E . Glycogen contents in the liver of mouse in each condition was shown by PAS ( periodic acid schiff ) staining . Scale bar , 500 μm .

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

E. Glycogen contents in the liver of mouse in each condition was shown by PAS (periodic acid schiff) staining. Scale bar, 500 μm.


Primary hepatocytes were isolated from 12 - week - old male Prom1 + / + ( WT ) or Prom1 - / - ( KO ) mice , serum - starved and stimulated with glucagon . F . Levels of the G6pc and Pck1 mRNAs 2 hours after glucagon stimulation ( 10 nM ) were determined by qRT - PCR . The level of each mRNA was normalized to the level of the 18S rRNA . For these experiments , primary hepatocytes were cultured under 3D conditions using a Matrigel matrix ( n = 3 mice / group ) .

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

Primary hepatocytes were isolated from 12-week-old male Prom1+/+ (WT) or Prom1-/- (KO) mice, serum-starved and stimulated with glucagon. F. Levels of the G6pc and Pck1 mRNAs 2 hours after glucagon stimulation (10 nM) were determined by qRT-PCR. The level of each mRNA was normalized to the level of the 18S rRNA. For these experiments, primary hepatocytes were cultured under 3D conditions using a Matrigel matrix (n = 3 mice/group).


Primary hepatocytes were isolated from 12 - week - old male Prom1 + / + ( WT ) or Prom1 - / - ( KO ) mice , serum - starved and stimulated with glucagon . G . Levels of phospho - PKA substrates ( p - PKA substrates ) , phospho - CREB ( p - CREB ) , CREB , phospho - Inositol trisphosphate receptor ( p - IP3R ) , IP3R , phospho - Hormone - Sensitive Lipase ( p - HSL ) , HSL , Prom1 and Gapdh 10 minutes after glucagon stimulation ( 10 nM ) were determined by immunoblotting .

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

Primary hepatocytes were isolated from 12-week-old male Prom1+/+ (WT) or Prom1-/- (KO) mice, serum-starved and stimulated with glucagon. G. Levels of phospho-PKA substrates (p-PKA substrates), phospho-CREB (p-CREB), CREB, phospho-Inositol trisphosphate receptor (p-IP3R), IP3R, phospho-Hormone-Sensitive Lipase (p-HSL), HSL, Prom1 and Gapdh 10 minutes after glucagon stimulation (10 nM) were determined by immunoblotting.


Primary hepatocytes were isolated from 12 - week - old male Prom1 + / + ( WT ) or Prom1 - / - ( KO ) mice , serum - starved and stimulated with glucagon . H . The nuclear localization of p - CREB 10 minutes after glucagon stimulation ( 10 nM ) was determined by immunofluorescence staining . Scale bar , 20 μm .

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

Primary hepatocytes were isolated from 12-week-old male Prom1+/+ (WT) or Prom1-/- (KO) mice, serum-starved and stimulated with glucagon. H. The nuclear localization of p-CREB 10 minutes after glucagon stimulation (10 nM) was determined by immunofluorescence staining. Scale bar, 20 μm.


Primary hepatocytes were isolated from 12 - week - old male Prom1 + / + ( WT ) or Prom1 - / - ( KO ) mice , serum - starved and stimulated with glucagon . I . Relative PKA activities 10 minutes after glucagon stimulation ( 10 nM ) were determined using the PKA assay kit in the absence or presence of 10 μM IBMX ( n = 3 mice / group ) .

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

Primary hepatocytes were isolated from 12-week-old male Prom1+/+ (WT) or Prom1-/- (KO) mice, serum-starved and stimulated with glucagon. I. Relative PKA activities 10 minutes after glucagon stimulation (10 nM) were determined using the PKA assay kit in the absence or presence of 10 μM IBMX (n = 3 mice/group).


Primary hepatocytes were isolated from 12 - week - old male Prom1 + / + ( WT ) or Prom1 - / - ( KO ) mice , serum - starved and stimulated with glucagon . J . The cAMP concentration 10 minutes after glucagon stimulation ( 10 nM ) was determined using the cAMP assay kit in the absence or presence of 10 μM IBMX ( n = 3 mice / group ) .

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

Primary hepatocytes were isolated from 12-week-old male Prom1+/+ (WT) or Prom1-/- (KO) mice, serum-starved and stimulated with glucagon. J. The cAMP concentration 10 minutes after glucagon stimulation (10 nM) was determined using the cAMP assay kit in the absence or presence of 10 μM IBMX (n = 3 mice/group).


12 - week - old male WT ( Prom1 + / + ) and KO ( Prom1 - / - ) mice were fasted for 4 hours and intraperitoneally injected with glucagon . ( A ) Blood glucose levels ( mg / dL ) were measured 0 , 15 , 30 , 60 and 120 minutes after glucagon stimulation ( 200 µg / kg body weight , n = 10 / group ) .

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 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

12-week-old male WT ( Prom1+/+) and KO (Prom1-/-) mice were fasted for 4 hours and intraperitoneally injected with glucagon. (A) Blood glucose levels (mg/dL) were measured 0, 15, 30, 60 and 120 minutes after glucagon stimulation (200 µg/kg body weight, n = 10/group).


12 - week - old male WT ( Prom1 + / + ) and KO ( Prom1 - / - ) mice were fasted for 4 hours and intraperitoneally injected with glucagon . ( B ) Levels of p - PKA substrates , p - CREB , CREB and GAPDH in the liver after a 5 minutes glucagon treatment were determined by immunoblotting ( 2 mg / kg body weight ) .

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

12-week-old male WT ( Prom1+/+) and KO (Prom1-/-) mice were fasted for 4 hours and intraperitoneally injected with glucagon. (B) Levels of p-PKA substrates, p-CREB, CREB and GAPDH in the liver after a 5 minutes glucagon treatment were determined by immunoblotting (2 mg/kg body weight).


12 - week - old male WT ( Prom1 + / + ) and KO ( Prom1 - / - ) mice were fasted for 4 hours and intraperitoneally injected with glucagon . ( C ) Relative PKA activities in the liver after a 5 minutes glucagon treatment were determined using the PKA assay kit ( 2 mg / kg body weight , n = 3 / group ) .

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 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 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

12-week-old male WT ( Prom1+/+) and KO (Prom1-/-) mice were fasted for 4 hours and intraperitoneally injected with glucagon. (C) Relative PKA activities in the liver after a 5 minutes glucagon treatment were determined using the PKA assay kit (2 mg/kg body weight, n = 3/group).


12 - week - old male WT ( Prom1 + / + ) and KO ( Prom1 - / - ) mice were fasted for 4 hours and intraperitoneally injected with glucagon . ( D ) The cAMP concentration in the liver after a 5 minutes glucagon treatment was determined using the cAMP assay kit ( 2 mg / kg body weight , n = 3 / group ) .

12-week-old male WT ( Prom1+/+) and KO (Prom1-/-) mice were fasted for 4 hours and intraperitoneally injected with glucagon. (D) The cAMP concentration in the liver after a 5 minutes glucagon treatment was determined using the cAMP assay kit (2 mg/kg body weight, n = 3/group).


Glucose disposal rates were measured in 12 - week - old male mice using glucose , pyruvate ( E ) tolerance tests ( n = 10 mice / group ) .

B-MEASURED_VAR 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 O O O

Glucose disposal rates were measured in 12-week-old male mice using glucose, pyruvate (E) tolerance tests (n = 10 mice/group).


Glucose disposal rates were measured in 12 - week - old male mice using insulin ( F ) tolerance tests ( n = 10 mice / group ) .

B-MEASURED_VAR 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

Glucose disposal rates were measured in 12-week-old male mice using insulin (F) tolerance tests (n = 10 mice/group).


G . Levels of p - AKT , AKT , p - ERK , ERK , IRβ and GAPDH were determined by immunoblotting after insulin stimulation ( 10 nM ) .

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

G. Levels of p-AKT, AKT, p-ERK, ERK, IRβ and GAPDH were determined by immunoblotting after insulin stimulation (10 nM).


Primary hepatocytes were isolated from 12 - week - old male WT ( Prom1 + / + ) and KO ( Prom1 - / - ) mice , serum - starved for 16 hours and stimulated with isoprenaline . ( H ) The nuclear localization of p - CREB after isoprenaline stimulation ( 10 μM ) for 10 minutes was determined by immunofluorescence staining . Scale bar , 20 μm .

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

Primary hepatocytes were isolated from 12-week-old male WT ( Prom1+/+) and KO (Prom1-/-) mice, serum-starved for 16 hours and stimulated with isoprenaline. (H) The nuclear localization of p-CREB after isoprenaline stimulation (10 μM) for 10 minutes was determined by immunofluorescence staining. Scale bar, 20 μm.


Primary hepatocytes were isolated from 12 - week - old male WT ( Prom1 + / + ) and KO ( Prom1 - / - ) mice , serum - starved for 16 hours and stimulated with isoprenaline . ( I ) Levels of p - PKA substrates , p - CREB , CREB , Prom1 and GAPDH after isoprenaline stimulation ( 10 μM ) for 0 , 10 or 20 minutes were determined by immunoblotting .

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

Primary hepatocytes were isolated from 12-week-old male WT ( Prom1+/+) and KO (Prom1-/-) mice, serum-starved for 16 hours and stimulated with isoprenaline. (I) Levels of p-PKA substrates, p-CREB, CREB, Prom1 and GAPDH after isoprenaline stimulation (10 μM) for 0, 10 or 20 minutes were determined by immunoblotting.


Primary hepatocytes were isolated from 12 - week - old male WT ( Prom1 + / + ) and KO ( Prom1 - / - ) mice , serum - starved for 16 hours and stimulated with isoprenaline . ( J ) The cAMP concentration after isoprenaline stimulation ( 10 μM ) for 10 minutes was determined using the cAMP assay kit in the presence of 10 μM IBMX ( n = 3 / group ) .

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

Primary hepatocytes were isolated from 12-week-old male WT ( Prom1+/+) and KO (Prom1-/-) mice, serum-starved for 16 hours and stimulated with isoprenaline. (J) The cAMP concentration after isoprenaline stimulation (10 μM) for 10 minutes was determined using the cAMP assay kit in the presence of 10 μM IBMX (n = 3/group).


WT ( Prom1 + / + ) and KO ( Prom1 - / - ) mice were fasted for 4 hours and intraperitoneally injected with epinephrine ( 3µg / 10g ) . ( K ) Blood glucose levels ( mg / dL ) were measured 0 , 15 , 30 , 60 and 120 minutes after epinephrine

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 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

WT (Prom1+/+) and KO (Prom1-/-) mice were fasted for 4 hours and intraperitoneally injected with epinephrine (3µg/10g). (K) Blood glucose levels (mg/dL) were measured 0, 15, 30, 60 and 120 minutes after epinephrine stimulation


WT ( Prom1 + / + ) and KO ( Prom1 - / - ) mice were fasted for 4 hours and intraperitoneally injected with epinephrine ( 3µg / 10g ) . ( L ) Levels of p - PKA substrates , p - CREB , CREB and GAPDH in the liver 15 minutes after epinephrine treatment were determined by immunoblotting .

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

WT (Prom1+/+) and KO (Prom1-/-) mice were fasted for 4 hours and intraperitoneally injected with epinephrine (3µg/10g). (L) Levels of p-PKA substrates, p-CREB, CREB and GAPDH in the liver 15 minutes after epinephrine treatment were determined by immunoblotting.


M - O . WT ( Prom1 + / + ) and KO ( Prom1 - / - ) mice were fasted for 4 hours and subjected to immobilization test . ( M ) Blood glucose levels ( mg / dL ) were measured 0 , 60 and 120 minutes after immobilization ( n = 10 / group ) .

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 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

M-O. WT (Prom1+/+) and KO (Prom1-/-) mice were fasted for 4 hours and subjected to immobilization test. (M) Blood glucose levels (mg/dL) were measured 0, 60 and 120 minutes after immobilization (n = 10/group).


WT ( Prom1 + / + ) and KO ( Prom1 - / - ) mice were fasted for 4 hours and subjected to immobilization test . ( N ) Levels of p - PKA substrates , p - CREB , CREB and GAPDH in the liver after a 30 minutes - immobilization were determined by immunoblotting .

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

WT (Prom1+/+) and KO (Prom1-/-) mice were fasted for 4 hours and subjected to immobilization test. (N) Levels of p-PKA substrates, p-CREB, CREB and GAPDH in the liver after a 30 minutes-immobilization were determined by immunoblotting.


WT ( Prom1 + / + ) and KO ( Prom1 - / - ) mice were fasted for 4 hours and subjected to immobilization test . ( O ) Serum epinephrine level of mice after a 30 minutes - immobilization was determined ( n = 3 / control group , 7 or 8 / immobilized group ) .

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 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

WT (Prom1+/+) and KO (Prom1-/-) mice were fasted for 4 hours and subjected to immobilization test. (O) Serum epinephrine level of mice after a 30 minutes-immobilization was determined (n = 3/control group, 7 or 8/immobilized group).


A . The expression of Prom1 , radixin , AKAP7 , AKAP8 , AKAP8I , AKAP9 , AKAP10 , AKAP12 or AKAP13 was silenced in WT hepatocytes using siRNAs . Hepatocytes were then serum - starved for 18 hours and stimulated with glucagon ( 10 nM ) for 10 minutes . The phosphorylation of PKA substrates was determined by immunoblotting .

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

A. The expression of Prom1, radixin, AKAP7, AKAP8, AKAP8I, AKAP9, AKAP10, AKAP12 or AKAP13 was silenced in WT hepatocytes using siRNAs. Hepatocytes were then serum-starved for 18 hours and stimulated with glucagon (10 nM) for 10 minutes. The phosphorylation of PKA substrates was determined by immunoblotting.


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 .

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

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 .

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

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 B-CONTROLLED_VAR O O O B-CONTROLLED_VAR 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 B-CONTROLLED_VAR O B-CONTROLLED_VAR 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 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 O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR 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 O O O O O B-MEASURED_VAR O O O B-MEASURED_VAR 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 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 O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR 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 O O O O O O O B-MEASURED_VAR O O O B-MEASURED_VAR 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 B-MEASURED_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 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 .

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

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 B-CONTROLLED_VAR 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 B-CONTROLLED_VAR 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 .

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

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 B-MEASURED_VAR 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 B-MEASURED_VAR 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 B-CONTROLLED_VAR O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O B-MEASURED_VAR O O B-MEASURED_VAR O B-MEASURED_VAR I-MEASURED_VAR O B-MEASURED_VAR 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 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 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 B-MEASURED_VAR O B-MEASURED_VAR O B-MEASURED_VAR O B-MEASURED_VAR I-MEASURED_VAR 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 B-MEASURED_VAR 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 B-CONTROLLED_VAR 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 .

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

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 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 B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O B-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR 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 B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O B-MEASURED_VAR O O O O B-MEASURED_VAR O B-MEASURED_VAR O B-MEASURED_VAR O O O B-MEASURED_VAR 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 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 B-CONTROLLED_VAR O O O O O O O B-CONTROLLED_VAR 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 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 B-MEASURED_VAR 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 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 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 B-MEASURED_VAR 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-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 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 B-CONTROLLED_VAR O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O 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).


( a ) ( top ) Experimental design . Cultured cortical neurons are stimulated with BDNF , KCl or no treatment ( Control ) and activated neurons are prepared in three specific time points for RNA - seq and ATAC - seq at early ( 1h ) and late time points ( 6 and 10h ) ; ( bottom ) Blot for pMAPK in neurons treated with KCl 55 mM and several concentrations of BDNF ( Con = no stimulation ) . 44 and 42 KDa bands indicated with lines . Nucleolin shown as internal control .

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

(a) (top) Experimental design. Cultured cortical neurons are stimulated with BDNF, KCl or no treatment (Control) and activated neurons are prepared in three specific time points for RNA-seq and ATAC-seq at early (1h) and late time points (6 and 10h); (bottom) Blot for pMAPK in neurons treated with KCl 55 mM and several concentrations of BDNF (Con = no stimulation). 44 and 42 KDa bands indicated with lines. Nucleolin shown as internal control.


( b ) Hierarchical clustering for all differentially - expressed genes ( DE - genes , adjusted P - value < 0 . 1 , using Benjamini Hochberg ' s correction ) using correlation of log2 - fold changes when compared to unstimulated control samples at each time point .

O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Hierarchical clustering for all differentially-expressed genes (DE-genes, adjusted P-value < 0.1, using Benjamini Hochberg's correction) using correlation of log2-fold changes when compared to unstimulated control samples at each time point.


( d ) Partitioning around medoids clustering of expression dynamics using genewise - scaled Z - scores of top 5 , 000 DE - genes from ( c ) with lowest adjusted p - value ( k = 10 clusters ) . Numbers indicate clusters . Left line plots indicate expression levels for signature genes selected for each cluster . 4 , 907 DE - genes are also selected with FDR = 1 % and more than 100 counts across samples . ( e ) Gene Ontology enrichment analysis for clusters shown in ( d ) . Each ribbon shows clusters with their respective significant gene groups using topGO ( Alexa et al , 2006 ) . Up to five significant terms per cluster are shown ( P - values adjusted with BH ) . Circle sizes indicate enrichment of ontology genes in each cluster versus all other clusters . Names on the right y - axis indicate ontology common names .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Partitioning around medoids clustering of expression dynamics using genewise-scaled Z-scores of top 5,000 DE-genes from (c) with lowest adjusted p-value (k = 10 clusters). Numbers indicate clusters. Left line plots indicate expression levels for signature genes selected for each cluster. 4,907 DE-genes are also selected with FDR=1% and more than 100 counts across samples. (e) Gene Ontology enrichment analysis for clusters shown in (d). Each ribbon shows clusters with their respective significant gene groups using topGO(Alexa et al, 2006). Up to five significant terms per cluster are shown (P-values adjusted with BH). Circle sizes indicate enrichment of ontology genes in each cluster versus all other clusters. Names on the right y-axis indicate ontology common names.


( a ) Hierarchical clustering for all differentially accessible peaks ( DA - peaks , adjusted P < 0 . 1 , using Benjamini Hochberg ' s correction ) using correlation of log2 - fold changes when compared versus matched control samples .

O O O O O O O O O O O O O O O O O O O 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) Hierarchical clustering for all differentially accessible peaks (DA-peaks, adjusted P < 0.1, using Benjamini Hochberg's correction) using correlation of log2-fold changes when compared versus matched control samples.


( d ) ( top ) Association between distal regulatory elements ( DREs ) and gene expression at the BDNF 1h time - point . Each point indicates the log2 - fold change of an ATAC - seq peak ( x - axis ) and the gene expression of a closest gene ( y - axis ) with distance between 2 - 20 Kbp . Colors indicate whether none ( gray ) , only the peak ( orange ) , or both peak and gene ( red ) show significant changes versus control neurons . ( bottom ) Enrichment for paired DA - peak and DE - gene in the four quadrants are summarized for BDNF and KCl . Asterisks indicate P values as corrected by a Benjamini Hochberg procedure ( * = P < 0 . 05 ; * * = P < 0 . 01 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) (top) Association between distal regulatory elements (DREs) and gene expression at the BDNF 1h time-point. Each point indicates the log2-fold change of an ATAC-seq peak (x-axis) and the gene expression of a closest gene (y-axis) with distance between 2-20 Kbp. Colors indicate whether none (gray), only the peak (orange), or both peak and gene (red) show significant changes versus control neurons. (bottom) Enrichment for paired DA-peak and DE-gene in the four quadrants are summarized for BDNF and KCl. Asterisks indicate P values as corrected by a Benjamini Hochberg procedure (* = P < 0.05; ** = P < 0.01).


( c ) RNA expression changes for TFs related to 8 - mer groups bZIP , CTCF , HIC1 , and EGR . Significant changes relative to control samples are shown with asterisks ( Wilcoxon rank sums test , two - sided ) .

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(c) RNA expression changes for TFs related to 8-mer groups bZIP, CTCF, HIC1, and EGR. Significant changes relative to control samples are shown with asterisks (Wilcoxon rank sums test, two-sided).


( c ) Chromosome 15 genome tracks neighboring Arc , displaying ATAC - seq read counts per million ( CPM ) ; RNA - seq CPM ; H3K27ac normalized signal upon KCl stimulation , H3K4me1 ( Malik et al , 2014 ) ; CTCF ( Sams et al , 2016 ) ; and Cortical neurons Hi - C data ( Bonev et al , 2017 ) . Red bars in ATAC - seq tracks indicate gained DA - peaks in BDNF , and red bars in RNA - seq tracks indicate Arc differential expression in BDNF and KCl 1h . Green blocks in Hi - C tracks indicate anchor points for the calculation of contact scores , using shaman ( Cohen et al , 2017 ) . Line and Spearman ' s rho value indicate counts rank - based correlation between highlighted peaks across all samples ( Appendix Figure S4c ) . TF module names indicate the presence of 8 - mers in those peaks .

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(c) Chromosome 15 genome tracks neighboring Arc, displaying ATAC-seq read counts per million (CPM); RNA-seq CPM; H3K27ac normalized signal upon KCl stimulation, H3K4me1(Malik et al, 2014); CTCF(Sams et al, 2016); and Cortical neurons Hi-C data(Bonev et al, 2017). Red bars in ATAC-seq tracks indicate gained DA-peaks in BDNF, and red bars in RNA-seq tracks indicate Arc differential expression in BDNF and KCl 1h. Green blocks in Hi-C tracks indicate anchor points for the calculation of contact scores, using shaman(Cohen et al, 2017). Line and Spearman's rho value indicate counts rank-based correlation between highlighted peaks across all samples (Appendix Figure S4c). TF module names indicate the presence of 8-mers in those peaks.


( a ) Associations between chromatin accessibility DA - peaks and GWAS summary statistics . All DA - peaks ( DA ) , gained DA - peaks ( gained ) , closing DA - peaks , and consensus peaks ( all ) are fitted to summary statistics data of multiple GWAS studies ( y - axis ) . Circle color indicates LDSC regression coefficient ( effect size ) , and circle size indicates association significance . Shadings indicate treatments . Time points are shown ( orange = 1h , brown = 6h ) . Red lines indicate LD score regression coefficient adjusted P < 0 . 1 after BH correction . Orange lines indicate P < 0 . 1 . ( BD = Bipolar Disorder ; SZ = Schizophrenia ; BD + SZ : BP and SZ samples combined ; MDD = Major Depressive Disorder ; ADHD = Attention deficit hyperactivity disorder ; AD = Alzheimer ' s Disease ) .

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(a) Associations between chromatin accessibility DA-peaks and GWAS summary statistics. All DA-peaks (DA), gained DA-peaks (gained), closing DA-peaks, and consensus peaks (all) are fitted to summary statistics data of multiple GWAS studies (y-axis). Circle color indicates LDSC regression coefficient (effect size), and circle size indicates association significance. Shadings indicate treatments. Time points are shown (orange = 1h, brown = 6h). Red lines indicate LD score regression coefficient adjusted P < 0.1 after BH correction. Orange lines indicate P < 0.1. (BD = Bipolar Disorder; SZ = Schizophrenia; BD+SZ: BP and SZ samples combined; MDD = Major Depressive Disorder; ADHD = Attention deficit hyperactivity disorder; AD = Alzheimer's Disease).


( b ) Correlations between differentially accessible elements conserved mouse and human regions across conditions ( N = 16 , 580 ) . Asterisks indicate significance of Pearson ' s R correlation after correction with BH - procedure .

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(b) Correlations between differentially accessible elements conserved mouse and human regions across conditions (N=16,580). Asterisks indicate significance of Pearson's R correlation after correction with BH-procedure.


( D ) Validation by western blot of changes in specific phosphorylation events on kinases . Left , Immunoblots of cortical lysates of CTL and micTNFα - KO at ZT6 for : ( top ) phosphorylated GSK3α / β ( pGSK3α at Ser21 and pGSK3β at Ser9 ) and total GSK3α ; and ( bottom ) phosphorylated MAPK ( pMAPK , MAPK3 at Thr203 / Tyr205 and MAPK1 at Thr183 / Tyr185 ) and total MAPK . Right , Ratio between phosphorylated and total kinase normalized to CTL at ZT18 and ZT6 . n = 5 mice per group . * * * p = 0 . 000009 ( GSK3α ) , multiple t - test with Benjamini - Hochberg correction .

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(D) Validation by western blot of changes in specific phosphorylation events on kinases. Left, Immunoblots of cortical lysates of CTL and micTNFα-KO at ZT6 for: (top) phosphorylated GSK3α/β (pGSK3α at Ser21 and pGSK3β at Ser9) and total GSK3α; and (bottom) phosphorylated MAPK (pMAPK, MAPK3 at Thr203/Tyr205 and MAPK1 at Thr183/Tyr185) and total MAPK. Right, Ratio between phosphorylated and total kinase normalized to CTL at ZT18 and ZT6. n=5 mice per group. ***p=0.000009 (GSK3α), multiple t-test with Benjamini-Hochberg correction.


( E ) Kinases with altered activity between CTL and micTNF - KOs predicted by robust kinase activity inference ( RoKAI ) at ZT18 and ZT6 . Graph shows the z - score value of each kinase assigned by RoKAI . Kinases above and below the dotted line represent prediction of up - and downregulated activity in micTNFα - KO , respectively . Kinases are color - coded according to kinase group as described in ( C ) .

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(E) Kinases with altered activity between CTL and micTNF-KOs predicted by robust kinase activity inference (RoKAI) at ZT18 and ZT6. Graph shows the z-score value of each kinase assigned by RoKAI. Kinases above and below the dotted line represent prediction of up- and downregulated activity in micTNFα-KO, respectively. Kinases are color-coded according to kinase group as described in (C).


( F Validation by western blot of altered activity of predicted kinases between CTL and micTNF - KOs . ( F ) Left , Immunoblots of cortical lysates of CTL and micTNFα - KO at ZT6 using antibodies specific to target phosphorylation motifs of MAPK / CDK ( PXS * P , S * PX ( K / R ) ) and PKC ( ( K / R ) XS * X ( K / R ) ) . Dashed boxes indicate quantified signal . Right , Ratio between phosphorylated substrates and loading control normalized to CTL at ZT18 and ZT6 . ( F , n = 5 mice per group . * p = 0 . 022 ( MAPK / CDK phospho sub ) , * p = 0 . 014 ( PKC phospho sub ) multiple t - test with Benjamini - Hochberg correction .

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(F Validation by western blot of altered activity of predicted kinases between CTL and micTNF-KOs. (F) Left, Immunoblots of cortical lysates of CTL and micTNFα-KO at ZT6 using antibodies specific to target phosphorylation motifs of MAPK/CDK (PXS*P, S*PX(K/R)) and PKC ((K/R)XS*X(K/R)). Dashed boxes indicate quantified signal. Right, Ratio between phosphorylated substrates and loading control normalized to CTL at ZT18 and ZT6. (F, n=5 mice per group. *p=0.022 (MAPK/CDK phospho sub), *p=0.014 (PKC phospho sub) multiple t-test with Benjamini-Hochberg correction.


G ) Validation by western blot of altered activity of predicted kinases between CTL and micTNF - KOs . ( G ) Left , Immunoblots show signal of phosphorylated threonine in MARKs activation loop . Right , Ratio between phosphorylated MARK activation loop and loading control normalized to CTL at ZT18 and ZT6 . G ) n = 5 mice per group . * * p = 0 . 0091 ( MARK ) , multiple t - test with Benjamini - Hochberg correction .

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G) Validation by western blot of altered activity of predicted kinases between CTL and micTNF-KOs. (G) Left, Immunoblots show signal of phosphorylated threonine in MARKs activation loop. Right, Ratio between phosphorylated MARK activation loop and loading control normalized to CTL at ZT18 and ZT6. G) n=5 mice per group. **p=0.0091 (MARK), multiple t-test with Benjamini-Hochberg correction.


A MOAP - 1 co - localizes with p62 in the cytoplasmic inclusion bodies upon proteasome inhibition . HCT116 cells were transfected with plasmid encoding GFP - MOAP - 1 or GFP only control . 14 hours later , cells were treated with the proteasome inhibitor MG132 ( 5 µM ) for 8 hours and analyzed by immunofluorescence ( IF ) with anti - GFP ( in green ) and anti - p62 ( in red ) antibodies . Data information : , nuclei were counterstained with DAPI ( blue )

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A MOAP-1 co-localizes with p62 in the cytoplasmic inclusion bodies upon proteasome inhibition. HCT116 cells were transfected with plasmid encoding GFP-MOAP-1 or GFP only control. 14 hours later, cells were treated with the proteasome inhibitor MG132 (5 µM) for 8 hours and analyzed by immunofluorescence (IF) with anti-GFP (in green) and anti-p62 (in red) antibodies. Data information: , nuclei were counterstained with DAPI (blue)


B Endogenous MOAP - 1 resides at the p62 bodies upon proteasome inhibition . HCT116 cells were treated with MG132 ( 5 µM ) for 8 hours and analyzed by IF with anti - MOAP - 1 ( in green ) and anti - p62 ( in red ) antibodies . Data information : nuclei were counterstained with DAPI ( blue )

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B Endogenous MOAP-1 resides at the p62 bodies upon proteasome inhibition. HCT116 cells were treated with MG132 (5 µM) for 8 hours and analyzed by IF with anti-MOAP-1 (in green) and anti-p62 (in red) antibodies. Data information: nuclei were counterstained with DAPI (blue)


C MOAP - 1 is recruited to the p62 bodies upon exposure to cellular stresses . LO2 hepatocytes were transfected with plasmid encoding Myc - MOAP - 1 . 14 hours later , cells were then treated with MG132 ( 5 µM ) , arsenic trioxide ( As2O3 , 10 µM ) or diethylnitrosamine ( DEN , 200 µM ) for 8 hours each . Cells were then processed for IF analysis with anti - Myc ( in green ) and anti - p62 ( in red ) antibodies . Data information : nuclei were counterstained with DAPI ( blue )

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C MOAP-1 is recruited to the p62 bodies upon exposure to cellular stresses. LO2 hepatocytes were transfected with plasmid encoding Myc-MOAP-1. 14 hours later, cells were then treated with MG132 (5 µM), arsenic trioxide (As2O3, 10 µM) or diethylnitrosamine (DEN, 200 µM) for 8 hours each. Cells were then processed for IF analysis with anti-Myc (in green) and anti-p62 (in red) antibodies. Data information: nuclei were counterstained with DAPI (blue)


D MOAP - 1 spontaneously localizes to the p62 bodies at resting state in the liver cancer cell lines , HepG2 , Huh - 1 , JHH5 and JHH7 . The liver cancer cell lines were transfected with the plasmid encoding Myc - MOAP - 1 . 14 hours later , transfected cells were subjected to IF analysis with anti - Myc ( in green ) and anti - p62 ( in red ) antibodies . Data information : nuclei were counterstained with DAPI ( blue )

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D MOAP-1 spontaneously localizes to the p62 bodies at resting state in the liver cancer cell lines, HepG2, Huh-1, JHH5 and JHH7. The liver cancer cell lines were transfected with the plasmid encoding Myc-MOAP-1. 14 hours later, transfected cells were subjected to IF analysis with anti-Myc (in green) and anti-p62 (in red) antibodies. Data information: nuclei were counterstained with DAPI (blue)


E Absence of the aggregated patterns of MOAP - 1 in the p62 deficient cells . WT and p62 KO HepG2 cells were transfected with plasmid encoding Myc - MOAP - 1 for 14 hours and the cells were subjected to IF analysis as in ( D ) . Data information : , nuclei were counterstained with DAPI ( blue )

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E Absence of the aggregated patterns of MOAP-1 in the p62 deficient cells. WT and p62 KO HepG2 cells were transfected with plasmid encoding Myc-MOAP-1 for 14 hours and the cells were subjected to IF analysis as in (D). Data information: , nuclei were counterstained with DAPI (blue)


F Western blotting analysis of p62 and Myc - MOAP - 1 protein levels in the WT and p62 KO HepG2 cells as described in ( E ) . Actin as loading control .

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F Western blotting analysis of p62 and Myc-MOAP-1 protein levels in the WT and p62 KO HepG2 cells as described in (E). Actin as loading control.


A MOAP - 1 downregulates the levels of the diethylnitrosamine ( DEN ) - induced p62 bodies in a time - dependent manner . LO2 cells were transfected with plasmid encoding Myc - MOAP - 1 . 14 hours later , cells were treated with DEN ( 200 µM ) for the indicated period of times , before being subjected to IF with anti - Myc ( in green ) and anti - p62 ( in red ) antibodies . Cells expressing Myc - MOAP - 1 were marked by dashed lines . Data information : nuclei were counterstained with DAPI ( blue )

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A MOAP-1 downregulates the levels of the diethylnitrosamine (DEN)-induced p62 bodies in a time-dependent manner. LO2 cells were transfected with plasmid encoding Myc-MOAP-1. 14 hours later, cells were treated with DEN (200 µM) for the indicated period of times, before being subjected to IF with anti-Myc (in green) and anti-p62 (in red) antibodies. Cells expressing Myc-MOAP-1 were marked by dashed lines. Data information: nuclei were counterstained with DAPI (blue)


B MOAP - 1 downregulates the p62 bodies in the resting HepG2 liver cancer cells . HepG2 were transfected with plasmid encoding Myc - MOAP - 1 . At the indicated time post - transfection , transfected cells were subjected to IF as in ( A ) . Data information : nuclei were counterstained with DAPI ( blue )

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B MOAP-1 downregulates the p62 bodies in the resting HepG2 liver cancer cells. HepG2 were transfected with plasmid encoding Myc-MOAP-1. At the indicated time post-transfection, transfected cells were subjected to IF as in (A). Data information: nuclei were counterstained with DAPI (blue)


C Loss of MOAP - 1 leads to spontaneous elevation of the basal levels of p62 bodies in the LO2 cells . WT , MOAP - 1 KO and p62 KO LO2 cells were subjected to IF with anti - p62 antibody ( in red ) . p62 KO cells were included as a negative control . Insets in the upper panels are enlarged in the lower panels . D Quantification of the p62 bodies in the WT and MOAP - 1 KO LO2 cells . ( Left panel ) number of p62 bodies per cell . n = 7 independently plated samples . ( Right panel ) size of p62 bodies . n = 442 and 1014 bodies detected in the WT and MOAP - 1 KO LO2 cells , respectively , from three independently plated samples . Data information : nuclei were counterstained with DAPI ( blue )

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C Loss of MOAP-1 leads to spontaneous elevation of the basal levels of p62 bodies in the LO2 cells. WT, MOAP-1 KO and p62 KO LO2 cells were subjected to IF with anti-p62 antibody (in red). p62 KO cells were included as a negative control. Insets in the upper panels are enlarged in the lower panels. D Quantification of the p62 bodies in the WT and MOAP-1 KO LO2 cells. (Left panel) number of p62 bodies per cell. n = 7 independently plated samples. (Right panel) size of p62 bodies. n = 442 and 1014 bodies detected in the WT and MOAP-1 KO LO2 cells, respectively, from three independently plated samples. Data information: nuclei were counterstained with DAPI (blue)


C Loss of MOAP - 1 leads to spontaneous elevation of the basal levels of p62 bodies in the LO2 cells . WT , MOAP - 1 KO and p62 KO LO2 cells were subjected to IF with anti - p62 antibody ( in red ) . p62 KO cells were included as a negative control . Insets in the upper panels are enlarged in the lower panels .

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C Loss of MOAP-1 leads to spontaneous elevation of the basal levels of p62 bodies in the LO2 cells. WT, MOAP-1 KO and p62 KO LO2 cells were subjected to IF with anti-p62 antibody (in red). p62 KO cells were included as a negative control. Insets in the upper panels are enlarged in the lower panels.


E Re - expression of MOAP - 1 reduces levels of p62 bodies in the MOAP - 1 deficient LO2 cells . MOAP - 1 KO LO2 cells were transfected with plasmid encoding Myc - MOAP - 1 for 24 hours and subjected to IF with anti - p62 ( in red ) and anti - Myc ( green ) antibodies . Cells expressing Myc - MOAP - 1 were marked by dashed lines . F Quantification of the p62 bodies in the MOAP - 1 KO LO2 cells re - introduced with Myc - MOAP - 1 . ( Left panel ) number of p62 bodies per cell . n = 7 independently plated samples of MOAP - 1 KO cells transfected with empty vector control ( Ctrl ) or plasmid encoding Myc - MOAP - 1 . ( Right panel ) size of p62 bodies . n = 178 and 69 bodies detected in the Ctrl and Myc - MOAP - 1 expressing cells , respectively , from three independently plated samples . Data information : , nuclei were counterstained with DAPI ( blue )

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E Re-expression of MOAP-1 reduces levels of p62 bodies in the MOAP-1 deficient LO2 cells. MOAP-1 KO LO2 cells were transfected with plasmid encoding Myc-MOAP-1 for 24 hours and subjected to IF with anti-p62 (in red) and anti-Myc (green) antibodies. Cells expressing Myc-MOAP-1 were marked by dashed lines. F Quantification of the p62 bodies in the MOAP-1 KO LO2 cells re-introduced with Myc-MOAP-1. (Left panel) number of p62 bodies per cell. n = 7 independently plated samples of MOAP-1 KO cells transfected with empty vector control (Ctrl) or plasmid encoding Myc-MOAP-1. (Right panel) size of p62 bodies. n = 178 and 69 bodies detected in the Ctrl and Myc-MOAP-1 expressing cells, respectively, from three independently plated samples. Data information: , nuclei were counterstained with DAPI (blue)


E Re - expression of MOAP - 1 reduces levels of p62 bodies in the MOAP - 1 deficient LO2 cells . MOAP - 1 KO LO2 cells were transfected with plasmid encoding Myc - MOAP - 1 for 24 hours and subjected to IF with anti - p62 ( in red ) and anti - Myc ( green ) antibodies . Cells expressing Myc - MOAP - 1 were marked by dashed lines .

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E Re-expression of MOAP-1 reduces levels of p62 bodies in the MOAP-1 deficient LO2 cells. MOAP-1 KO LO2 cells were transfected with plasmid encoding Myc-MOAP-1 for 24 hours and subjected to IF with anti-p62 (in red) and anti-Myc (green) antibodies. Cells expressing Myc-MOAP-1 were marked by dashed lines.


G Increased level of p62 bodies in MOAP - 1 KO MEFs upon treatment with arsenic trioxide . WT and MOAP - 1 KO MEFs were treated with arsenic trioxide ( As2O3 , 10 µM ) for the indicated durations and subjected to IF with anti - p62 antibody ( in red ) . H Quantification of the p62 bodies in the WT and MOAP - 1 KO MEFs treated with arsenic trioxide . ( Left panel ) number of p62 bodies per cell . n ≥ 6 independently plated samples . ( Right panel ) size of p62 bodies . n = 10 ( WT , basal ) , 17 ( MOAP - 1 KO , basal ) , 104 ( WT , As2O3 2h ) , 137 ( MOAP - 1 KO , As2O3 2h ) , 605 ( WT , As2O3 4h ) , 977 ( MOAP - 1 KO , As2O3 4h ) , 721 ( WT , As2O3 6h ) and 487 bodies ( MOAP - 1 KO , As2O3 6h ) from three independently plated samples . Data information : nuclei were counterstained with DAPI ( blue )

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G Increased level of p62 bodies in MOAP-1 KO MEFs upon treatment with arsenic trioxide. WT and MOAP-1 KO MEFs were treated with arsenic trioxide (As2O3, 10 µM) for the indicated durations and subjected to IF with anti-p62 antibody (in red). H Quantification of the p62 bodies in the WT and MOAP-1 KO MEFs treated with arsenic trioxide. (Left panel) number of p62 bodies per cell. n ≥ 6 independently plated samples. (Right panel) size of p62 bodies. n = 10 (WT, basal), 17 (MOAP-1 KO, basal), 104 (WT, As2O3 2h), 137 (MOAP-1 KO, As2O3 2h), 605 (WT, As2O3 4h), 977 (MOAP-1 KO, As2O3 4h), 721 (WT, As2O3 6h) and 487 bodies (MOAP-1 KO, As2O3 6h) from three independently plated samples. Data information: nuclei were counterstained with DAPI (blue)


G Increased level of p62 bodies in MOAP - 1 KO MEFs upon treatment with arsenic trioxide . WT and MOAP - 1 KO MEFs were treated with arsenic trioxide ( As2O3 , 10 µM ) for the indicated durations and subjected to IF with anti - p62 antibody ( in red ) .

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G Increased level of p62 bodies in MOAP-1 KO MEFs upon treatment with arsenic trioxide. WT and MOAP-1 KO MEFs were treated with arsenic trioxide (As2O3, 10 µM) for the indicated durations and subjected to IF with anti-p62 antibody (in red).


I MOAP - 1 deficiency does not alter total protein levels of p62 in the LO2 cells . Western blotting analysis of p62 protein levels in the WT , MOAP - 1 KO and p62 KO LO2 cells lysed in 1x Laemmli sample buffer containing 2 % SDS . Actin as loading control .

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I MOAP-1 deficiency does not alter total protein levels of p62 in the LO2 cells. Western blotting analysis of p62 protein levels in the WT, MOAP-1 KO and p62 KO LO2 cells lysed in 1x Laemmli sample buffer containing 2% SDS. Actin as loading control.


J MOAP - 1 deficiency does not affect total protein levels of p62 in MEFs under basal and As2O3 - treated conditions . Western blotting analysis of p62 and MOAP - 1 in the WT and MOAP - 1 KO MEFs treated with As2O3 as indicated . MOAP - 1 was immunoprecipitated from the lysates . Actin as loading control .

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J MOAP-1 deficiency does not affect total protein levels of p62 in MEFs under basal and As2O3-treated conditions. Western blotting analysis of p62 and MOAP-1 in the WT and MOAP-1 KO MEFs treated with As2O3 as indicated. MOAP-1 was immunoprecipitated from the lysates. Actin as loading control.


K Loss of MOAP - 1 does not alter protein turnover of p62 in the LO2 cells . WT and MOAP - 1 KO LO2 cells stably expressing Halo - tagged p62 were subjected to TMR labelling and harvested at the time points post - labelling as indicated . TMR signals of Halo - p62 were visualized by a Bio - rad Imager at 565 nM wavelength , whereas the total Halo - p62 protein was detected by Western blotting using anti - Halo or anti - p62 antibodies . L Quantification of the TMR signals in the WT and MOAP - 1 KO LO2 cells by densitometric analysis . Average integrated density values of the TMR signals from three independent experiments were presented , relative to the levels at 0 hour .

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K Loss of MOAP-1 does not alter protein turnover of p62 in the LO2 cells. WT and MOAP-1 KO LO2 cells stably expressing Halo-tagged p62 were subjected to TMR labelling and harvested at the time points post-labelling as indicated. TMR signals of Halo-p62 were visualized by a Bio-rad Imager at 565 nM wavelength, whereas the total Halo-p62 protein was detected by Western blotting using anti-Halo or anti-p62 antibodies. L Quantification of the TMR signals in the WT and MOAP-1 KO LO2 cells by densitometric analysis. Average integrated density values of the TMR signals from three independent experiments were presented, relative to the levels at 0 hour.


K Loss of MOAP - 1 does not alter protein turnover of p62 in the LO2 cells . WT and MOAP - 1 KO LO2 cells stably expressing Halo - tagged p62 were subjected to TMR labelling and harvested at the time points post - labelling as indicated . TMR signals of Halo - p62 were visualized by a Bio - rad Imager at 565 nM wavelength , whereas the total Halo - p62 protein was detected by Western blotting using anti - Halo or anti - p62 antibodies .

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K Loss of MOAP-1 does not alter protein turnover of p62 in the LO2 cells. WT and MOAP-1 KO LO2 cells stably expressing Halo-tagged p62 were subjected to TMR labelling and harvested at the time points post-labelling as indicated. TMR signals of Halo-p62 were visualized by a Bio-rad Imager at 565 nM wavelength, whereas the total Halo-p62 protein was detected by Western blotting using anti-Halo or anti-p62 antibodies.


A Overexpression of MOAP - 1 downregulates levels of p62 bodies in the ATG5 - depleted cells . HepG2 cells were infected with lentivirus encoding short hairpin RNA for ATG5 ( shATG5 ) or scramble control ( shCtrl ) for 48 hours before being transfected with Myc - MOAP - 1 or empty vector ( EV ) control for 24 hours and subjected to IF analysis using anti - p62 ( in red ) and anti - Myc ( in green ) antibodies . Cells expressing Myc - MOAP - 1 were marked by dashed lines . B Quantification of the p62 bodies in the ATG5 - depleted cells transfected with Myc - MOAP - 1 or empty vector . ( Upper panel ) number of p62 bodies per cell . n ≥ 3 independently plated samples . ( Lower panel ) size of p62 bodies . n = 135 , 133 and 65 bodies in the shCtrl + EV , shATG5 + EV and shATG5 + Myc - MOAP - 1 cells , respectively , from three independently plated samples . Data information : nuclei were counterstained with DAPI ( blue )

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A Overexpression of MOAP-1 downregulates levels of p62 bodies in the ATG5-depleted cells. HepG2 cells were infected with lentivirus encoding short hairpin RNA for ATG5 (shATG5) or scramble control (shCtrl) for 48 hours before being transfected with Myc-MOAP-1 or empty vector (EV) control for 24 hours and subjected to IF analysis using anti-p62 (in red) and anti-Myc (in green) antibodies. Cells expressing Myc-MOAP-1 were marked by dashed lines. B Quantification of the p62 bodies in the ATG5-depleted cells transfected with Myc-MOAP-1 or empty vector. (Upper panel) number of p62 bodies per cell. n ≥ 3 independently plated samples. (Lower panel) size of p62 bodies. n = 135, 133 and 65 bodies in the shCtrl+EV, shATG5+EV and shATG5+Myc-MOAP-1 cells, respectively, from three independently plated samples. Data information: nuclei were counterstained with DAPI (blue)


A Overexpression of MOAP - 1 downregulates levels of p62 bodies in the ATG5 - depleted cells . HepG2 cells were infected with lentivirus encoding short hairpin RNA for ATG5 ( shATG5 ) or scramble control ( shCtrl ) for 48 hours before being transfected with Myc - MOAP - 1 or empty vector ( EV ) control for 24 hours and subjected to IF analysis using anti - p62 ( in red ) and anti - Myc ( in green ) antibodies . Cells expressing Myc - MOAP - 1 were marked by dashed lines .

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A Overexpression of MOAP-1 downregulates levels of p62 bodies in the ATG5-depleted cells. HepG2 cells were infected with lentivirus encoding short hairpin RNA for ATG5 (shATG5) or scramble control (shCtrl) for 48 hours before being transfected with Myc-MOAP-1 or empty vector (EV) control for 24 hours and subjected to IF analysis using anti-p62 (in red) and anti-Myc (in green) antibodies. Cells expressing Myc-MOAP-1 were marked by dashed lines.


C Western blotting of the lysates from HepG2 cells infected with lentivirus encoding shATG5 or shCtrl and transfected with Myc - MOAP - 1 .

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C Western blotting of the lysates from HepG2 cells infected with lentivirus encoding shATG5 or shCtrl and transfected with Myc-MOAP-1.


D Inhibition of the auto - lysosomal degradation pathway does not prevent MOAP - 1 - mediated reduction of the p62 bodies . MOAP - 1 KO LO2 cells were pre - treated with auto - lysosome inhibitors chloroquine ( CQ ) ( 10 µM ) or BafA1 ( 1 µM ) for 3 hours , before being transfected with Myc - MOAP - 1 for 24 hours in the presence of CQ or BafA1 and subjected to immunofluorescence using anti - p62 ( in red ) and anti - Myc ( in green ) antibodies . Cells expressing Myc - MOAP - 1 were marked by dashed lines . E Quantification of the p62 bodies in the MOAP - 1 KO LO2 cells pre - treated with CQ or BafA1 before being transfected with Myc - MOAP - 1 or empty vector control . ( Upper panel ) number of p62 bodies per cell . n = 5 independently plated samples . ( Lower panel ) size of p62 bodies . n = 362 ( Ctrl , BafA1 ) , 226 ( Myc - MOAP - 1 , BafA1 ) , 544 ( Ctrl , CQ ) and 266 bodies ( Myc - MOAP - 1 , CQ ) , from three independently plated samples . Data information : nuclei were counterstained with DAPI ( blue ) ,

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D Inhibition of the auto-lysosomal degradation pathway does not prevent MOAP-1-mediated reduction of the p62 bodies. MOAP-1 KO LO2 cells were pre-treated with auto-lysosome inhibitors chloroquine (CQ) (10 µM) or BafA1 (1 µM) for 3 hours, before being transfected with Myc-MOAP-1 for 24 hours in the presence of CQ or BafA1 and subjected to immunofluorescence using anti-p62 (in red) and anti-Myc (in green) antibodies. Cells expressing Myc-MOAP-1 were marked by dashed lines. E Quantification of the p62 bodies in the MOAP-1 KO LO2 cells pre-treated with CQ or BafA1 before being transfected with Myc-MOAP-1 or empty vector control. (Upper panel) number of p62 bodies per cell. n = 5 independently plated samples. (Lower panel) size of p62 bodies. n = 362 (Ctrl, BafA1), 226 (Myc-MOAP-1, BafA1), 544 (Ctrl, CQ) and 266 bodies (Myc-MOAP-1, CQ), from three independently plated samples. Data information: nuclei were counterstained with DAPI (blue),


F Western blotting of lysates from MOAP - 1 KO LO2 cells pre - treated with CQ or BafA1 and transfected with Myc - MOAP - 1 .

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F Western blotting of lysates from MOAP-1 KO LO2 cells pre-treated with CQ or BafA1 and transfected with Myc-MOAP-1.


A Larger RFP - p62 bodies in the MOAP - 1 deficient cells . WT and MOAP - 1 KO LO2 cells were transfected with expression vector encoding RFP - tagged p62 . 16 hours later , cells were subjected to live imaging analysis . Z - stacks of the confocal images were flattened as maximal intensity projections . Scale bar : 5 µm . B Quantification of size of RFP - p62 bodies in WT and MOAP - 1 KO LO2 cells . n = 129 and 161 bodies in the WT and MOAP - 1 KO cells , respectively , from three independently plated samples . * * * P < 0 . 001 , student ' s t - test .

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A Larger RFP-p62 bodies in the MOAP-1 deficient cells. WT and MOAP-1 KO LO2 cells were transfected with expression vector encoding RFP-tagged p62. 16 hours later, cells were subjected to live imaging analysis. Z-stacks of the confocal images were flattened as maximal intensity projections. Scale bar: 5 µm. B Quantification of size of RFP-p62 bodies in WT and MOAP-1 KO LO2 cells. n = 129 and 161 bodies in the WT and MOAP-1 KO cells, respectively, from three independently plated samples. ***P<0.001, student's t-test.


A Larger RFP - p62 bodies in the MOAP - 1 deficient cells . WT and MOAP - 1 KO LO2 cells were transfected with expression vector encoding RFP - tagged p62 . 16 hours later , cells were subjected to live imaging analysis . Z - stacks of the confocal images were flattened as maximal intensity projections . Scale bar : 5 µm .

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A Larger RFP-p62 bodies in the MOAP-1 deficient cells. WT and MOAP-1 KO LO2 cells were transfected with expression vector encoding RFP-tagged p62. 16 hours later, cells were subjected to live imaging analysis. Z-stacks of the confocal images were flattened as maximal intensity projections. Scale bar: 5 µm.


C Western analysis of lysates of the WT and MOAP - 1 KO LO2 cells transfected with expression vector encoding RFP - tagged p62 as in ( A ) .

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C Western analysis of lysates of the WT and MOAP-1 KO LO2 cells transfected with expression vector encoding RFP-tagged p62 as in (A).


D , E RFP - p62 bodies exhibited fusion ( D ) and fission ( E ) characteristics of liquid droplets . WT and MOAP - 1 KO LO2 cells were transfected with expression vector encoding RFP - tagged p62 and 14 hours later , cells were analyzed by live - imaging to visualize the dynamics of RFP - p62 bodies . Scale bar : 2 µm

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D, E RFP-p62 bodies exhibited fusion (D) and fission (E) characteristics of liquid droplets. WT and MOAP-1 KO LO2 cells were transfected with expression vector encoding RFP-tagged p62 and 14 hours later, cells were analyzed by live-imaging to visualize the dynamics of RFP-p62 bodies. Scale bar: 2 µm


D , E RFP - p62 bodies exhibited fusion ( D ) and fission ( E ) characteristics of liquid droplets . WT and MOAP - 1 KO LO2 cells were transfected with expression vector encoding RFP - tagged p62 and 14 hours later , cells were analyzed by live - imaging to visualize the dynamics of RFP - p62 bodies . Scale bar : 2

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F Loss of MOAP - 1 leads to slower exchange of p62 in the RFP - p62 bodies . WT and MOAP - 1 KO LO2 cells transfected with expression vector encoding RFP - tagged p62 . 14 hours later , cells were subjected to the fluorescence recovery after photobleaching ( FRAP ) assay . Scale bar : 2 µm . G Quantification of the rate of fluorescence recovery of RFP - p62 in WT and MOAP - 1 KO cells . n = 5 samples .

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F Loss of MOAP-1 leads to slower exchange of p62 in the RFP-p62 bodies. WT and MOAP-1 KO LO2 cells transfected with expression vector encoding RFP-tagged p62. 14 hours later, cells were subjected to the fluorescence recovery after photobleaching (FRAP) assay. Scale bar: 2 µm. G Quantification of the rate of fluorescence recovery of RFP-p62 in WT and MOAP-1 KO cells. n = 5 samples.


F Loss of MOAP - 1 leads to slower exchange of p62 in the RFP - p62 bodies . WT and MOAP - 1 KO LO2 cells transfected with expression vector encoding RFP - tagged p62 . 14 hours later , cells were subjected to the fluorescence recovery after photobleaching ( FRAP ) assay . Scale bar : 2 µm .

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F Loss of MOAP-1 leads to slower exchange of p62 in the RFP-p62 bodies. WT and MOAP-1 KO LO2 cells transfected with expression vector encoding RFP-tagged p62. 14 hours later, cells were subjected to the fluorescence recovery after photobleaching (FRAP) assay. Scale bar: 2 µm.


H Overexpression of MOAP - 1 but not GFP leads to increased exchange of p62 in the RFP - p62 bodies . HepG2 cells were transfected with expression vector encoding RFP - tagged p62 and GFP - tagged MOAP - 1 or GFP only control and 14 hours later , cells were subjected to FRAP assay . Scale bar : 2 µm . I Quantification of the rate of fluorescence recovery of RFP - p62 in HepG2 cells co - expressing GFP - MOAP - 1 or GFP . n = 5 samples .

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H Overexpression of MOAP-1 but not GFP leads to increased exchange of p62 in the RFP-p62 bodies. HepG2 cells were transfected with expression vector encoding RFP-tagged p62 and GFP-tagged MOAP-1 or GFP only control and 14 hours later, cells were subjected to FRAP assay. Scale bar: 2 µm. I Quantification of the rate of fluorescence recovery of RFP-p62 in HepG2 cells co-expressing GFP-MOAP-1 or GFP. n = 5 samples.


H Overexpression of MOAP - 1 but not GFP leads to increased exchange of p62 in the RFP - p62 bodies . HepG2 cells were transfected with expression vector encoding RFP - tagged p62 and GFP - tagged MOAP - 1 or GFP only control and 14 hours later , cells were subjected to FRAP assay . Scale bar : 2 µm .

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H Overexpression of MOAP-1 but not GFP leads to increased exchange of p62 in the RFP-p62 bodies. HepG2 cells were transfected with expression vector encoding RFP-tagged p62 and GFP-tagged MOAP-1 or GFP only control and 14 hours later, cells were subjected to FRAP assay. Scale bar: 2 µm.


J Re - introduction of MOAP - 1 disrupts the spherical structure of p62 bodies in the MOAP - 1 deficient LO2 cells . MOAP - 1 KO LO2 cells were transfected with expression vector encoding Myc - MOAP - 1 and harvested at 14 - or 24 - hours post - transfection for IF analysis using anti - p62 ( in red ) and anti - Myc ( in green ) antibodies . Insets were enlarged in the lower panels . Scale bar : 2 µm .

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

J Re-introduction of MOAP-1 disrupts the spherical structure of p62 bodies in the MOAP-1 deficient LO2 cells. MOAP-1 KO LO2 cells were transfected with expression vector encoding Myc-MOAP-1 and harvested at 14- or 24-hours post-transfection for IF analysis using anti-p62 (in red) and anti-Myc (in green) antibodies. Insets were enlarged in the lower panels. Scale bar: 2 µm.


A MOAP - 1 associates with p62 and their interaction is enhanced by DEN - mediated stress . LO2 cells transfected with Myc - MOAP - 1 were treated with or without diethylnitrosamine ( DEN ) ( 200 µM ) for 12 h and subjected to Duolink proximity ligation assay using anti - p62 and anti - Myc antibodies . Red dots represent MOAP - 1 / p62 interaction .

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

A MOAP-1 associates with p62 and their interaction is enhanced by DEN-mediated stress. LO2 cells transfected with Myc-MOAP-1 were treated with or without diethylnitrosamine (DEN) (200 µM) for 12 h and subjected to Duolink proximity ligation assay using anti-p62 and anti-Myc antibodies. Red dots represent MOAP-1/p62 interaction.