G . U2OS / GFP - FAM111A cell lines that were treated or not with DOX were stained with RFC1 antibody , pre - extracted and fixed , and stained with DAPI . RFC1 and DAPI signal intensities were analyzed by QIBC .

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G. U2OS/GFP-FAM111A cell lines that were treated or not with DOX were stained with RFC1 antibody, pre-extracted and fixed, and stained with DAPI. RFC1 and DAPI signal intensities were analyzed by QIBC.


U2OS / GFP - FAM111A cell lines that were treated or not with DOX were pre - extracted and fixed , and stained with DAPI . DAPI signal intensities were analyzed by QIBC . H . cells were stained with RPB1 antibody .

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U2OS/GFP-FAM111A cell lines that were treated or not with DOX were pre-extracted and fixed, and stained with DAPI. DAPI signal intensities were analyzed by QIBC. H. cells were stained with RPB1 antibody.


I . Immunoblot analysis of U2OS / GFP - FAM111A cell lines treated or not with DOX in the absence or presence of Z - VAD - FMK .

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I. Immunoblot analysis of U2OS/GFP-FAM111A cell lines treated or not with DOX in the absence or presence of Z-VAD-FMK.


Immunoblot analysis of cell lines treated or not with DOX in the absence or presence of Z - VAD - FMK . J . using U2OS cell lines conditionally expressing ectopic untagged FAM111A alleles .

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Immunoblot analysis of cell lines treated or not with DOX in the absence or presence of Z-VAD-FMK. J. using U2OS cell lines conditionally expressing ectopic untagged FAM111A alleles.


K . Purified recombinant FLAG - FAM111A proteins were incubated at indicated temperatures for 4 h , and FAM111A auto - proteolytic activity was analyzed by immunoblotting .

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K. Purified recombinant FLAG-FAM111A proteins were incubated at indicated temperatures for 4 h, and FAM111A auto-proteolytic activity was analyzed by immunoblotting.


L . GFP IPs from U2OS cell lines expressing GFP - FAM111A WT or D528G mutant were analyzed by mass spectrometry . Volcano plot shows enrichment of individual proteins ( WT / D528G ratio ) plotted against the P value . Dashed lines indicate the significance thresholds ( FDR < 0 . 05 ; s0 = 1 ) .

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L. GFP IPs from U2OS cell lines expressing GFP-FAM111A WT or D528G mutant were analyzed by mass spectrometry. Volcano plot shows enrichment of individual proteins (WT/D528G ratio) plotted against the P value. Dashed lines indicate the significance thresholds (FDR<0.05; s0=1).


B . U2OS / GFP - FAM111A WT cells treated or not with DOX were subjected to GFP IP followed by immunoblotting with indicated antibodies . C . As in ( B ) , using U2OS / GFP - FAM111B WT cells .

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B. U2OS/GFP-FAM111A WT cells treated or not with DOX were subjected to GFP IP followed by immunoblotting with indicated antibodies. C. As in (B), using U2OS/GFP-FAM111B WT cells.


D . Purified recombinant FLAG - FAM111A and FLAG - FAM111B proteins were incubated separately or mixed , subjected to IP with FAM111A antibody and immunoblotted with FLAG antibody .

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D. Purified recombinant FLAG-FAM111A and FLAG-FAM111B proteins were incubated separately or mixed, subjected to IP with FAM111A antibody and immunoblotted with FLAG antibody.


E . Immunoblot analysis of U2OS / GFP - FAM111B cell lines treated or not with DOX in the absence or presence of Z - VAD - FMK .

E. Immunoblot analysis of U2OS/GFP-FAM111B cell lines treated or not with DOX in the absence or presence of Z-VAD-FMK.


F . Cells in ( E ) were pulse - labeled with EdU , stained with DAPI and analyzed for DAPI and EdU signal intensity using QIBC .

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F. Cells in (E) were pulse-labeled with EdU, stained with DAPI and analyzed for DAPI and EdU signal intensity using QIBC.


Cells analyzed using QIBC . G . Quantification of data in ( F ) for S phase ( EdU - positive ) cells ( red bars , mean ( A . U . , arbitrary units ) ; n > 2000 cells per condition ) .

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Cells analyzed using QIBC. G. Quantification of data in (F) for S phase (EdU-positive) cells (red bars, mean (A.U., arbitrary units); n>2000 cells per condition).


Cells stained with DAPI and analyzed for DAPI signal intensity using QIBC . H . cells were pulse - labeled with EU .

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Cells stained with DAPI and analyzed for DAPI signal intensity using QIBC. H. cells were pulse-labeled with EU.


Cells analyzed using QIBC . I . Quantification of EU incorporation in cells in ( H ) ( red bars , mean ; n > 2000 cells per condition ) .

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Cells analyzed using QIBC. I. Quantification of EU incorporation in cells in (H) (red bars, mean; n>2000 cells per condition).


J . U2OS / GFP - FAM111B cell lines ( Figure 5E ) were fixed at the indicated times after DOX treatment and stained with crystal violet .

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J. U2OS/GFP-FAM111B cell lines (Figure 5E) were fixed at the indicated times after DOX treatment and stained with crystal violet.


K . Cells in ( E ) were fixed , immunostained with γ - H2AX antibody and subjected to QIBC analysis of γ - H2AX signal intensity ( red bars , mean ; n > 2000 cells per condition ) .

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K. Cells in (E) were fixed, immunostained with γ-H2AX antibody and subjected to QIBC analysis of γ-H2AX signal intensity (red bars, mean; n>2000 cells per condition).


L . Immunoblot analysis of purified recombinant FLAG - FAM111B proteins and their auto - cleavage products .

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L. Immunoblot analysis of purified recombinant FLAG-FAM111B proteins and their auto-cleavage products.


C . Aliquots of the supernatants of the indicated HEK293T transfectants ( all expressing λ chains ) were resolved under non - reducing conditions and blotted with anti - µ antibodies . Wild type µs chains are secreted only as pentamers and hexamers , whilst mutants lacking C575 release mostly µs2L2 subunits ( indicated with a red arrow ) as does the double C414A - C575A . L566A releases instead mainly µs2L2 subunits of faster migration ( blue arrow ) . C414 mutants release no hexamers , but smaller assemblies , including fast migrating µs2L2 subunits .

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C. Aliquots of the supernatants of the indicated HEK293T transfectants (all expressing λ chains) were resolved under non-reducing conditions and blotted with anti-µ antibodies. Wild type µs chains are secreted only as pentamers and hexamers, whilst mutants lacking C575 release mostly µs2L2 subunits (indicated with a red arrow) as does the double C414A-C575A. L566A releases instead mainly µs2L2 subunits of faster migration (blue arrow). C414 mutants release no hexamers, but smaller assemblies, including fast migrating µs2L2 subunits.


E . Aliquots of the supernatants were treated with or without Endo - H and electrophoresed under reducing conditions . The blot was decorated with anti - µ antibodies . N563 - glycans acquire Endo - H resistance in µsL566A and µsC575A , but remain largely sensitive in wild type µs .

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E. Aliquots of the supernatants were treated with or without Endo-H and electrophoresed under reducing conditions. The blot was decorated with anti-µ antibodies. N563-glycans acquire Endo-H resistance in µsL566A and µsC575A, but remain largely sensitive in wild type µs.


A . Aliquots of the lysates from J558L transfectants expressing wild type µs or µsC575A chains were resolved under non - reducing conditions before or after removal of N - glycans by PNGaseF . Blots were decorated with anti - µ antibodies . Blue and red arrows point to the closed ( oxidized ) and open µs2L2 species respectively .

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A. Aliquots of the lysates from J558L transfectants expressing wild type µs or µsC575A chains were resolved under non-reducing conditions before or after removal of N-glycans by PNGaseF. Blots were decorated with anti-µ antibodies. Blue and red arrows point to the closed (oxidized) and open µs2L2 species respectively.


Intracellular µs2L2 species purified from the murine hybridoma B1 . 8 were subjected to the sequential alkylation and analyzed by mass spectrometry In the MS / MS spectrum Y and B ions are shown in blue and red , respectively .

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Intracellular µs2L2 species purified from the murine hybridoma B1.8 were subjected to the sequential alkylation and analyzed by mass spectrometry In the MS/MS spectrum Y and B ions are shown in blue and red, respectively.


A . I . 29µ + B lymphoma cells were treated with LPS to induce differentiation towards IgM secretion . At the indicated times , cells were lysed in TritonX114 to separate soluble and integral membrane proteins ( aqueous and detergent phases , respectively ) . Aliquots from the two phases were resolved electrophoretically and the blot was decorated by anti - µ antibodies . The blue arrow points at a band likely corresponding to µs2L2C575OX , the intensity of which increases upon LPS stimulation .

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A. I.29µ+ B lymphoma cells were treated with LPS to induce differentiation towards IgM secretion. At the indicated times, cells were lysed in TritonX114 to separate soluble and integral membrane proteins (aqueous and detergent phases, respectively). Aliquots from the two phases were resolved electrophoretically and the blot was decorated by anti-µ antibodies. The blue arrow points at a band likely corresponding to µs2L2C575OX, the intensity of which increases upon LPS stimulation.


C . µ - chains with oxidized C575 bind AMS after reduction and migrate more slowly than those which were reduced at the time of lysis and hence alkylated by NEM .

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C. µ-chains with oxidized C575 bind AMS after reduction and migrate more slowly than those which were reduced at the time of lysis and hence alkylated by NEM.


D . The bar graphs show the levels of C575 and C414 oxidation , expressed as the ratio between IAA and NEM modified peptides .

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D. The bar graphs show the levels of C575 and C414 oxidation, expressed as the ratio between IAA and NEM modified peptides.


ERp44KO HeLa cells expressing Ig - λ chains were transfected with wild type ( µ ) , L566A or C575A mutant µs chains and Halo - ERp44 , or Halo - ERp44 ∆ RDEL , as indicated . Empty vectors ( - or EV ) served as controls . A . Aliquots from the spent media ( Extracellular ) were blotted under non - reducing conditions and decorated with anti - µ antibodies . The blue arrow points at the faster migrating µs2L2 released by cells expressing wild type µs or L566A chains . Expression of HaloERp44 restores C575 - dependent retention , and wild type µs are secreted only as polymers ( black arrow ) . Traces of covalent HaloERp44 - µ2L2 complexes ( black asterisks ) are detected in the media of cells expressing HaloERp44 ∆ RDEL and µs or L566A . The slightly faster mobility of µs2L2 and µsL complexes released by cells expressing ERp44 ∆ RDEL suggest that the latter inhibits processing of the N563 glycan , likely hindering it to the Golgi enzymes upon non - covalent binding . Data information : All membranes were decorated with anti - μ antibodies . Black roundish spots seen in the left panels are artifacts that formed during blotting procedures and signal acquisition .

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ERp44KO HeLa cells expressing Ig-λ chains were transfected with wild type (µ), L566A or C575A mutant µs chains and Halo-ERp44, or Halo-ERp44∆RDEL, as indicated. Empty vectors (- or EV) served as controls. A. Aliquots from the spent media (Extracellular) were blotted under non-reducing conditions and decorated with anti-µ antibodies. The blue arrow points at the faster migrating µs2L2 released by cells expressing wild type µs or L566A chains . Expression of HaloERp44 restores C575-dependent retention, and wild type µs are secreted only as polymers (black arrow). Traces of covalent HaloERp44-µ2L2 complexes (black asterisks) are detected in the media of cells expressing HaloERp44∆RDEL and µs or L566A. The slightly faster mobility of µs2L2 and µsL complexes released by cells expressing ERp44∆RDEL suggest that the latter inhibits processing of the N563 glycan, likely hindering it to the Golgi enzymes upon non-covalent binding. Data information: All membranes were decorated with anti-μ antibodies. Black roundish spots seen in the left panels are artifacts that formed during blotting procedures and signal acquisition.


ERp44KO HeLa cells expressing Ig - λ chains were transfected with wild type ( µ ) , L566A or C575A mutant µs chains and Halo - ERp44 , or Halo - ERp44 ∆ RDEL , as indicated . Empty vectors ( - or EV ) served as controls . B . The corresponding cell lysates are shown in this panel . The short black arrow points at the polymers formed in ERp44KO HeLa cells upon expression of wild type HaloERp44 . The black asterisks indicate covalent complexes that this transgene forms with C575 containing µ2L2 . Traces of intracellular µs2L2 - ERp44 ∆ RDEL complexes are visible in µs and L566A but absent from C575A expressing cells . Data information : All membranes were decorated with anti - μ antibodies . Black roundish spots seen in the left panels are artifacts that formed during blotting procedures and signal acquisition .

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ERp44KO HeLa cells expressing Ig-λ chains were transfected with wild type (µ), L566A or C575A mutant µs chains and Halo-ERp44, or Halo-ERp44∆RDEL, as indicated. Empty vectors (- or EV) served as controls. B. The corresponding cell lysates are shown in this panel. The short black arrow points at the polymers formed in ERp44KO HeLa cells upon expression of wild type HaloERp44. The black asterisks indicate covalent complexes that this transgene forms with C575 containing µ2L2. Traces of intracellular µs2L2-ERp44∆RDEL complexes are visible in µs and L566A but absent from C575A expressing cells. Data information: All membranes were decorated with anti-μ antibodies. Black roundish spots seen in the left panels are artifacts that formed during blotting procedures and signal acquisition.


C . Lysates of the ERp44KO transfectants were precipitated with NP - sepharose , resolved under non reducing conditions and the blot decorated sequentially with anti - µ , anti - ERp44 or anti - Halo . The two black asterisks point at bands corresponding to covalent complexes containing µs2L2 and endogenous or Halo - tagged ERp44 .

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C. Lysates of the ERp44KO transfectants were precipitated with NP-sepharose, resolved under non reducing conditions and the blot decorated sequentially with anti-µ, anti-ERp44 or anti-Halo. The two black asterisks point at bands corresponding to covalent complexes containing µs2L2 and endogenous or Halo-tagged ERp44.


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 .

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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 . 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 .

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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 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 .

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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 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 ) .

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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 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 .

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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 cells and 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 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 ) .

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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 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 .

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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.


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 .

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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.


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 ) .

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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).


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 .

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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.


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 ) .

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 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 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 . 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 ) .

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 O O O O O O O O O O O

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 . 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 .

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 B-MEASURED_VAR I-MEASURED_VAR 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

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 levels ( mg / dL ) of mice after fasted for 24 hours ( n = 12 or 14 / 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

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


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

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


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


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 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 O O O O O O O O O O O O O O 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 O 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 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. 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 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

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 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 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. 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 O O O O O O O O O O O O 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. 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 O O O O O O O O O O O O O O O O O O O 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 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 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 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 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 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 ) .

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 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. (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 ) .

O O O O O O O O O O O O O O O O O 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 ) .

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 O 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 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

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 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

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

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

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 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 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 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 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 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

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 O O O O O O O O O O O O O O O O O O O O O O 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 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 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 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 O O O O O O O O O O O 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 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 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 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 O O O O O O O O O O O O O O O O O O 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. (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 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

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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. (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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O

(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 .

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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 ) 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 ) .

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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 ) 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.