0.0119, Erbb4#1 P = 0.0197, Erbb4#2 P = 0.0512; Cdkn1a panel: Stat5b#1 P = 0.0091, Stat5b#2 P = 0.0107, Erbb4#1 P = 0.0235, Erbb4#2 P = 0.048). Data information: For all boxplots the central band represents the median, the box the interquartile range and whiskers the whole range of values.


A - B : Proximity ligation assay ( PLA ) of association of dynamin - 2 with ERBB4 in primary mouse cardiomyocytes treated with either DMSO or the dynamin inhibitor dynasore for 5 hours . Panel A depicts a z projection of a stack of confocal images . PLA interactions are shown in red , nuclear stain DAPI in blue . Panel B depicts quantification of the data . One dot in the boxplot corresponds to the number of PLA signals in µm2 in one cell ( Control : n = 6 , DMSO buffer : n = 11 , dynasore : n = 11 ; technical replicates from one of two biological replicate experiments ) . Non - parametric Kruskal - Wallis ANOVA and the Dunn ' s multicomparison test with multiple test correction was used for statistics . Scale bar 20µm . Data information : For all boxplots the central band represents the median , the box the interquartile range and whiskers the whole range of values .

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

A-B: Proximity ligation assay (PLA) of association of dynamin-2 with ERBB4 in primary mouse cardiomyocytes treated with either DMSO or the dynamin inhibitor dynasore for 5 hours. Panel A depicts a z projection of a stack of confocal images. PLA interactions are shown in red, nuclear stain DAPI in blue. Panel B depicts quantification of the data. One dot in the boxplot corresponds to the number of PLA signals in µm2 in one cell (Control: n = 6, DMSO buffer: n=11, dynasore: n=11 ; technical replicates from one of two biological replicate experiments). Non-parametric Kruskal-Wallis ANOVA and the Dunn's multicomparison test with multiple test correction was used for statistics. Scale bar 20µm. Data information: For all boxplots the central band represents the median, the box the interquartile range and whiskers the whole range of values.


G : Real - time RT - PCR analysis of expression of the indicated STAT5b target genes in primary mouse cardiomyocytes treated for 1 - 3 hours with the DMSO buffer , the ERBB kinase inhibitor AG1478 , or dynasore , and stimulated or not for 30 minutes with NRG - 1 . One dot corresponds to the mean of technical repeats in one experiment and the whiskers the standard deviation ( for each group in Igf - 1 and Cdkn1a panel : n = 6 , for each group expect the dynasore group in Myc panel : n = 4 , for the dynasore group in Myc panel : n = 3 ; biological replicate experiments ) . One - way ANOVA and the Dunnett ' s multicomparison test with multiple test correction was used for statistics . * , P < 0 . 05 ; * * P < 0 . 01 ; * * * , P < 0 . 001 ; against DMSO + NRG - 1 treatment ( Igf - 1 panel : DMSO without NRG - 1 P = 0 . 0041 , AG1478 P = 0 . 0082 , dynasore P = 6 . 61e - 06 ; Myc panel : DMSO without NRG - 1 P = 0 . 0386 , AG1478 P = 0 . 0037 , dynasore P = 0 . 0064 ; Cdkn1a panel : DMSO without NRG - 1 P = 0 . 0123 , AG1478 P = 0 . 0064 , dynasore P = 0 . 0092 ) . Data information : For all boxplots the central band represents the median , the box the interquartile range and whiskers the whole range of values .

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

G: Real-time RT-PCR analysis of expression of the indicated STAT5b target genes in primary mouse cardiomyocytes treated for 1-3 hours with the DMSO buffer, the ERBB kinase inhibitor AG1478, or dynasore, and stimulated or not for 30 minutes with NRG-1. One dot corresponds to the mean of technical repeats in one experiment and the whiskers the standard deviation (for each group in Igf-1 and Cdkn1a panel: n = 6, for each group expect the dynasore group in Myc panel: n=4, for the dynasore group in Myc panel: n=3 ; biological replicate experiments). One-way ANOVA and the Dunnett's multicomparison test with multiple test correction was used for statistics. *, P < 0.05; ** P < 0.01; ***, P < 0.001; against DMSO + NRG-1 treatment (Igf-1 panel: DMSO without NRG-1 P = 0.0041, AG1478 P = 0.0082, dynasore P = 6.61e-06; Myc panel: DMSO without NRG-1 P = 0.0386, AG1478 P = 0.0037, dynasore P = 0.0064; Cdkn1a panel: DMSO without NRG-1 P = 0.0123, AG1478 P = 0.0064, dynasore P = 0.0092). Data information: For all boxplots the central band represents the median, the box the interquartile range and whiskers the whole range of values.


H - I : Confocal images ( H ) and quantification ( I ) of immunofluorescence staining of myosin heavy chain in primary mouse cardiomyocytes . The cells were treated with either DMSO or dynasore and stimulated with NRG - 1 for two days . The myosin immunoreactivity was used to quantify cell area . One dot in the boxplot corresponds to one cell ( DMSO : n = 22 , dynasore : n = 28 ; technical replicates combined from three biological replicate experiments ) . Non - parametric Mann - Whitney U - test was used for statistics . Scale bar 20µm . Data information : For all boxplots the central band represents the median , the box the interquartile range and whiskers the whole range of values .

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

H-I: Confocal images (H) and quantification (I) of immunofluorescence staining of myosin heavy chain in primary mouse cardiomyocytes. The cells were treated with either DMSO or dynasore and stimulated with NRG-1 for two days. The myosin immunoreactivity was used to quantify cell area. One dot in the boxplot corresponds to one cell (DMSO: n = 22, dynasore: n = 28; technical replicates combined from three biological replicate experiments). Non-parametric Mann-Whitney U-test was used for statistics. Scale bar 20µm. Data information: For all boxplots the central band represents the median, the box the interquartile range and whiskers the whole range of values.


Confocal images ( A and quantification ( B , of immunofluorescence staining of myosin heavy chain ( green ) and DAPI ( blue ) in the hearts of 4 dpf zebrafish embryos injected with either the buffer control or NRG - 1 into the pericardial sac at 2 dpf . The myosin immunoreactivity was used to quantify both the average thickness of the ventricular wall as well as the cross - sectional area of the ventricles . The number of nuclei was estimated with the DAPI stain . The amount nuclei ( red ) in the ventricle and the ventricle area ( yellow ) was quantified to differentiate between hypertrophic and hyperplastic growth . One dot in the boxplots corresponds to one heart ( Control ( A - B ) : n = 10 , NRG - 1 ( A - B ) : n = 7 biological replicates from two replicate experiments ) . Unpaired two - tailed T - test was used for statistics . Scale bars 20µm . Data information : For all boxplots the central band represents the median , the box the interquartile range and whiskers the whole range of values .

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

Confocal images (A and quantification (B, of immunofluorescence staining of myosin heavy chain (green) and DAPI (blue) in the hearts of 4 dpf zebrafish embryos injected with either the buffer control or NRG-1 into the pericardial sac at 2 dpf. The myosin immunoreactivity was used to quantify both the average thickness of the ventricular wall as well as the cross-sectional area of the ventricles. The number of nuclei was estimated with the DAPI stain. The amount nuclei (red) in the ventricle and the ventricle area (yellow) was quantified to differentiate between hypertrophic and hyperplastic growth. One dot in the boxplots corresponds to one heart (Control (A-B): n = 10, NRG-1 (A-B): n = 7 biological replicates from two replicate experiments). Unpaired two-tailed T-test was used for statistics. Scale bars 20µm. Data information: For all boxplots the central band represents the median, the box the interquartile range and whiskers the whole range of values.


Western analysis of Stat5 , Akt and Erk phosphorylation in zebrafish embryos . The embryos were treated as in A . ( Control : n = 15 , NRG - 1 : n = 16 ; biological replicates combined from three replicate experiments ) .

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

Western analysis of Stat5, Akt and Erk phosphorylation in zebrafish embryos. The embryos were treated as in A. (Control: n = 15, NRG-1: n = 16; biological replicates combined from three replicate experiments).


densitometric quantification of Stat5 , Akt and Erk phosphorylation in zebrafish embryos . The embryos were treated as in A . One dot in the boxplots corresponds to the relative densitometric value of one pooled sample of 3 - 4 zebrafish embryos ( Control : n = 15 , NRG - 1 : n = 16 ; biological replicates combined from three replicate experiments ) . Unpaired two - tailed T - test with Welch ' s correction ( pStat5 ) or unpaired two - tailed T - test ( pErk , pAkt ) was used for statistics . Data information : For all boxplots the central band represents the median , the box the interquartile range and whiskers the whole range of values .

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

densitometric quantification of Stat5, Akt and Erk phosphorylation in zebrafish embryos. The embryos were treated as in A. One dot in the boxplots corresponds to the relative densitometric value of one pooled sample of 3-4 zebrafish embryos (Control: n = 15, NRG-1: n = 16; biological replicates combined from three replicate experiments). Unpaired two-tailed T-test with Welch's correction (pStat5) or unpaired two-tailed T-test (pErk, pAkt) was used for statistics. Data information: For all boxplots the central band represents the median, the box the interquartile range and whiskers the whole range of values.


Western analysis ( C ) of Stat5 , Akt and Erk phosphorylation in zebrafish embryos . The embryos were treated as in A . ( DMSO ( Stat5 ) n = 13 , AG1478 ( Stat5 ) n = 7 , Lapatinib ( Stat5 ) : n = 4 , Gefitinib ( Stat5 ) n = 6 , DMSO ( Akt ) n = 9 , AG1478 ( Akt ) n = 3 , Lapatinib ( Akt ) n = 3 , Gefitinib ( Akt ) n = 3 , DMSO ( Erk ) n = 13 , AG1478 ( Erk ) n = 6 , Lapatinib ( Erk ) n = 4 , Gefitinib ( Erk ) n = 8 ; biological replicates combined from three replicate experiments ) .

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

Western analysis (C) of Stat5, Akt and Erk phosphorylation in zebrafish embryos. The embryos were treated as in A. (DMSO (Stat5) n = 13, AG1478 (Stat5) n = 7, Lapatinib (Stat5): n = 4, Gefitinib (Stat5) n = 6, DMSO (Akt) n = 9, AG1478 (Akt) n = 3, Lapatinib (Akt) n = 3, Gefitinib (Akt) n = 3, DMSO (Erk) n = 13, AG1478 (Erk) n = 6, Lapatinib (Erk) n = 4, Gefitinib (Erk) n = 8 ; biological replicates combined from three replicate experiments).


densitometric quantification ( D ) of Stat5 , Akt and Erk phosphorylation in zebrafish embryos . The embryos were treated as in A . One dot in the boxplots corresponds to the relative densitometric value of one pooled sample of 5 zebrafish embryos ( DMSO ( Stat5 ) n = 13 , AG1478 ( Stat5 ) n = 7 , Lapatinib ( Stat5 ) : n = 4 , Gefitinib ( Stat5 ) n = 6 , DMSO ( Akt ) n = 9 , AG1478 ( Akt ) n = 3 , Lapatinib ( Akt ) n = 3 , Gefitinib ( Akt ) n = 3 , DMSO ( Erk ) n = 13 , AG1478 ( Erk ) n = 6 , Lapatinib ( Erk ) n = 4 , Gefitinib ( Erk ) n = 8 ; biological replicates combined from three replicate experiments ) . One - way ANOVA and the Dunnett ' s multicomparison test ( pStat5 , pAkt ) or non - parametric Kruskal - Wallis with Dunn ' s multicomparison test ( pErk ) was used for statistics . Data information : For all boxplots the central band represents the median , the box the interquartile range and whiskers the whole range of values .

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

densitometric quantification (D) of Stat5, Akt and Erk phosphorylation in zebrafish embryos. The embryos were treated as in A. One dot in the boxplots corresponds to the relative densitometric value of one pooled sample of 5 zebrafish embryos (DMSO (Stat5) n = 13, AG1478 (Stat5) n = 7, Lapatinib (Stat5): n = 4, Gefitinib (Stat5) n = 6, DMSO (Akt) n = 9, AG1478 (Akt) n = 3, Lapatinib (Akt) n = 3, Gefitinib (Akt) n = 3, DMSO (Erk) n = 13, AG1478 (Erk) n = 6, Lapatinib (Erk) n = 4, Gefitinib (Erk) n = 8 ; biological replicates combined from three replicate experiments). One-way ANOVA and the Dunnett's multicomparison test (pStat5, pAkt) or non-parametric Kruskal-Wallis with Dunn's multicomparison test (pErk) was used for statistics. Data information: For all boxplots the central band represents the median, the box the interquartile range and whiskers the whole range of values.


E - F : Confocal images ( E ) and quantification ( F ) of immunofluorescence staining of myosin heavy chain in hearts of 4 dpf zebrafish embryos treated with DMSO or dynasore for 2 days . The myosin immunoreactivity was used to quantify the average thickness of the ventricular wall and the cross - sectional area of the ventricles . One dot in the boxplots corresponds to one heart ( DMSO ( thickness , ejection fraction ) n = 11 , dynasore ( thickness , ejection fraction ) n = 12 , DMSO ( area ) n = 15 , dynasore ( area ) n = 14 ; biological replicates ) . Unpaired two - tailed T - test was used for statistics . Scale bar 50µm . Data information : For all boxplots the central band represents the median , the box the interquartile range and whiskers the whole range of values .

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

E-F: Confocal images (E) and quantification (F) of immunofluorescence staining of myosin heavy chain in hearts of 4 dpf zebrafish embryos treated with DMSO or dynasore for 2 days. The myosin immunoreactivity was used to quantify the average thickness of the ventricular wall and the cross-sectional area of the ventricles. One dot in the boxplots corresponds to one heart (DMSO (thickness, ejection fraction) n = 11, dynasore (thickness, ejection fraction) n = 12, DMSO (area) n = 15, dynasore (area) n = 14; biological replicates). Unpaired two-tailed T-test was used for statistics. Scale bar 50µm. Data information: For all boxplots the central band represents the median, the box the interquartile range and whiskers the whole range of values.


I - J : Western analysis ( I ) and densitometric quantification ( J ) of Stat5 , Akt and Erk phosphorylation in zebrafish embryos . The embryos were treated as in E . One dot in the boxplots corresponds to the relative densitometric value of one pooled sample of 5 zebrafish embryos ( for each group n = 5 ; biological replicates ) . Unpaired two - tailed T - test was used for statistics . Data information : For all boxplots the central band represents the median , the box the interquartile range and whiskers the whole range of values .

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

I-J: Western analysis (I) and densitometric quantification (J) of Stat5, Akt and Erk phosphorylation in zebrafish embryos. The embryos were treated as in E. One dot in the boxplots corresponds to the relative densitometric value of one pooled sample of 5 zebrafish embryos (for each group n = 5; biological replicates). Unpaired two-tailed T-test was used for statistics. Data information: For all boxplots the central band represents the median, the box the interquartile range and whiskers the whole range of values.


A - B : Confocal images ( A ) and quantification ( B ) of immunofluorescence staining of myosin heavy chain and Stat5b in hearts of 4 dpf zebrafish embryos injected with CRISPR / Cas9 and either control or stat5b - targeting gRNA at one - cell stage . The myosin immunoreactivity was used to quantify the average thickness of the ventricular wall and the cross - sectional area of the ventricles . Stat5b staining intensity was also quantified . One dot in the boxplots corresponds to one heart ( control gRNA : n = 14 , stat5b gRNA n = 13 ; biological replicates from one of three replicate experiments ) . Unpaired two - tailed T - test was used for statistics . Scale bar 50µm . Data information : For all boxplots the central band represents the median , the box the interquartile range and whiskers the whole range of values .

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

A-B: Confocal images (A) and quantification (B) of immunofluorescence staining of myosin heavy chain and Stat5b in hearts of 4 dpf zebrafish embryos injected with CRISPR/Cas9 and either control or stat5b-targeting gRNA at one-cell stage. The myosin immunoreactivity was used to quantify the average thickness of the ventricular wall and the cross-sectional area of the ventricles. Stat5b staining intensity was also quantified. One dot in the boxplots corresponds to one heart (control gRNA: n = 14, stat5b gRNA n = 13; biological replicates from one of three replicate experiments). Unpaired two-tailed T-test was used for statistics. Scale bar 50µm. Data information: For all boxplots the central band represents the median, the box the interquartile range and whiskers the whole range of values.


quantification of the ejection fraction ( D ) . The 4 dpf embryos were treated as in A . Ejection fraction was quantified from the videos . One dot in the boxplots corresponds to ejection fraction of one heart ( for each group n = 22 ; biological replicates from one of three replicate experiments ) . Unpaired two - tailed T - test was used for statistics . Data information : For all boxplots the central band represents the median , the box the interquartile range and whiskers the whole range of values .

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

quantification of the ejection fraction (D). The 4 dpf embryos were treated as in A. Ejection fraction was quantified from the videos. One dot in the boxplots corresponds to ejection fraction of one heart (for each group n = 22; biological replicates from one of three replicate experiments). Unpaired two-tailed T-test was used for statistics. Data information: For all boxplots the central band represents the median, the box the interquartile range and whiskers the whole range of values.


C - D : Immunohistochemical analysis ( C ) and quantification ( D ) of STAT5 activation in sections representing normal myocardia or pathological cardiac hypertrophy ( n = 3 , aortic stenosis ; n = 2 , idiopathic cardiomyopathy ) . Phospho - STAT5b staining intensity was quantified . One dot in the boxplot corresponds to one subject ( normal : n = 6 , hypertrophic n = 13 ; biological replicates ) . In the boxplot the central band represents the median , the box the interquartile range and whiskers the whole range of values . Unpaired two - tailed T - test was used for statistics . Scale bar 50µm .

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

C-D: Immunohistochemical analysis (C) and quantification (D) of STAT5 activation in sections representing normal myocardia or pathological cardiac hypertrophy (n = 3, aortic stenosis; n = 2, idiopathic cardiomyopathy). Phospho-STAT5b staining intensity was quantified. One dot in the boxplot corresponds to one subject (normal: n = 6, hypertrophic n = 13; biological replicates). In the boxplot the central band represents the median, the box the interquartile range and whiskers the whole range of values. Unpaired two-tailed T-test was used for statistics. Scale bar 50µm.


( A ) Live imaging of MTOC dynamics during meiosis I . Z - projection images ( top ) show MTOCs ( mNG - Cep192 , green ) and chromosomes ( H2B - mCherry , magenta ) . Central MTOCs are magnified ( squares ) . 3D - reconstructed spindles are aligned with the spindle axis ( middle ) . Central MTOCs are indicated with arrowheads . Time in h : mm . The kymograph ( bottom ) shows projected signals on the spindle axis for all time points .

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

(A) Live imaging of MTOC dynamics during meiosis I. Z-projection images (top) show MTOCs (mNG-Cep192, green) and chromosomes (H2B-mCherry, magenta). Central MTOCs are magnified (squares). 3D-reconstructed spindles are aligned with the spindle axis (middle). Central MTOCs are indicated with arrowheads. Time in h:mm. The kymograph (bottom) shows projected signals on the spindle axis for all time points.


( B ) Density maps of MTOCs ( left ) and chromosomes ( right ) along the spindle axis in a representative oocyte . The color represents the percentage of MTOCs or chromosome volumes , respectively , coded from dark ( 0 % ) to white ( 50 % or more of the total volume ) . ( C ) Average density maps of 21 oocytes mapped as in ( B ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Density maps of MTOCs (left) and chromosomes (right) along the spindle axis in a representative oocyte. The color represents the percentage of MTOCs or chromosome volumes, respectively, coded from dark (0%) to white (50% or more of the total volume). (C) Average density maps of 21 oocytes mapped as in (B).


( D ) Dynamics of MTOC sorting and chromosome congression . Ratio of MTOCs ( left ) or chromosomes ( right ) in the polar region versus those in the middle region of the spindle are plotted over time .

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

(D) Dynamics of MTOC sorting and chromosome congression. Ratio of MTOCs (left) or chromosomes (right) in the polar region versus those in the middle region of the spindle are plotted over time.


( E ) Dynamics of spindle elongation . The spindle was visualized with EGFP - Map4 The length / width ratio of the spindle was measured after 3D reconstruction ( n = 13 oocytes from 3 independent experiments ) .

(E) Dynamics of spindle elongation. The spindle was visualized with EGFP-Map4 The length/width ratio of the spindle was measured after 3D reconstruction (n=13 oocytes from 3 independent experiments).


( F ) Percentage of oocytes carrying one or more MTOCs in the middle region of the spindle . Oocytes were fixed and stained for MTOCs ( pericentrin , green ) , chromosomes ( Hoechst33342 , magenta ) and microtubules ( α - tubulin , grey ) . n = 21 , 28 , 24 oocytes . A representative oocyte fixed at 5 hours after NEBD is shown . Arrowheads indicate central MTOCs .

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

(F) Percentage of oocytes carrying one or more MTOCs in the middle region of the spindle. Oocytes were fixed and stained for MTOCs (pericentrin, green), chromosomes (Hoechst33342, magenta) and microtubules (α-tubulin, grey). n=21, 28, 24 oocytes. A representative oocyte fixed at 5 hours after NEBD is shown. Arrowheads indicate central MTOCs.


( A ) Live imaging of MTOC and kinetochore dynamics . MTOCs ( mNG - Cep192 , green ) and kinetochores ( tdTomato - CENP - C , magenta ) in B6D2F1 oocytes are shown . Time in h : mm after NEBD . Circles indicate central MTOCs , which are positioned in the middle region of the spindle later than 4 hours after NEBD . Arrows indicate displacement of the MTOCs . Scale bar , 10 μm .

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

(A) Live imaging of MTOC and kinetochore dynamics. MTOCs (mNG-Cep192, green) and kinetochores (tdTomato-CENP-C, magenta) in B6D2F1 oocytes are shown. Time in h:mm after NEBD. Circles indicate central MTOCs, which are positioned in the middle region of the spindle later than 4 hours after NEBD. Arrows indicate displacement of the MTOCs. Scale bar, 10 μm.


( B ) Tracking analysis of central MTOCs . In the left graph , the positions of central MTOCs along the spindle axis are shown over time . MTOC positions were used to calculate distance to the spindle equator . In the right graph , lines showing temporal changes in MTOC - equator distance are aligned based on the time when the MTOC reached a position closest to the spindle equator . Horizontal lines at 7 and at − 7 μm denote the thresholds used for the definition of the middle and polar regions of the spindle .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Tracking analysis of central MTOCs. In the left graph, the positions of central MTOCs along the spindle axis are shown over time. MTOC positions were used to calculate distance to the spindle equator. In the right graph, lines showing temporal changes in MTOC-equator distance are aligned based on the time when the MTOC reached a position closest to the spindle equator. Horizontal lines at 7 and at −7 μm denote the thresholds used for the definition of the middle and polar regions of the spindle.


( C ) Central MTOCs originate from spindle poles and transiently stay in the middle region of the spindle . trajectories of central MTOCs ( n = 25 MTOCs of 8 oocytes from 3 independent experiments ) were analyzed and their temporal changes in MTOC - equator distance are shown ( Left graph ) . Central MTOCs were categorized into two groups : ( 1 ) ones that came from polar regions of the spindle ( n = 21 , middle graph ) and ( 2 ) others that stayed in the middle region throughout the period before reaching a position closest to the equator ( n = 4 , right graph ) .

(C) Central MTOCs originate from spindle poles and transiently stay in the middle region of the spindle. trajectories of central MTOCs (n=25 MTOCs of 8 oocytes from 3 independent experiments) were analyzed and their temporal changes in MTOC-equator distance are shown (Left graph). Central MTOCs were categorized into two groups: (1) ones that came from polar regions of the spindle (n=21, middle graph) and (2) others that stayed in the middle region throughout the period before reaching a position closest to the equator (n=4, right graph).


( E ) Destination of central MTOCs . The tracks of central MTOCs that originated from polar regions were used ( n = 21 MTOCs ) . The pole of origin was defined as Pole 1 , while the other pole was defined as Pole 2 . MTOC positions along the spindle axis ( positive values for those closer to Pole 1 ) over time are shown . Horizontal lines at 7 and at − 7 μm denote the thresholds used for the definition of the middle and polar regions of the spindle . Note that central MTOCs moved back to the original pole ( Pole 1 ) or switched their positions to the opposite pole ( Pole 2 ) . ( F ) Destination of central MTOCs that originated from a polar region was categorized as in ( E ) .

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

(E) Destination of central MTOCs. The tracks of central MTOCs that originated from polar regions were used (n=21 MTOCs). The pole of origin was defined as Pole 1, while the other pole was defined as Pole 2. MTOC positions along the spindle axis (positive values for those closer to Pole 1) over time are shown. Horizontal lines at 7 and at −7 μm denote the thresholds used for the definition of the middle and polar regions of the spindle. Note that central MTOCs moved back to the original pole (Pole 1) or switched their positions to the opposite pole (Pole 2). (F) Destination of central MTOCs that originated from a polar region was categorized as in (E).