B . To selectively label intracellular HC575A , were treated with 1 nM Halo - tag TMR Direct Ligand for 24 hours . left panel shows aliquots of lysates of indicated transfectants , resolved under non - reducing conditions decorated with anti - λ antibodies . In two right panels , samples were precipitated with NP - Sepharose electrophoresed under non reducing conditions . signal for Halo - tag TMR was checked directly in gel . arrows point at various assemblies between µ , HC575A λ chains .

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B. To selectively label intracellular HC575A, cells were treated with 1 nM Halo-tag TMR Direct Ligand for 24 hours. The left panel shows aliquots of the lysates of the indicated transfectants, resolved under non-reducing conditions and decorated with anti-λ antibodies. In the two right panels, samples were precipitated with NP-Sepharose and electrophoresed under non reducing conditions. The signal for the Halo-tag TMR was checked directly in the gel. The arrows point at the various assemblies between µ, HC575A and λ chains.


C . experiment shown aims to determine whether ERp44 can bind also to a single C575 , or rather to non - native bonds . Aliquots of lysates from indicated ERp44KO transfectants were precipitated with NP - Sepharose , resolved under non reducing conditions blot decorated with anti - λ antibodies or anti - ERp44 as indicated . Black asterisks point to covalent complexes of Halo - ERp44 with µs2L2 .

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C. The experiment shown aims to determine whether ERp44 can bind also to a single C575, or rather to non-native bonds. Aliquots of the lysates from the indicated ERp44KO transfectants were precipitated with NP-Sepharose, resolved under non reducing conditions and the blot decorated with anti-λ antibodies or anti-ERp44 as indicated. Black asterisks point to covalent complexes of Halo-ERp44 with µs2L2.


A Western blots of acid - extracted histones from activated CD4 + T showing global histone lactylation H3K18 lactylation in presence absence of glucose at 24 hours after stimulation of . One of three similar experiments is shown .

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A Western blots of acid-extracted histones from activated CD4+ T cells showing global histone lactylation and H3K18 lactylation levels in the presence and absence of glucose at 24 hours after stimulation of cells. One of three similar experiments is shown.


C , D Western blots of acid - extracted histones from activated CD4 + T in presence of glycolysis inhibitor 2 - DG PDK inhibitor DCA , respectively . were analysed for global lactylation H3K18 lactylation 24 hours after activation of TCR with anti - CD3 ( 5 µg / ml ) .

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C, D Western blots of acid-extracted histones from activated CD4+ T cells in the presence of glycolysis inhibitor 2-DG and PDK inhibitor DCA, respectively. Cells were analysed for global lactylation and H3K18 lactylation 24 hours after activation of TCR with anti-CD3 (5 µg/ml).


E Immunoblotting of acid - extracted lactylated histones from anti - CD3 - activated CD4 + T in presence of extracellular lactate ( 25 mM ) at 24 hours of culture . One representative out of three experiments is shown . For immunoblotting experiments , ex vivo purified , non - activated CD4 + T were used as control lymphocytes .

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E Immunoblotting of acid-extracted lactylated histones from anti-CD3-activated CD4+ T cells in the presence of extracellular lactate (25 mM) at 24 hours of cell culture. One representative out of three experiments is shown. For all immunoblotting experiments, ex vivo purified, non-activated CD4+ T cells were used as control lymphocytes.


F ChIP - sequncing showing H3K18 lactylation signals in control Th17 lactate - treated Th17 lymphocytes ( 25 mM ) . Biological duplicates are shown .

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F ChIP-sequncing showing H3K18 lactylation signals in control Th17 cells and lactate-treated Th17 lymphocytes (25 mM). Biological duplicates are shown.


A - I . Correlative electron microscopy ( CLEM ) showing Prom1 at plasma of hepatocytes . hepatocytes were isolated from 12 - week - old WT ( Prom1 + / + ) or KO ( Prom1cre - ert2 - nlacz ) mice . boxed areas in panel ( A , D , G ) ( B , E , H ) were magnified presented as panel ( B , E , H ) ( C , F , I ) , respectively . Scale bar ; 5 μm ( A , D G ) , 1 μm ( B , E H ) , 0 . 2 μm ( C , F I ) .

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A-I. Correlative light electron microscopy (CLEM) showing Prom1 at the plasma membrane of primary hepatocytes. Primary hepatocytes were isolated from 12-week-old male WT (Prom1+/+) or KO ( Prom1cre-ert2-nlacz) mice. The boxed areas in panel (A, D, G) and (B, E, H) were magnified and presented as panel (B, E, H) and (C, F, I), respectively. Scale bar; 5 μm (A, D and G), 1 μm (B, E and H), and 0.2 μm (C, F and I).


J . Immunofluorescent of Prom1 , CK19 HNF4α in two serial cryosections of mouse liver . boxed in top panel was magnified presented as bottom panel . Asterisks ( * ) in top panel indicate cholangiocytes of bile bucts triangles ( Γ ) in bottom panel indicate hepatocytes . Scale bar , 100 μm .

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J. Immunofluorescent staining of Prom1, CK19 and HNF4α in two serial cryosections of mouse liver. The boxed area in the top panel was magnified and presented as the bottom panel. Asterisks (*) in the top panel indicate cholangiocytes of the bile bucts and triangles (Γ) in the bottom panel indicate hepatocytes. Scale bar, 100 μm.


hepatocytes were isolated from 12 - week - old Prom1 + / + ( WT ) or Prom1 - / - ( KO ) mice serum - starved . ( K ) Differentially expressed genes showing more than a 2 - fold change with P < 0 . 05 ( 118 DEGs ) induced ( n = 55 ) or repressed ( n = 63 ) by Prom1 knockout ( n = 2 / group ) . G6pc , glucose - 6 - phosphatase catalytic subunit ; Pck1 , phosphoenolpyruvate carboxykinase 1 .

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Primary hepatocytes were isolated from 12-week-old male Prom1+/+ (WT) or Prom1-/- (KO) mice and serum-starved. (K) Differentially expressed genes showing more than a 2-fold change with P < 0.05 (118 DEGs) induced (n=55) or repressed (n=63) by Prom1 knockout (n = 2/group). G6pc, glucose-6-phosphatase catalytic subunit; Pck1, phosphoenolpyruvate carboxykinase 1.


hepatocytes were isolated from 12 - week - old Prom1 + / + ( WT ) or Prom1 - / - ( KO ) mice serum - starved . ( L ) Relative expression of G6pc Pck1 mRNAs after serum starvation for 16 hours were determined by qRT - PCR ( n = 3 / group ) .

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Primary hepatocytes were isolated from 12-week-old male Prom1+/+ (WT) or Prom1-/- (KO) mice and serum-starved. (L) Relative expression levels of G6pc and Pck1 mRNAs after serum starvation for 16 hours were determined by qRT-PCR (n = 3/group).


hepatocytes were isolated from 12 - week - old Prom1 + / + ( WT ) or Prom1 - / - ( KO ) mice serum - starved . ( M ) of Prom1 , PCK , G6PC GAPDH were determined by immunoblotting .

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Primary hepatocytes were isolated from 12-week-old male Prom1+/+ (WT) or Prom1-/- (KO) mice and serum-starved. (M) Levels of the Prom1, PCK, G6PC and GAPDH proteins were determined by immunoblotting.


hepatocytes were isolated from 12 - week - old Prom1 + / + ( WT ) or Prom1 - / - ( KO ) mice serum - starved . ( N ) Prom1 + / + or Prom1 - / - hepatocytes serum - starved for 8 hours were incubated with 2 - [ 3H ] deoxyglucose for 10 minutes . Glucose uptake was assessed by calculating amount of - associated radioactivity normalized to amount of ( n = 3 mice / group ) .

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Primary hepatocytes were isolated from 12-week-old male Prom1+/+ (WT) or Prom1-/- (KO) mice and serum-starved. (N) Prom1+/+ or Prom1-/- hepatocytes serum-starved for 8 hours were incubated with 2-[3H]deoxyglucose for 10 minutes. Glucose uptake was assessed by calculating the amount of cell-associated radioactivity normalized to the amount of protein (n = 3 mice/group).


A . of G6pc Pck1 mRNAs in liver of mice after 18 hours fasting were determined by qRT - PCR . level of each mRNA was normalized to level of 18s rRNA ( n = 3 / fed , 7 or 8 / fasted ) .

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A. Levels of G6pc and Pck1 mRNAs in the liver of mice after 18 hours fasting were determined by qRT-PCR. The level of each mRNA was normalized to the level of 18s rRNA (n = 3/fed, 7 or 8/fasted).


B . of phospho - PKA substrates ( p - PKA substrates ) , phospho - CREB ( p - CREB ) , CREB , G6pc Pck , Prom1 GAPDH were determined after 18 hours fasting . Band for p - CREB was quantified using Photoshop software .

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B. Levels of phospho-PKA substrates (p-PKA substrates), phospho-CREB (p-CREB), CREB, G6pc Pck, Prom1 and GAPDH were determined after 18 hours fasting. Band intensity for p-CREB was quantified using Photoshop software.


C . Blood glucose ( mg / dL ) of mice after fasted for 24 hours ( n = 12 or 14 / group ) .

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C. Blood glucose levels (mg/dL) of mice after fasted for 24 hours (n = 12 or 14/group).


D . Serum glucagon level in fed or fasted mice ( n = 3 / fed , 7 or 8 / fasted ) .

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D. Serum glucagon level in the fed or fasted mice (n = 3/fed, 7 or 8/fasted).


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

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


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

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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).


hepatocytes were isolated from 12 - week - old Prom1 + / + ( WT ) or Prom1 - / - ( KO ) mice , serum - starved stimulated with glucagon . G . 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 Gapdh 10 minutes after glucagon stimulation ( 10 nM ) were determined by immunoblotting .

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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.


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

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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.


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

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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).


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

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

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

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

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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 WT ( Prom1 + / + ) KO ( Prom1 - / - ) mice were fasted for 4 hours intraperitoneally injected with glucagon . ( D ) cAMP in liver after a 5 minutes glucagon treatment was determined using cAMP kit ( 2 mg / kg , n = 3 / group ) .

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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 mice using glucose , pyruvate ( E ) tolerance ( n = 10 mice / group ) .

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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 mice using insulin ( F ) tolerance ( n = 10 mice / group ) .

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


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

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


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

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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.


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

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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.


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

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

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

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

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

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-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

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 + / + ) KO ( Prom1 - / - ) mice were fasted for 4 hours subjected to immobilization . ( 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 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

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 . 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 stimulated with glucagon ( 10 nM ) for 10 minutes . phosphorylation of PKA substrates was determined by immunoblotting .

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 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 radixin expression were silenced in WT ( Prom1 + / + ) 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 stimulated with glucagon ( 10 nM ) for 10 minutes . ( B ) localization of p - CREB after glucagon stimulation was determined by immunofluorescence . Blue ; DAPI , Red ; p - CREB . of with p - CREB among more than 300 in each group was also statistically analyzed ( Figure EV3G ) . Scale bar , 20 μm .

B-CONTROLLED_VAR I-CONTROLLED_VAR 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 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 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 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

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 radixin expression were silenced in WT ( Prom1 + / + ) 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 stimulated with glucagon ( 10 nM ) for 10 minutes . ( C ) of p - PKA substrates , p - CREB , CREB , p - IP3R , IP3R , Prom1 , radixin GAPDH after glucagon stimulation ( 10 minutes ) were determined by immunoblotting .

B-CONTROLLED_VAR I-CONTROLLED_VAR 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 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 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

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 of Prom1 in mouse liver tranduced with adenoviral particles . Confocal microscope 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 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 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 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 intraperitoneally injected with glucagon . ( E ) Blood glucose ( mg / dL ) were measured 0 , 15 , 30 , 60 120 minutes after glucagon administration ( 200 µg / kg , 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 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

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 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 intraperitoneally injected with glucagon . ( F ) of p - PKA , p - CREB , CREB , radixin GAPDH in liver after a 10 - minutes glucagon stimulation ( 2 mg / kg ) 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 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 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 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 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 intraperitoneally injected with glucagon . ( G ) Glucose disposal rates in sh - Con or sh - Rdx ( radixin ) mice were measured using glucose , insulin pyruvate tolerance ( 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 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 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

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).


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

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


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

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 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 B-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O B-MEASURED_VAR 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 ) radixin ( green ) . lower panels represent magnification of square frame in upper panels . Scale bar , 20 μm .

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

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

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

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

O O O O O O O B-CONTROLLED_VAR 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 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 . localization of radixin actin in WT ( Prom1 + / + ) KO ( Prom1 - / - ) hepatocytes was determined by immunofluorescence for radixin phalloidin . Scale bar , 20 μm .

O O O O B-MEASURED_VAR 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 B-MEASURED_VAR 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 + / + ) KO ( Prom1 - / - ) mouse liver samples . of Prom1 , radixin ( RDX ) , flotillin transferrin receptor ( TfR ) 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 B-CONTROLLED_VAR 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 I-MEASURED_VAR I-MEASURED_VAR O B-MEASURED_VAR 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 + / + ) KO ( Prom1 - / - ) mouse hepatocytes after cholesterol depletion by methyl - β - cyclodextrin ( mβCD , 30 mM ) for 30 minutes at 4 oC . of Prom1 , radixin , flotillin transferrin receptor 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 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 B-MEASURED_VAR O B-MEASURED_VAR O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR 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.


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

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


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

O O O B-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 . molecular interaction between PROM1 - FLAG Myc - radixin in presence of increasing amounts of Myc - FERM was determined by co - immunoprecipitation with an anti - FLAG antibody .

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

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


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


WT 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 stimulated with glucagon ( 10 nM ) for 10 minutes . of p - PKA substrates , p - CREB , CREB , FERM - Myc 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 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

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 wild type mice were infected with an adenovirus harboring GFP or FERM for 3 days , fasted for 4 hours intraperitoneally injected with glucagon . ( G ) Blood glucose ( mg / dL ) were measured 0 , 15 , 30 , 60 120 minutes after glucagon stimulation ( 0 . 2 mg / kg , 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

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 wild type mice were infected with an adenovirus harboring GFP or FERM for 3 days , fasted for 4 hours intraperitoneally injected with glucagon . ( H ) of p - CREB , CREB , p - PKA substrates , FERM in liver 10 minutes after glucagon stimulation ( 2 mg / kg ) 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 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 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 wild type mice were infected with an adenovirus harboring GFP or FERM for 3 days , fasted for 4 hours intraperitoneally injected with glucagon . ( I ) Glucose disposal rates in GFP - or FERM - overexpressing mice were measured using glucose , pyruvate insulin tolerance ( 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

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

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(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 hepatocytes for 24 hours . Hepatocytes were further serum - starved for 18 hours stimulated with glucagon ( 10 nM ) for 10 minutes . of p - PKA substrates , p - CREB , CREB , radixin , Myc - radixin GAPDH were determined by immunoblotting .

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

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

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

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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 WT ( Prom1 + / + ) KO ( Prom1 - / - ) mice were fed a high - fat diet for 8 ( Diet - Induced Obesity ; DIO ) , 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 intraperitoneally injected with glucagon ( 100 µg / kg ) . Blood glucose ( mg / dL ) were measured 0 , 15 , 30 , 60 120 minutes after glucagon stimulation ( GST ) . Glucose disposal rates were measured using pyruvate tolerance ( PTT ) ( n = 10 mice / group ) .

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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 ) activated neurons are prepared in three specific time points for - seq ATAC - seq at early ( 1h ) late time points ( 6 10h ) ; ( bottom ) Blot for pMAPK in neurons treated with KCl 55 mM several concentrations of BDNF ( Con = no stimulation ) . 44 42 KDa bands indicated with lines . Nucleolin shown as internal control .

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(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 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 .

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(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 plots indicate expression for signature genes selected for each cluster . 4 , 907 DE - genes are also selected with FDR = 1 % more than 100 across samples . ( e ) Gene Ontology enrichment 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 other clusters . Names on right y - axis indicate ontology common names .

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(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 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 .

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

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(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 ) expression changes for TFs related to 8 - mer groups bZIP , CTCF , HIC1 , EGR . Significant changes relative to control samples are shown with asterisks ( Wilcoxon rank sums , 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 per million ( CPM ) ; - seq CPM ; H3K27ac normalized signal upon KCl stimulation , H3K4me1 ( Malik et al , 2014 ) ; CTCF ( Sams et al , 2016 ) ; Cortical neurons Hi - C data ( Bonev et al , 2017 ) . Red bars in ATAC - seq tracks indicate gained DA - peaks in BDNF , red bars in - seq tracks indicate Arc differential expression in BDNF KCl 1h . Green blocks in Hi - C tracks indicate anchor points for calculation of contact scores , using shaman ( Cohen et al , 2017 ) . Spearman ' s rho value indicate rank - correlation between highlighted peaks across samples ( Appendix Figure S4c ) . TF module names indicate 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 accessibility DA - peaks GWAS summary statistics . DA - peaks ( DA ) , gained DA - peaks ( gained ) , closing DA - peaks , consensus peaks ( ) are fitted to summary statistics data of multiple GWAS studies ( y - axis ) . Circle color indicates LDSC regression coefficient ( effect ) , circle 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 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 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 micTNFα - KO at ZT6 for : ( top ) phosphorylated GSK3α / β ( pGSK3α at Ser21 pGSK3β at Ser9 ) total GSK3α ; ( bottom ) phosphorylated MAPK ( pMAPK , MAPK3 at Thr203 / Tyr205 MAPK1 at Thr183 / Tyr185 ) total MAPK . Right , between phosphorylated total kinase normalized to CTL at ZT18 ZT6 . n = 5 mice per group . * * * p = 0 . 000009 ( GSK3α ) , multiple t - 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 micTNF - KOs predicted by robust kinase activity inference ( RoKAI ) at ZT18 ZT6 . Graph shows z - score value of each kinase assigned by RoKAI . Kinases above below dotted represent prediction of up - 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 micTNF - KOs . ( F ) Left , Immunoblots of cortical lysates of CTL micTNFα - KO at ZT6 using antibodies specific to target phosphorylation motifs of MAPK / CDK ( PXS * P , S * PX ( K / R ) ) PKC ( ( K / R ) XS * X ( K / R ) ) . Dashed boxes indicate quantified signal . Right , between phosphorylated substrates loading control normalized to CTL at ZT18 ZT6 . ( F , n = 5 mice per group . * p = 0 . 022 ( MAPK / CDK phospho sub ) , * p = 0 . 014 ( PKC phospho sub ) multiple t - 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 micTNF - KOs . ( G ) Left , Immunoblots show signal of phosphorylated threonine in MARKs activation loop . Right , between phosphorylated MARK activation loop loading control normalized to CTL at ZT18 ZT6 . G ) n = 5 mice per group . * * p = 0 . 0091 ( MARK ) , multiple t - 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 inclusion bodies upon proteasome inhibition . HCT116 were transfected with plasmid encoding GFP - MOAP - 1 or GFP only control . 14 hours later , were treated with proteasome inhibitor MG132 ( 5 µM ) for 8 hours analyzed by immunofluorescence ( IF ) with anti - GFP ( in green ) 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 p62 bodies upon proteasome inhibition . HCT116 were treated with MG132 ( 5 µM ) for 8 hours analyzed by IF with anti - MOAP - 1 ( in green ) 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 p62 bodies upon exposure to stresses . LO2 hepatocytes were transfected with plasmid encoding Myc - MOAP - 1 . 14 hours later , were then treated with MG132 ( 5 µM ) , arsenic trioxide ( As2O3 , 10 µM ) or diethylnitrosamine ( DEN , 200 µM ) for 8 hours each . were then processed for IF with anti - Myc ( in green ) 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 p62 bodies at resting state in liver cancer lines , HepG2 , Huh - 1 , JHH5 JHH7 . liver cancer lines were transfected with plasmid encoding Myc - MOAP - 1 . 14 hours later , transfected were subjected to IF with anti - Myc ( in green ) anti - p62 ( in red ) antibodies . Data information : nuclei were counterstained with DAPI ( blue )

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

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 aggregated patterns of MOAP - 1 in p62 deficient . WT p62 KO HepG2 were transfected with plasmid encoding Myc - MOAP - 1 for 14 hours were subjected to IF 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 of p62 Myc - MOAP - 1 in WT p62 KO HepG2 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 of diethylnitrosamine ( DEN ) - induced p62 bodies in a time - dependent manner . LO2 were transfected with plasmid encoding Myc - MOAP - 1 . 14 hours later , were treated with DEN ( 200 µM ) for indicated period of times , before being subjected to IF with anti - Myc ( in green ) anti - p62 ( in red ) antibodies . 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 p62 bodies in resting HepG2 liver cancer . HepG2 were transfected with plasmid encoding Myc - MOAP - 1 . At indicated time post - transfection , transfected 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 basal of p62 bodies in LO2 . WT , MOAP - 1 KO p62 KO LO2 were subjected to IF with anti - p62 antibody ( in red ) . p62 KO were included as a negative control . Insets in upper panels are enlarged in lower panels . D of p62 bodies in WT MOAP - 1 KO LO2 . ( Left panel ) number of p62 bodies per . n = 7 independently plated samples . ( Right panel ) of p62 bodies . n = 442 1014 bodies detected in WT MOAP - 1 KO LO2 , 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 basal of p62 bodies in LO2 . WT , MOAP - 1 KO p62 KO LO2 were subjected to IF with anti - p62 antibody ( in red ) . p62 KO were included as a negative control . Insets in upper panels are enlarged in 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 of p62 bodies in MOAP - 1 deficient LO2 . MOAP - 1 KO LO2 were transfected with plasmid encoding Myc - MOAP - 1 for 24 hours subjected to IF with anti - p62 ( in red ) anti - Myc ( green ) antibodies . expressing Myc - MOAP - 1 were marked by dashed lines . F of p62 bodies in MOAP - 1 KO LO2 re - introduced with Myc - MOAP - 1 . ( Left panel ) number of p62 bodies per . n = 7 independently plated samples of MOAP - 1 KO transfected with empty vector control ( Ctrl ) or plasmid encoding Myc - MOAP - 1 . ( Right panel ) of p62 bodies . n = 178 69 bodies detected in Ctrl Myc - MOAP - 1 expressing , respectively , from three independently plated samples . Data information : , nuclei were counterstained with DAPI ( blue )

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

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 of p62 bodies in MOAP - 1 deficient LO2 . MOAP - 1 KO LO2 were transfected with plasmid encoding Myc - MOAP - 1 for 24 hours subjected to IF with anti - p62 ( in red ) anti - Myc ( green ) antibodies . 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 MOAP - 1 KO MEFs were treated with arsenic trioxide ( As2O3 , 10 µM ) for indicated durations subjected to IF with anti - p62 antibody ( in red ) . H of p62 bodies in WT MOAP - 1 KO MEFs treated with arsenic trioxide . ( Left panel ) number of p62 bodies per . n ≥ 6 independently plated samples . ( Right panel ) 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 ) 487 bodies ( MOAP - 1 KO , As2O3 6h ) from three independently plated samples . Data information : nuclei were counterstained with DAPI ( blue )

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

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 MOAP - 1 KO MEFs were treated with arsenic trioxide ( As2O3 , 10 µM ) for indicated durations subjected to IF with anti - p62 antibody ( in red ) .

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

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 of p62 in LO2 . Western blotting of p62 in WT , MOAP - 1 KO p62 KO LO2 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 of p62 in MEFs under basal As2O3 - treated conditions . Western blotting of p62 MOAP - 1 in WT MOAP - 1 KO MEFs treated with As2O3 as indicated . MOAP - 1 was immunoprecipitated from 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 turnover of p62 in LO2 . WT MOAP - 1 KO LO2 stably expressing Halo - tagged p62 were subjected to TMR labelling harvested at time points post - labelling as indicated . TMR signals of Halo - p62 were visualized by a Bio - rad Imager at 565 nM wavelength , whereas total Halo - p62 was detected by Western blotting using anti - Halo or anti - p62 antibodies . L of TMR signals in WT MOAP - 1 KO LO2 by densitometric . Average integrated density values of TMR signals from three independent experiments were presented , relative to at 0 hour .

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

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 turnover of p62 in LO2 . WT MOAP - 1 KO LO2 stably expressing Halo - tagged p62 were subjected to TMR labelling harvested at time points post - labelling as indicated . TMR signals of Halo - p62 were visualized by a Bio - rad Imager at 565 nM wavelength , whereas total Halo - p62 was detected by Western blotting using anti - Halo or anti - p62 antibodies .

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

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 of p62 bodies in ATG5 - depleted . HepG2 were infected with lentivirus encoding short hairpin 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 subjected to IF using anti - p62 ( in red ) anti - Myc ( in green ) antibodies . expressing Myc - MOAP - 1 were marked by dashed lines . B of p62 bodies in ATG5 - depleted transfected with Myc - MOAP - 1 or empty vector . ( Upper panel ) number of p62 bodies per . n ≥ 3 independently plated samples . ( Lower panel ) of p62 bodies . n = 135 , 133 65 bodies in shCtrl + EV , shATG5 + EV shATG5 + Myc - MOAP - 1 , respectively , from three independently plated samples . Data information : nuclei were counterstained with DAPI ( blue )

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

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 of p62 bodies in ATG5 - depleted . HepG2 were infected with lentivirus encoding short hairpin 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 subjected to IF using anti - p62 ( in red ) anti - Myc ( in green ) antibodies . expressing Myc - MOAP - 1 were marked by dashed lines .

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

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 lysates from HepG2 infected with lentivirus encoding shATG5 or shCtrl transfected with Myc - MOAP - 1 .

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

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

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

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 pre - treated with CQ or BafA1 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 MOAP - 1 deficient . WT MOAP - 1 KO LO2 were transfected with expression vector encoding RFP - tagged p62 . 16 hours later , were subjected to live imaging . Z - stacks of confocal were flattened as maximal projections . Scale bar : 5 µm . B of of RFP - p62 bodies in WT MOAP - 1 KO LO2 . n = 129 161 bodies in WT MOAP - 1 KO , respectively , from three independently plated samples . * * * P < 0 . 001 , student ' s t - .

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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 MOAP - 1 deficient . WT MOAP - 1 KO LO2 were transfected with expression vector encoding RFP - tagged p62 . 16 hours later , were subjected to live imaging . Z - stacks of confocal were flattened as maximal projections . Scale bar : 5 µm .

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

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

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

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

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.