( A ) GFP - PAQR3 transfected HeLa were fixed for immunofluorescence with indicated antibodies before or after glucose starvation ( GS ) for 4 h . nuclei were stained with Hoechst 33342 . Scale bar : 10 μm .

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(A) GFP-PAQR3 transfected HeLa cells were fixed for immunofluorescence staining with indicated antibodies before or after glucose starvation (GS) for 4 h. The nuclei were stained with Hoechst 33342. Scale bar: 10 μm.


( B ) Immunoblotting ( IB ) of wild type ( WT ) PAQR3 - deficient MEFs after GS for different time as indicated .

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(B) Immunoblotting (IB) analysis of wild type (WT) and PAQR3-deficient MEFs after GS for different time as indicated.


( C ) WT PAQR3 - deficient MEFs were treated with 80 mM chloroquine ( CQ ) for 2 h to induce LC3 - II accumulation . Whole - lysates were harvested for IB of LC3 - II .

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(C) WT and PAQR3-deficient MEFs were treated with 80 mM chloroquine (CQ) for 2 h to induce LC3-II accumulation. Whole-cell lysates were harvested for IB analysis of LC3-II.


( D ) Autophagosome - like structures in WT PAQR3 - deleted MEFs were detected after GS for 4 h by transmission electron microscopy . * indicates autophagosome - like structures . N , nucleus . ( E ) Quantitative of autophagic vacuoles in transmission electron microscopy . Thirty were quantified from each independent experiment ， which was repeated for three times with similar results . Values are presented as mean ± SD , * * * p < 0 . 001 .

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(D) Autophagosome-like structures in WT and PAQR3-deleted MEFs were detected after GS for 4 h by transmission electron microscopy. * indicates autophagosome-like structures. N, nucleus.(E) Quantitative analysis of autophagic vacuoles in transmission electron microscopy images. Thirty cells were quantified from each independent experiment，which was repeated for three times with similar results. Values are presented as mean ± SD, ***p < 0.001.


( F ) Immunofluorescence of LC3 in WT or PAQR3 - deficient MEFs after GS for 4 h . arrowhead indicates representative LC3 - positive . Scale bar : 10 μm .

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(F) Immunofluorescence staining of LC3 in WT or PAQR3-deficient MEFs after GS for 4 h. The arrowhead indicates representative LC3-positive puncta. Scale bar: 10 μm.


( G , H ) HeLa were infected by PAQR3 knockdown or overexpression lentivirus together with their respective control . After GS for 1 h , lysates were harvested for IB .

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(G, H) HeLa cells were infected by PAQR3 knockdown or overexpression lentivirus together with their respective control virus. After GS for 1 h, the cell lysates were harvested for IB.


( A ~ C ) HeLa stably expressing GFP - DFCP1 were transfected with or without PAQR3 - specific shRNA . GFP signals were observed in these before or after glucose starvation ( GS , shown in A ) . knockdown efficiency of PAQR3 is shown in B statistics of GFP - positive per is illustrated in C . Values are presented as mean ± SD , * * * P < 0 . 001 . Scale bar : 10 μm .

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(A~C) HeLa cells stably expressing GFP-DFCP1 were transfected with or without PAQR3-specific shRNA. GFP signals were observed in these cells before or after glucose starvation (GS, shown in A). The knockdown efficiency of PAQR3 is shown in B and the statistics of GFP-positive puncta per cell is illustrated in C. Values are presented as mean ± SD, *** P < 0.001. Scale bar: 10 μm.


( D ) GFP - WIPI1 was transfected into WT or PAQR3 - deficient MEFs . 24 h later , were incubated under GS for 4 h , followed by observation . Scale bar : 10 μm .

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(D) GFP-WIPI1 was transfected into WT or PAQR3-deficient MEFs. 24 h later, the cells were incubated under GS for 4 h, followed by fluorescence observation. Scale bar: 10 μm.


( E ) Different VPS34 complexes of WT or PAQR3 knockout HeLa were immunoprecipitated by ATG14L UVRAG antibodies respectively in normal medium ( NM ) or GS . Then PI ( 3 ) P level was determined by a quantitative ELISA . PI ( 3 ) P level was normalized to amount of ATG14L or UVRAG used in ( n = 5 ; * * * p < 0 . 001 ; ns , not significant ) .

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(E) Different VPS34 complexes of WT or PAQR3 knockout HeLa cells were immunoprecipitated by ATG14L and UVRAG antibodies respectively in normal medium (NM) or GS. Then PI(3)P level was determined by a quantitative ELISA assay. The PI(3)P level was normalized to the amount of ATG14L or UVRAG used in the assay (n = 5; ***p < 0.001; ns, not significant).


( F ) HeLa infected with control or PAQR3 - overexpressed lentivirus were treated with GS for 4 h . PI ( 3 ) P were determined as in Figure 2E ( n = 5 ; * * p < 0 . 01 ; ns , not significant ) .

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(F) HeLa cells infected with control or PAQR3-overexpressed lentivirus were treated with GS for 4 h. PI(3)P levels were determined as in Figure 2E(n = 5; **p < 0.01; ns, not significant).


( G , H ) PAQR3 knockout HeLa were transfected with plasmids of multiple PAQR3 deletion mutants . At 24 h after transfection , lysates were used to immunoprecipitate VPS34 by ATG14L antibody , followed by immunoblotting . activity of ATG14L - linked VPS34 activity was detected by a quantitative ELISA . PI ( 3 ) P level was normalized to amount of ATG14L ( n = 5 ; * * p < 0 . 01 ) .

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(G, H) PAQR3 knockout HeLa cells were transfected with plasmids of multiple PAQR3 deletion mutants. At 24 h after the transfection, the cell lysates were used to immunoprecipitate VPS34 complex by ATG14L antibody, followed by immunoblotting. The activity of ATG14L-linked VPS34 activity was detected by a quantitative ELISA assay. The PI(3)P level was normalized to the amount of ATG14L (n = 5; **p < 0.01).


( A ) Interaction between endogenous PAQR3 components of VPS34 in HEK293T . lysates were used for immunoprecipitation ( IP ) immunoblotting ( IB ) with antibodies as indicated .

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(A) Interaction between endogenous PAQR3 and the components of VPS34 complex in HEK293T cells. The cell lysates were used for immunoprecipitation (IP) and immunoblotting (IB) with the antibodies as indicated.


( B ) Cytosol Golgi fractionations were isolated from livers of PAQR3 - deleted mice their littermate controls . Equal amounts of cytosol Golgi fractions were subjected to IB .

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(B) Cytosol and Golgi fractionations were isolated from the livers of PAQR3-deleted mice and their littermate controls. Equal protein amounts of cytosol and Golgi fractions were subjected to IB.


( C ) HEK293T were transiently transfected with Myc - tagged PAQR3 . At 24 h after transfection , were treated with normal medium ( NM ) ， acid starvation ( AS ) or glucose starvation ( GS ) , HBSS solution , or rapamycin ( Rapa , 50 nM ) for 4 h respectively . lysates were then used in IP IB .

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(C) HEK293T cells were transiently transfected with Myc-tagged PAQR3. At 24 h after transfection, the cells were treated with normal medium (NM)，amino acid starvation (AS) or glucose starvation (GS), HBSS solution, or rapamycin (Rapa, 50 nM) for 4 h respectively. The cell lysates were then used in IP and IB.


( D ) Four VPS34 complexes of MEFs were immunopurified using indicated antibodies under NM , AS or GS conditions . relative abundance of VPS34 - binding partners was determined by IB . Each IP was normalized to amount of VPS34 .

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(D) Four VPS34 complexes of MEFs were immunopurified using the indicated antibodies under NM, AS or GS conditions. The relative abundance of VPS34-binding partners was determined by IB. Each IP was normalized to the amount of VPS34.


( E ) HEK293T were transfected with plasmids as indicated lysates were used in IP IB .

(E) HEK293T cells were transfected with the plasmids as indicated and the cell lysates were used in IP and IB.


( F ) lysates of WT or PAQR3 knockout MEFs were used in IP IB .

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(F) The lysates of WT or PAQR3 knockout MEFs were used in IP and IB.


( G ) MEFs were infected with control or PAQR3 - expressing lentivirus . Then a quantitative immunodepletion was performed with increasing amounts of indicated antibodies . Supernatants of MEF lysates after immunodepletion ( Post - IP ) were examined to determine level of VPS34 .

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(G) MEFs were infected with control or PAQR3-expressing lentivirus. Then a quantitative immunodepletion assay was performed with increasing amounts of the indicated antibodies. Supernatants of MEF lysates after immunodepletion (Post-IP) were examined to determine the level of VPS34 complex proteins.


( A ) A two - step co - IP in HEK293T to determine ternary containing PAQR3 , Beclin1 ATG14L . procedures of two - step co - IP are outlined in top . samples were subjected to immunoprecipitation ( IP ) immunoblotting ( IB ) with antibodies as indicated .

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(A) A two-step co-IP assay in HEK293T cells to determine the ternary complex containing PAQR3, Beclin1 and ATG14L. The procedures of the two-step co-IP are outlined in the top. The samples were subjected to immunoprecipitation (IP) and immunoblotting (IB) with the antibodies as indicated.


( B ) HeLa were transiently transfected with plasmids as indicated . 24 h after transfection , were treated with or without glucose starvation ( GS ) for 4 h . lysates were used in IP IB .

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(B) HeLa cells were transiently transfected with the plasmids as indicated. 24 h after transfection, the cells were treated with or without glucose starvation (GS) for 4 h. The cell lysates were used in IP and IB.


( C , D ) Different truncations of ATG14L ( C ) were co - transfected with Myc - tagged PAQR3 into HEK293T as indicated , followed by IP IB .

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(C, D) Different truncations of ATG14L (C) were co-transfected with Myc-tagged PAQR3 into HEK293T cells as indicated, followed by IP and IB.


( C , D ) Different truncations of Beclin1 ( D ) were co - transfected with Myc - tagged PAQR3 into HEK293T as indicated , followed by IP IB .

(C, D) Different truncations of Beclin1 (D) were co-transfected with Myc-tagged PAQR3 into HEK293T cells as indicated, followed by IP and IB.


( E ) Purified GST or GST - fused PAQR3 ( 1 - 71 ) ( GST fused NH2 - terminal 71aa of PAQR3 ) on glutathione agarose beads was mixed with purified His tagged as indicated . After incubation at 4 ° C for 3 h , samples were washed extensively subjected to IB .

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(E) Purified GST or GST-fused PAQR3 (1-71) (GST fused NH2-terminal 71aa of PAQR3) on glutathione agarose beads was mixed with purified His tagged proteins as indicated. After incubation at 4 °C for 3 h, the samples were washed extensively and subjected to IB.


( F , G ) Different PAQR3 deletion constructs were co - transfected with Flag - tagged Beclin1 ( F ) into HEK293T . were then subjected to IP IB .

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(F, G) Different PAQR3 deletion constructs were co-transfected with Flag-tagged Beclin1 (F) into HEK293T cells. The cells were then subjected to IP and IB.


( F , G ) Different PAQR3 deletion constructs were co - transfected with GFP - fused ATG14L ( G ) into HEK293T . were then subjected to IP IB .

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(F, G) Different PAQR3 deletion constructs were co-transfected with GFP-fused ATG14L (G) into HEK293T cells. The cells were then subjected to IP and IB.


( A ) HeLa were cultured in normal medium ( NM ) or glucose starvation ( GS ) for 4 h . starved lysates were treated with calf intestinal phosphatase ( CIP ) to remove phosphate groups analyzed by immunoblotting ( IB ) with Phos - tag gel or regular SDS - PAGE .

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(A) HeLa cells were cultured in normal medium (NM) or glucose starvation (GS) for 4 h. The starved lysates were treated with calf intestinal phosphatase (CIP) to remove phosphate groups and analyzed by immunoblotting (IB) with Phos-tag gel or regular SDS-PAGE.


( B ) lysates from HeLa were collected analyzed after GS for indicated time ( lane 1 ~ 5 ) , or GS for 2 h followed by 2 h chase in NM ( lane 6 ) . lysates were subjected to Phos - tag gel regular SDS PAGE respectively .

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(B) The cell lysates from HeLa cells were collected and analyzed after GS for indicated time (lane 1~5), or GS for 2 h followed by 2 h chase in NM (lane 6). The cell lysates were subjected to Phos-tag gel and regular SDS PAGE respectively.


( C ) WT or AMPKa1 / 2 double knockout MEFs were incubated under NM or GS for 4 h , followed by IB with Phos - tag gel or regular SDS - PAGE .

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(C) WT or AMPKa1/2 double knockout MEFs were incubated under NM or GS for 4 h, followed by IB with Phos-tag gel or regular SDS-PAGE.


( D ) HeLa were incubated with compound C ( 20 mM , 30 min ) with or without GS , followed by IB with Phos - tag gel or regular SDS - PAGE .

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(D) HeLa cells were incubated with compound C (20 mM, 30 min) with or without GS, followed by IB with Phos-tag gel or regular SDS-PAGE.


( E ) purified His - tagged NH2 - terminal 71 acid of PAQR3 was incubated with or without purified Flag - tagged ATG14L . Then complexes were treated with WT AMPK or DN - AMPK ( dominant negative AMPK ) for indicated time in vitro . PAQR3 phosphorylation was examined by Phos - tag gel .

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(E) Bacterial purified His-tagged NH2-terminal 71 amino acid of PAQR3 was incubated with or without purified Flag-tagged ATG14L. Then the complexes were treated with WT AMPK or DN-AMPK (dominant negative AMPK) for the indicated time in vitro. PAQR3 phosphorylation was examined by Phos-tag gel.


( F ) HeLa infected with control or ATG14L - expressing lentivirus were incubated under GS for different time as indicated . Whole lysates were analyzed by both Phos - tag gel regular SDS - PAGE .

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(F) HeLa cells infected with control or ATG14L-expressing lentivirus were incubated under GS for different time as indicated. Whole cell lysates were analyzed by both Phos-tag gel and regular SDS-PAGE.


( G ) HeLa were infected by lentivirus expressing control shRNA or ATG14L - specific shRNA . Then ATG14L knockdown were stably re - expressed with shRNA - resistant ATG14L ( rATG14L ) or Beclin1 incubated under NM or GS for 4 h . Whole lysates were analyzed by both Phos - tag gel regular SDS - PAGE .

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(G) HeLa cells were infected by lentivirus expressing control shRNA or ATG14L-specific shRNA. Then the ATG14L knockdown cells were stably re-expressed with shRNA-resistant ATG14L (rATG14L) or Beclin1 and incubated under NM or GS for 4 h. Whole cell lysates were analyzed by both Phos-tag gel and regular SDS-PAGE.


( A ) purified His - tagged WT or T32A NH2 - terminal 71aa of PAQR3 was incubated with or without Flag - tagged ATG14L purified from HEK293T . Then complexes were incubated with AMPK for indicated time subjected to Phos - tag gel or regular SDS - PAGE .

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(A) Bacterial purified His-tagged WT or T32A NH2-terminal 71aa of PAQR3 was incubated with or without Flag-tagged ATG14L purified from HEK293T cells. Then the complexes were incubated with AMPK for the indicated time and subjected to Phos-tag gel or regular SDS-PAGE.


( B ) PAQR3 - deficient HeLa were transfected with WT or T32A mutants of PAQR3 plasmids . 24 h later , were incubated with normal medium ( NM ) or glucose starvation ( GS ) for 4 h , followed by immunoblotting ( IB ) in Phos - tag gel or regular SDS - PAGE .

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(B) PAQR3-deficient HeLa cells were transfected with WT or T32A mutants of PAQR3 plasmids. 24 h later, the cells were incubated with normal medium (NM) or glucose starvation (GS) for 4 h, followed by immunoblotting (IB) in Phos-tag gel or regular SDS-PAGE.


( C ) HeLa were incubated with NM or GS for 1 h or treated with compound C ( C . C , 20 mM , 30 min ) prior to GS for 1 h . In parallel , 25 mM 2 - DG or 1 mM AICAR was added in NM for 1 h . lysates were analyzed by IB .

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(C) HeLa Cells were incubated with NM or GS for 1 h or treated with compound C (C.C, 20 mM, 30 min) prior to GS for 1 h. In parallel, 25 mM 2-DG or 1 mM AICAR was added in NM for 1 h. Cell lysates were analyzed by IB.


( D ) HEK293T were transfected with Myc - tagged PAQR3 . 24 h after transfection , were treated with GS for 4 h . Then lysates were used in IB immunoprecipitation ( IP ) with antibodies as indicated . immunoprecipitates were treated with CIP to remove phosphate groups analyzed by IB in Phos - tag gel or regular SDS - PAGE .

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(D) HEK293T cells were transfected with Myc-tagged PAQR3. 24 h after transfection, the cells were treated with GS for 4 h. Then the cell lysates were used in IB and immunoprecipitation (IP) with the antibodies as indicated. The immunoprecipitates were treated with CIP to remove phosphate groups and analyzed by IB in Phos-tag gel or regular SDS-PAGE.


( E ) WT , phosphorylation defective ( T32A ) , or phospho - mimetic ( T32E ) PAQR3 were transfected in presence or absence of constitutively active AMPK ( CA - AMPK ) into PAQR3 - deficient HeLa as indicated . 24 h later , without AMPK transfection were treated with GS for 4 h . 10 % of lysates were subjected to IB to detect PAQR3 expression level ( shown in Appendix Fig S4A ) , remaining lysates were used to immunoprecipitate VPS34 complexes by ATG14L antibody , followed by VPS34 activity measurement by a quantitative PI ( 3 ) P ELISA . PI ( 3 ) P level was normalized to amount of ATG14L ( n = 5 ; * p < 0 . 05 ; * * p < 0 . 01 as compared to first group ) .

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(E) WT, phosphorylation defective (T32A), or phospho-mimetic (T32E) PAQR3 were transfected in the presence or absence of constitutively active AMPK (CA-AMPK) into PAQR3-deficient HeLa cells as indicated. 24 h later, the cells without AMPK transfection were treated with GS for 4 h. 10% of the cell lysates were subjected to IB to detect PAQR3 expression level (shown in Appendix Fig S4A), the remaining cell lysates were used to immunoprecipitate VPS34 complexes by ATG14L antibody, followed by VPS34 activity measurement by a quantitative PI(3)P ELISA assay. The PI(3)P level was normalized to the amount of ATG14L (n = 5; *p < 0.05; **p < 0.01 as compared to the first group).


( F ) PAQR3 - deficient HeLa were infected with lentivirus expressing WT or T32A PAQR3 . Then PAQR3 expression were examined by IB .

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(F) PAQR3-deficient HeLa cells were infected with lentivirus expressing WT or T32A PAQR3. Then PAQR3 expression levels were examined by IB.


( G ) Both WT HeLa PAQR3 knockout HeLa infected with lentivirus expressing WT or T32A PAQR3 were treated with GS for 2 h or 4 h respectively . lysates were analyzed by IB .

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(G) Both WT HeLa cells and PAQR3 knockout HeLa cells infected with lentivirus expressing WT or T32A PAQR3 were treated with GS for 2 h or 4 h respectively. Cell lysates were analyzed by IB.


( A , B ) Representative immunohistochemical of LC3 in skeletal muscle ( A ) liver ( B ) after 80 min exercise in WT PAQR3 - deleted mice . Scale bar in ( A ) : 20 μm . Scale bar in ( B ) : 50 μm .

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(A, B) Representative immunohistochemical images of LC3 staining in skeletal muscle (A) and liver (B) after 80 min exercise in WT and PAQR3-deleted mice. Scale bar in (A): 20 μm. Scale bar in (B): 50 μm.


( C , D ) Immunoblotting of skeletal muscle ( C ) liver ( D ) from WT or PAQR3 knockout mice at or after 80 min exercise .

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(C, D) Immunoblotting analysis of skeletal muscle (C) and liver (D) from WT or PAQR3 knockout mice at rest or after 80 min exercise.


( E ) Abnormal limb - clasping of PAQR3 knockout mice when suspended by its tail . scoring of limb clasping phenotype is shown on right ( n = 7 for each group ; * * p < 0 . 01 ) .

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(E) Abnormal limb-clasping of PAQR3 knockout mice when suspended by its tail. Quantification scoring of the limb clasping phenotype is shown on the right (n = 7 for each group; **p < 0.01).


( F ) Motor performance was measured using an accelerating rotarod apparatus , time of each mouse spent on rotating rod until it fell off was recorded . Average time on rod for each group is shown ( n = 7 for each group ; * * p < 0 . 01 ) .

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(F) Motor performance was measured using an accelerating rotarod apparatus, and the time of each mouse spent on the rotating rod until it fell off was recorded. Average time on the rod for each group is shown (n = 7 for each group; **p < 0.01).


( G ) Co - immunoprecipitation of Beclin1 or ATG14L with their binding partners in liver tissues from mice of indicated genotype . Immunoprecipitation ( IP ) immunoblotting ( IB ) were performed using antibodies as indicated .

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(G) Co-immunoprecipitation of Beclin1 or ATG14L with their binding partners in liver tissues from mice of indicated genotype. Immunoprecipitation (IP) and immunoblotting (IB) were performed using the antibodies as indicated.


( H ) supernatants of liver lysates form WT or PAQR3 knockout mice were subjected to gel filtration , followed by IB .

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(H) The supernatants of liver lysates form WT or PAQR3 knockout mice were subjected to gel filtration analysis, followed by IB.


( I ) VPS34 complexes immunopurified with Beclin1 or ATG14L antibody in mouse liver were subjected to a quantitative PI ( 3 ) P ELISA ( n = 5 ; * * p < 0 . 01 ) .

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(I) VPS34 complexes immunopurified with Beclin1 or ATG14L antibody in mouse liver were subjected to a quantitative PI(3)P ELISA assay (n = 5; **p < 0.01).


A . Labeling of non - permeabilized ( top ) permeabilized ( bottom ) demonstrate that surface labeling does not label internal . GluA2 labeling ( green ) is visible both on surface internally , while DARPP32 labeling ( red ) is only visible internally .

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A. Labeling of non-permeabilized (top) and permeabilized (bottom) cells demonstrate that surface labeling does not label internal proteins. GluA2 labeling (green) is visible both on the surface and internally, while DARPP32 labeling (red) is only visible internally.


B . Top . Representative of surface GluA2 DARPP32 expression in striatal neurons from co - cultures with either no treatment or 20 min 2APB dantrolene . Bottom . Quantified expression of GluA2 under both conditions , n = 9 replicates of 10 each , Student ' s t .

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B. Top. Representative images of surface GluA2 and DARPP32 expression in striatal neurons from co-cultures with either no treatment or 20 min 2APB and dantrolene. Bottom. Quantified expression of GluA2 under both conditions, n = 9 replicates of 10 cells each, Student's t test.


C . Representative of surface GluA1 DARPP32 expression in striatal neurons from co - cultures with either no treatment , 30 min DHPG , or 2APB dantrolene pretreatment followed by DHPG . D . Quantified expression of GluA1 under each condition , n = 9 replicates of 10 , p < 0 . 001 , ANOVA . E . Representative of surface GluA2 DARPP32 expression in striatal neurons from co - cultures under same conditions as in C . F . Quantified expression of GluA2 under each condition , n = 12 replicates of 10 each , p < 0 . 001 , ANOVA . G . Representative of surface myc - GluA2 , internal myc - GluA2 , DARPP32 expression in striatal neurons from co - cultures under same conditions as in C . H . Quantified expression of surface myc : total myc under each condition , n = 4 replicates of 10 each , p < 0 . 05 , ANOVA . error bars represent s . e . m . Abbr . A1 = GluA1 , A2 = GluA2 , D = DARPP32 , s - myc = surface myc , i - myc = internal myc , m = merge .

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C. Representative images of surface GluA1 and DARPP32 expression in striatal neurons from co-cultures with either no treatment, 30 min DHPG, or 2APB and dantrolene pretreatment followed by DHPG. D. Quantified expression of GluA1 under each condition, n = 9 replicates of 10 cells, p < 0.001, ANOVA. E. Representative images of surface GluA2 and DARPP32 expression in striatal neurons from co-cultures under the same conditions as in C. F. Quantified expression of GluA2 under each condition, n = 12 replicates of 10 cells each, p < 0.001, ANOVA. G. Representative images of surface myc-GluA2, internal myc-GluA2, and DARPP32 expression in striatal neurons from co-cultures under the same conditions as in C. H. Quantified expression of surface myc:total myc under each condition, n = 4 replicates of 10 cells each, p < 0.05, ANOVA. All error bars represent s.e.m. Abbr. A1 = GluA1, A2 = GluA2, D = DARPP32, s-myc = surface myc, i-myc = internal myc, m = merge.


A . Left . Representative of surface GluA2 DARPP32 expression in striatal neurons from co - cultures treated either for 30 min with DHPG ( DHPG ) or for 30 min with DHPG pretreated with Xestospungin C S107 ( XeC / S107 + DHPG ) . Right . Quantified expression of surface GluA2 under each condition , n = 3 replicates of 10 or 20 each , p < 0 . 0001 , Student ' s t .

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A. Left. Representative images of surface GluA2 and DARPP32 expression in striatal neurons from co-cultures treated either for 30 min with DHPG (DHPG) or for 30 min with DHPG and pretreated with Xestospungin C and S107 (XeC/S107 + DHPG). Right. Quantified expression of surface GluA2 under each condition, n = 3 replicates of 10 or 20 cells each, p < 0.0001, Student's t test.


B . Left . Representative of surface GluA2 DARPP32 expression in striatal neurons from co - cultures treated either for 30 min with DHPG ( DHPG ) , or for 30 min with DHPG pretreated with S107 ( S107 + DHPG ) , or for 30 min with DHPG pretreated with Xestospungin C ( XeC + DHPG ) . Right . Quantified expression of surface GluA2 under each condition , n = 4 replicates of 10 each , ANOVA .

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B. Left. Representative images of surface GluA2 and DARPP32 expression in striatal neurons from co-cultures treated either for 30 min with DHPG (DHPG), or for 30 min with DHPG and pretreated with S107 (S107 + DHPG), or for 30 min with DHPG and pretreated with Xestospungin C (XeC + DHPG). Right. Quantified expression of surface GluA2 under each condition, n = 4 replicates of 10 cells each, ANOVA.


C . Top . Representative of surface GluA2 DARPP32 expression in striatal neurons from co - cultures under control or 10 min Thapsigargin ( TG ) treatment . Bottom . Quantified expression of surface GluA2 under each condition , n = 3 replicates of 10 each , Student ' s t . D . Top . Representative of surface GluA2 DARPP32 expression in striatal neurons from co - cultures with treatment with either no DHPG or with DHPG for 30 min , after 15 min of Thapsigargin . Bottom . Quantified expression of surface GluA2 under each condition , n = 4 replicates of 10 each , Student ' s t . E . Top . Representative of surface GluA2 DARPP32 expression in striatal neurons from co - cultures with either no DHPG or with DHPG for 30 min , after 5 min of BAPTA . Bottom . Quantified expression of GluA2 under each condition , n = 3 replicates of 10 each , p < 0 . 001 , Student ' s t . F . Top . Representative of surface GluA2 DARPP32 expression in striatal neurons from co - cultures with either no DHPG or with DHPG for 30 min after 5 min of BAPTA - AM . Bottom . Quantified expression of GluA2 under each condition , n = 3 replicates of 10 each , Student ' s t . error bars represent s . e . m .

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C. Top. Representative images of surface GluA2 and DARPP32 expression in striatal neurons from co-cultures under control or 10 min Thapsigargin (TG) treatment. Bottom. Quantified expression of surface GluA2 under each condition, n = 3 replicates of 10 cells each, Student's t test. D. Top. Representative images of surface GluA2 and DARPP32 expression in striatal neurons from co-cultures with treatment with either no DHPG or with DHPG for 30 min, after 15 min of Thapsigargin. Bottom. Quantified expression of surface GluA2 under each condition, n = 4 replicates of 10 cells each, Student's t test. E. Top. Representative images of surface GluA2 and DARPP32 expression in striatal neurons from co-cultures with either no DHPG or with DHPG for 30 min, after 5 min of BAPTA. Bottom. Quantified expression of GluA2 under each condition, n = 3 replicates of 10 cells each, p < 0.001, Student's t test. F. Top. Representative images of surface GluA2 and DARPP32 expression in striatal neurons from co-cultures with either no DHPG or with DHPG for 30 min after 5 min of BAPTA-AM. Bottom. Quantified expression of GluA2 under each condition, n = 3 replicates of 10 cells each, Student's t test. All error bars represent s.e.m.


A . Top . Representative image of a radiograph of mature immature bands of GluA2 after no treatment , DHPG , or 2APB / dan followed by DHPG incubated with Endo H . Bottom . Quantified graph of of mature , endo H insensitive GluA2 band to total GluA2 for each condition . For DHPG , n = 16 ; for DHPG + 2APB / dan , n = 11 , p < 0 . 05 , ANOVA .

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A. Top. Representative image of a radiograph of mature and immature bands of GluA2 after no treatment, DHPG, or 2APB/dan followed by DHPG and incubated with Endo H. Bottom. Quantified graph of the ratio of the mature, endo H insensitive GluA2 band to the total GluA2 for each condition. For DHPG, n = 16; for DHPG + 2APB/dan, n = 11, p < 0.05, ANOVA.


B . Top . Representative image of a radiograph of mature immature bands of GluA1 after no treatment , DHPG , or 2APB / dan followed by DHPG incubated with Endo H . Bottom . Quantified graph of of mature , endo H insensitive GluA1 band to total GluA1 for each condition , For DHPG , n = 4 ; for DHPG + 2APB / dan , n = 4 , ANOVA .

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B. Top. Representative image of a radiograph of mature and immature bands of GluA1 after no treatment, DHPG, or 2APB/dan followed by DHPG and incubated with Endo H. Bottom. Quantified graph of the ratio of the mature, endo H insensitive GluA1 band to the total GluA1 for each condition, For DHPG, n = 4; for DHPG + 2APB/dan, n = 4, ANOVA.


C . Representative of surface GluA2 DARPP32 expression in striatal neurons from co - cultures with either no DHPG or 30 min DHPG after 15 min of brefeldin A treatment . D . Quantified expression of surface GluA2 under each condition , n = 3 replicates of 10 each , Student ' s t . error bars represent s . e . m .

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C. Representative images of surface GluA2 and DARPP32 expression in striatal neurons from co-cultures with either no DHPG or 30 min DHPG after 15 min of brefeldin A treatment. D. Quantified expression of surface GluA2 under each condition, n = 3 replicates of 10 cells each, Student's t test. All error bars represent s.e.m.


A Top . Representative immunoblots from GluA2 coimmunoprecipitations probed for Sec23 GluA2 after no treatment , 10 min DHPG , 2APB / dantrolene followed by 10 min DHPG . Bottom . of Sec23 normalized to GluA2 under each of these conditions . For 10 min DHPG , n = 8 ; for 10 min DHPG + 2APB / dan , n = 6 , p < 0 . 05 , ANOVA .

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A Top. Representative immunoblots from GluA2 coimmunoprecipitations probed for Sec23 and GluA2 after no treatment, 10 min DHPG, and 2APB/dantrolene followed by 10 min DHPG. Bottom. Quantification of Sec23 normalized to GluA2 under each of these conditions. For 10 min DHPG, n = 8; for 10 min DHPG + 2APB/dan, n = 6, p < 0.05, ANOVA.


B . Top . Representative immunoblots from GluA2 coimmunoprecipitations probed for Sec23 GluA2 after no treatment , 10 min DHPG , cycloheximide followed by 10 min DHPG . Bottom . of Sec23 normalized to GluA2 under each of these conditions , For 10 min DHPG , n = 8 ; for 10 min DHPG + CHX , n = 5 , p < 0 . 05 , ANOVA .

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B. Top. Representative immunoblots from GluA2 coimmunoprecipitations probed for Sec23 and GluA2 after no treatment, 10 min DHPG, and and cycloheximide followed by 10 min DHPG. Bottom. Quantification of Sec23 normalized to GluA2 under each of these conditions, For 10 min DHPG, n = 8; for 10 min DHPG + CHX, n = 5, p < 0.05, ANOVA.


C . Top . Representative immunoblots from GluA2 co - IPs probed for Sec23 GluA2 after no treatment , 30 min DHPG , 2APB / dantrolene followed by 30 min DHPG . Bottom . of Sec23 normalized to GluA2 under each of these conditions . For 30 min DHPG , n = 17 ; For 30 min DHPG + 2APB / dan , n = 14 , p < 0 . 05 , ANOVA .

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C. Top. Representative immunoblots from GluA2 co-IPs probed for Sec23 and GluA2 after no treatment, 30 min DHPG, and 2APB/dantrolene followed by 30 min DHPG. Bottom. Quantification of Sec23 normalized to GluA2 under each of these conditions. For 30 min DHPG, n = 17; For 30 min DHPG + 2APB/dan, n = 14, p < 0.05, ANOVA.


D . Top . Representative immunoblots from GluA2 coimmunoprecipitations probed for Sec23 GluA2 after no treatment , 30 min DHPG , cycloheximide followed by 30 min DHPG . Bottom . of Sec23 normalized to GluA2 under each of these conditions . For 30 min DHPG , n = 12 ; For 30 min DHPG + CHX , n = 11 p < 0 . 01 , ANOVA .

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D. Top. Representative immunoblots from GluA2 coimmunoprecipitations probed for Sec23 and GluA2 after no treatment, 30 min DHPG, and cycloheximide followed by 30 min DHPG. Bottom. Quantification of Sec23 normalized to GluA2 under each of these conditions. For 30 min DHPG, n = 12; For 30 min DHPG + CHX, n = 11 p < 0.01, ANOVA.


E . Top . Representative immunoblot demonstrating IgG GluA2 coIP after no treatment 30 min DHPG . Bottom . of Sec23 after subtracting non - specific signal , n = 3 , p < 0 . 01 , Student ' s t .

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E. Top. Representative immunoblot demonstrating IgG and GluA2 coIP after no treatment and 30 min DHPG. Bottom. Quantification of Sec23 after subtracting the non-specific signal, n = 3, p < 0.01, Student's t test.


F . Representative immunoblot of a control coIP that demonstrates specificity of GluA2 coIP . error bars represent s . e . m .

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F. Representative immunoblot of a control coIP that demonstrates the specificity of the GluA2 coIP. All error bars represent s.e.m.


A . Representative immunoblots from GluA2 coIP probed for Sec23 , Sec13 , GluA2 after no DHPG , 10 , 30 , 60 minutes of treatment with DHPG . B . Graph depicting kinetics of association between GluA2 Sec23 as well as GluA2 Sec13 after treatment with DHPG for same points in time as in A . For Sec23 , 10 min DHPG , n = 16 ; 30 min DHPG , n = 15 ; 60 min DHPG , n = 7 , p < 0 . 0001 , ANOVA . For Sec13 , 10 min DHPG , n = 7 ; 30 min DHPG , n = 14 ; 60 min DHPG , n = 6 , p < 0 . 01 , ANOVA .

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A. Representative immunoblots from GluA2 coIP probed for Sec23, Sec13, and GluA2 after no DHPG, 10, 30, and 60 minutes of treatment with DHPG. B. Graph depicting the kinetics of the association between GluA2 and Sec23 as well as GluA2 and Sec13 after treatment with DHPG for the same points in time as in A. For Sec23, 10 min DHPG, n = 16; 30 min DHPG, n = 15; 60 min DHPG, n = 7, p < 0.0001, ANOVA. For Sec13, 10 min DHPG, n = 7; 30 min DHPG, n = 14; 60 min DHPG, n = 6, p < 0.01, ANOVA.


C . Representative of total expression of Sec23 ( top ) Sec13 ( bottom ) . Expression do not change in presence of DHPG . error bars represent s . e . m .

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C. Representative images of total expression levels of Sec23 (top) and Sec13 (bottom). Expression levels do not change in the presence of DHPG. All error bars represent s.e.m.


A . traces of mEPSCs of both control naspm treated neurons after TTX , TTX + DHPG , TTX + DHPG + 2APB / dan treatment .

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A. Individual traces of mEPSCs of both control and naspm treated neurons after TTX, TTX + DHPG, and TTX + DHPG + 2APB/dan treatment.


B . Averaged mEPSCs an enlarged time scale from under same conditions as A . Traces from that were not treated with naspm are in black naspm treated traces are in grey .

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B. Averaged mEPSCs and an enlarged time scale from cells under the same conditions as A. Traces from cells that were not treated with naspm are in black and naspm treated traces are in grey.


C . Average amplitude of mEPSCs ( pA ) for both control ( bars ) naspm treated ( black bars ) neurons under each of conditions , TTX , TTX + DHPG , TTX + DHPG + 2APB / dantrolene . TTX , n = 26 ; TTX + naspm , n = 17 ; TTX + DHPG , n = 18 ; TTX + DHPG + naspm , n = 23 ; TTX + DHPG + 2APB / dan , n = 32 ; TTX + DHPG + 2APB / dan + naspm , n = 36 , p < 0 . 005 , ANOVA . D . Average frequency of mEPSC ( Hz ) for both control ( bars ) naspm treated ( black bars ) neurons under each of conditions , TTX , TTX + DHPG , TTX + DHPG + 2APB / dantrolene . TTX , n = 26 ; TTX + naspm , n = 12 ; TTX + DHPG , n = 18 ; TTX + DHPG + naspm , n = 23 ; TTX + DHPG + 2APB / dan , n = 32 ; TTX + DHPG + 2APB / dan + naspm , n = 36 , p < 0 . 0001 , ANOVA .

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C. Average amplitude of mEPSCs (pA) for both control (white bars) and naspm treated (black bars) neurons under each of the conditions, TTX, TTX + DHPG, and TTX + DHPG + 2APB/dantrolene. TTX, n = 26; TTX + naspm, n = 17; TTX + DHPG, n = 18; TTX + DHPG + naspm, n = 23; TTX + DHPG + 2APB/dan, n = 32; TTX + DHPG + 2APB/dan + naspm, n = 36, p < 0.005, ANOVA. D. Average frequency of mEPSC (Hz) for both control (white bars) and naspm treated (black bars) neurons under each of the conditions, TTX, TTX + DHPG, and TTX + DHPG + 2APB/dantrolene. TTX, n = 26; TTX + naspm, n = 12; TTX + DHPG, n = 18; TTX + DHPG + naspm, n = 23; TTX + DHPG + 2APB/dan, n = 32; TTX + DHPG + 2APB/dan + naspm, n = 36, p < 0.0001, ANOVA.


E . Average amplitude of mEPSCs ( pA ) for only naspm treated neurons under each of conditions , TTX , TTX + DHPG , TTX + DHPG + 2APB / dantrolene . F . Average frequency of mEPSC ( Hz ) for only naspm treated neurons under each of conditions , TTX , TTX + DHPG , TTX + DHPG + 2APB / dantrolene .

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E. Average amplitude of mEPSCs (pA) for only naspm treated neurons under each of the conditions, TTX, TTX + DHPG, and TTX + DHPG + 2APB/dantrolene. F. Average frequency of mEPSC (Hz) for only naspm treated neurons under each of the conditions, TTX, TTX + DHPG, and TTX + DHPG + 2APB/dantrolene.


G . Average rise time of mEPSC ( ms ) for both control ( bars ) naspm treated ( black bars ) neurons under each of conditions , TTX , TTX + DHPG , TTX + DHPG + 2APB / dantrolene . H . Average decay time of mEPSC ( ms ) for both control ( bars ) naspm treated ( black bars ) neurons under each of conditions , TTX , TTX + DHPG , TTX + DHPG + 2APB / dantrolene . error bars represent s . e . m .

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G. Average rise time of mEPSC (ms) for both control (white bars) and naspm treated (black bars) neurons under each of the conditions, TTX, TTX + DHPG, and TTX + DHPG + 2APB/dantrolene. H. Average decay time of mEPSC (ms) for both control (white bars) and naspm treated (black bars) neurons under each of the conditions, TTX, TTX + DHPG, and TTX + DHPG + 2APB/dantrolene. All error bars represent s.e.m.


Gata6 - WT Gata6 - KOmye peritoneal macrophages ( pMɸ ) were unstimulated ( - ) or stimulated with TLR2L Pam3CSK4 ( 500 ng . ml - 1 ) , TLR3L Poly I : C ( 1 µg . ml - 1 ) , TLR4L purified LPS ( 100 ng . ml - 1 ) , TLR5L Flagellin ( 100 ng . ml - 1 ) , TLR7 8L R848 ( 1 µg . ml - 1 ) or TLR9L CpG ODN1826 ( 5 µM ) . Culture supernatants were collected 24 h after start of stimulation IL - 1β TNF ELISA were performed . n = 5 , two - way ANOVA with Tukeys multiple comparison post - .

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Gata6-WT and Gata6-KOmye peritoneal macrophages (pMɸ) were unstimulated (-) or stimulated with TLR2L Pam3CSK4 (500 ng.ml-1), TLR3L Poly I:C (1 µg.ml-1), TLR4L purified LPS (100 ng.ml-1), TLR5L Flagellin (100 ng.ml-1), TLR7 and 8L R848 (1 µg.ml-1) or TLR9L CpG ODN1826 (5 µM). Culture supernatants were collected 24 h after the start of stimulation and IL-1β and TNF ELISA were performed. n=5, two-way ANOVA analysis with Tukeys multiple comparison post-test.


IL - 1β ( C ) TNF ( D ) ELISA of Gata6 - WT Gata6 - KOmye pMɸ stimulated for 24 h with indicated LPS concentrations . n = 3 , two - way ANOVA with Sidak ' s multiple comparison post - .

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IL-1β (C) and TNF (D) ELISA of Gata6-WT and Gata6-KOmye pMɸ stimulated for 24 h with the indicated LPS concentrations. n=3, two-way ANOVA analysis with Sidak's multiple comparison post-test.


IL - 1β ( E ) TNF ( F ) ELISA from Gata6 - WT Gata6 - KOmye pMɸ stimulated with LPS ( 100 ng . ml - 1 ) for indicated times . n = 3 , two - way ANOVA with Sidak ' s multiple comparison post - .

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IL-1β (E) and TNF (F) ELISA from Gata6-WT and Gata6-KOmye pMɸ stimulated with LPS (100 ng.ml-1) for the indicated times. n=3, two-way ANOVA analysis with Sidak's multiple comparison post-test.


IL - 1β ELISA of Gata6 - WT Gata6 - KOmye pMɸ stimulated for 18 h with 100 ng . ml - 1 LPS or recombinant IL - 1 receptor antagonist ( rIL - 1ra ) , n = 4 to 5 , two - way ANOVA with Tukey ' s multiple comparison post - .

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IL-1β ELISA of Gata6-WT and Gata6-KOmye pMɸ stimulated for 18 h with 100 ng.ml-1 LPS or recombinant IL-1 receptor antagonist (rIL-1ra), n=4 to 5, two-way ANOVA analysis with Tukey's multiple comparison post-test.


IL - 1β ELISA of Gata6 - WT Gata6 - KOmye pMɸ stimulated for 18 h with 100 ng . ml - 1 LPS or 100 ng . ml - 1 recombinant TNF ( recTNF ) , n = 4 to 5 , two - way ANOVA with Tukey ' s multiple comparison post - .

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IL-1β ELISA of Gata6-WT and Gata6-KOmye pMɸ stimulated for 18 h with 100 ng.ml-1 LPS or 100 ng.ml-1 recombinant TNF (recTNF), n=4 to 5, two-way ANOVA analysis with Tukey's multiple comparison post-test.


Normalised expression of Tlr Cd14 genes following microarray performed on unstimulated - sorted Gata6 - WT Gata6 - KOmye pMɸ . Data are shown as mean + / - from three biological replicates . Statistical significance was determined using empirical Bayesian statistic corrected for false discovery rate by Benjamin - Hochberg procedure . n . d = non detectable .

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Normalised expression of Tlr and Cd14 genes following microarray analysis performed on unstimulated cell-sorted Gata6-WT and Gata6-KOmye pMɸ. Data are shown as mean +/- SEM from three biological replicates. Statistical significance was determined using empirical Bayesian statistic corrected for false discovery rate by the Benjamin-Hochberg procedure. n.d = non detectable.


Mean ( MFI ) of extracellular TLR2 , TLR4 CD14 expression on naïve Gata6 - WT Gata6 - KOmye pMɸ . n = 4 to 7 mice per group .

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Mean fluorescence intensity (MFI) of extracellular TLR2, TLR4 and CD14 expression on naïve Gata6-WT and Gata6-KOmye pMɸ. n = 4 to 7 individual mice per group.


Il1b Tnf mRNA relative expression of Gata6 - WT Gata6 - KOmye pMɸ stimulated with 100 ng . ml - 1 LPS for 3 h 6 h respectively . Data shown are representative of at least 3 independent experiments .

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Il1b and Tnf mRNA relative expression of Gata6-WT and Gata6-KOmye pMɸ stimulated with 100 ng.ml-1 LPS for 3 h and 6 h respectively. Data shown are representative of at least 3 independent experiments.


IL - 1β Western blot ( D ) of Gata6 - WT Gata6 - KOmye pMɸ stimulated with 100 ng . ml - 1 LPS for 3 h 6 h respectively . Data shown are representative of at least 3 independent experiments . Western blot was performed on whole lysates .

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IL-1β Western blot protein analysis (D) of Gata6-WT and Gata6-KOmye pMɸ stimulated with 100 ng.ml-1 LPS for 3 h and 6 h respectively. Data shown are representative of at least 3 independent experiments. Western blot was performed on whole cell lysates.


Representative dot plot , mean ( MFI ) of pro - IL - 1β + Gata6 - WT Gata6 - KOmye pMФ flow cytometry 3 h after stimulation with 100 ng . ml - 1 LPS . n = at least 3 independent experiments .

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Representative dot plot, percentage and mean fluorescence intensity (MFI) analysis of pro-IL-1β+ Gata6-WT and Gata6-KOmye pMФ flow cytometry analysis 3 h after stimulation with 100 ng.ml-1 LPS. n = at least 3 independent experiments.


Nlrp3 mRNA relative expression of Gata6 - WT Gata6 - KOmye pMɸ stimulated with 100 ng . ml - 1 LPS for 3 h . Data shown are pooled from 3 independent experiments .

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Nlrp3 mRNA relative expression of Gata6-WT and Gata6-KOmye pMɸ stimulated with 100 ng.ml-1 LPS for 3 h. Data shown are pooled from 3 independent experiments.


Western blot of Gata6 - WT Gata6 - KOmye pMɸ stimulated with 100 ng . ml - 1 LPS for 6 h . Data shown are representative of at least 3 independent experiments . Western blot was performed on whole lysates .

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Western blot protein analysis of Gata6-WT and Gata6-KOmye pMɸ stimulated with 100 ng.ml-1 LPS for 6 h. Data shown are representative of at least 3 independent experiments. Western blot was performed on whole cell lysates.


IL - 1β ELISA of supernatants collected from Gata6 - WT Gata6 - KOmye pMɸ stimulated with 100 ng . ml - 1 LPS either vehicle control ( Vh , DMSO ) or 10 µM MCC950 for 24 h ( n = 5 ) . Data shown in ( H ) are pooled from 5 independent replicates .

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IL-1β ELISA of supernatants collected from Gata6-WT and Gata6-KOmye pMɸ stimulated with 100 ng.ml-1 LPS and either vehicle control (Vh, DMSO) or 10 µM MCC950 for 24 h (n=5). Data shown in (H) are pooled from 5 independent replicates.


Western blot ( I ) of supernatants collected from Gata6 - WT Gata6 - KOmye pMɸ stimulated with 100 ng . ml - 1 LPS either vehicle control ( Vh , DMSO ) or 10 µM MCC950 for 24 h ( n = 5 ) .

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Western blot protein analysis (I) of supernatants collected from Gata6-WT and Gata6-KOmye pMɸ stimulated with 100 ng.ml-1 LPS and either vehicle control (Vh, DMSO) or 10 µM MCC950 for 24 h (n=5).


Caspase1 ( Casp1 ) mRNA relative expression of Gata6 - WT Gata6 - KOmye pMɸ stimulated with 100 ng . ml - 1 LPS for 3 h 6 h respectively . Data shown are pooled from 3 independent experiments .

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Caspase1 (Casp1) mRNA relative expression of Gata6-WT and Gata6-KOmye pMɸ stimulated with 100 ng.ml-1 LPS for 3 h and 6 h respectively. Data shown are pooled from 3 independent experiments.


Western blot ( K ) of Gata6 - WT Gata6 - KOmye pMɸ stimulated with 100 ng . ml - 1 LPS for 3 h 6 h respectively . Data shown are pooled from 3 independent experiments .

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Western blot protein analysis (K) of Gata6-WT and Gata6-KOmye pMɸ stimulated with 100 ng.ml-1 LPS for 3 h and 6 h respectively. Data shown are pooled from 3 independent experiments.


IL - 1β ELISA of Gata6 - WT Gata6 - KOmye pMɸ stimulated with 100 ng . ml - 1 LPS either vehicle control ( Vh , DMSO ) or Ac - YVAD - cmk for 24 h . Data shown are pooled of 5 independent replicates .

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IL-1β ELISA of Gata6-WT and Gata6-KOmye pMɸ stimulated with 100 ng.ml-1 LPS and either vehicle control (Vh, DMSO) or Ac-YVAD-cmk for 24 h. Data shown are pooled of 5 independent replicates.


IL - 1β ELISA of Gata6 - WT Gata6 - KOmye pMɸ stimulated with 100 ng . ml - 1 LPS for 3 h , followed by a 30 min with either vehicle control ( Vh ) , 5 mM ATP or 20 µM Nigericin . Data shown are pooled of 5 independent replicates .

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IL-1β ELISA of Gata6-WT and Gata6-KOmye pMɸ stimulated with 100 ng.ml-1 LPS for 3 h, followed by a 30 min pulse with either vehicle control (Vh), 5 mM ATP or 20 µM Nigericin. Data shown are pooled of 5 independent replicates.


Representative picture of confocal immunofluorescence of Gata6 - WT Gata6 - KOmye pMФ stimulated with 100 ng . ml - 1 LPS for 3 h , followed by a 30 min with 5 mM ATP . arrows show ASC specks . Scale bar = 10 µm .

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Representative picture of confocal immunofluorescence analysis of Gata6-WT and Gata6-KOmye pMФ stimulated with 100 ng.ml-1 LPS for 3 h, followed by a 30 min pulse with 5 mM ATP. The white arrows show ASC specks. Scale bar = 10 µm.


IL - 1β ELISA of supernatants of Gata6 - WT Gata6 - KOmye pMɸ in monoculture or co - cultured ( 1 : 1 ) stimulated for 24 h with 100 ng . ml - 1 LPS . Data shown are pooled from 3 independent experiments . One - way ANOVA statistical with Tukey ' s multiple comparison was performed .

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IL-1β ELISA analysis of supernatants of Gata6-WT and Gata6-KOmye pMɸ in monoculture or co-cultured (ratio 1:1) and stimulated for 24 h with 100 ng.ml-1 LPS. Data shown are pooled from 3 independent experiments. One-way ANOVA statistical analysis with Tukey's multiple comparison test was performed.


IL - 1β ELISA of supernatants of Gata6 - WT Gata6 - KOmye pMɸ co - cultured in same well ( co - culture ) or using Transwell system ( 1 : 1 ) stimulated for 24 h with 100 ng . ml - 1 LPS . Data shown are pooled from 3 independent experiments . One - way ANOVA statistical with Tukey ' s multiple comparison was performed .

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IL-1β ELISA analysis of supernatants of Gata6-WT and Gata6-KOmye pMɸ co-cultured in the same well (co-culture) or using Transwell system (ratio 1:1) and stimulated for 24 h with 100 ng.ml-1 LPS. Data shown are pooled from 3 independent experiments. One-way ANOVA statistical analysis with Tukey's multiple comparison test was performed.


Microarray of Ptgis , Tbxas1 , Ptges2 Ptgs1 expression from Gata6 - WT Gata6 - KOmye pMɸ isolated from naïve mice . Data is shown as mean + / - from three biological replicates . Statistical significance was determined using empirical Bayesian statistic corrected for false discovery rate by Benjamin - Hochberg procedure .

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Microarray analysis of Ptgis, Tbxas1, Ptges2 and Ptgs1 expression from Gata6-WT and Gata6-KOmye pMɸ isolated from naïve mice. Data is shown as mean +/- SEM from three biological replicates. Statistical significance was determined using empirical Bayesian statistic corrected for false discovery rate by the Benjamin-Hochberg procedure.


Western blot of Ptgis level of unstimulated pMФ from Gata6 - WT Gata6 - KOmye mice .

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Western blot analysis of Ptgis protein level of unstimulated pMФ from Gata6-WT and Gata6-KOmye mice.


Mass spectrometry of 6 - keto - PGF1α , TXB2 PGE2 content of Gata6 - WT Gata6 - KOmye pMɸ unstimulated ( - ) or stimulated for 3 h with 100 ng . ml - 1 LPS . n = 6 . Two - way ANOVA statistical with Tukey ' s multiple comparison post - was performed .

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Mass spectrometry analysis of 6-keto-PGF1α, TXB2 and PGE2 content of Gata6-WT and Gata6-KOmye pMɸ unstimulated (-) or stimulated for 3 h with 100 ng.ml-1 LPS. n = 6. Two-way ANOVA statistical analysis with Tukey's multiple comparison post-test was performed.


IL - 10 ELISA of supernatants of Gata6 - WT Gata6 - KOmye pMɸ unstimulated ( - ) or stimulated with 100 ng . ml - 1 LPS . Data shown are representative of at least 5 independent experiments .

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IL-10 ELISA of supernatants of Gata6-WT and Gata6-KOmye pMɸ unstimulated (-) or stimulated with 100 ng.ml-1 LPS. Data shown are representative of at least 5 independent experiments.


IL - 10 ( B ) IL - 1β ( C ) ELISA of supernatants of Gata6 - WT Gata6 - KOmye pMɸ unstimulated ( - ) or stimulated with 100 ng . ml - 1 LPS , beraprost ( 1 10 µM ) , cicaprost ( 1 10 µM ) , Iloprost ( 1 or 10 nM ) or vehicle control ( Vh , DMSO ) . Data shown are representative of at least 3 independent experiments .

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IL-10 (B) and IL-1β (C) ELISA of supernatants of Gata6-WT and Gata6-KOmye pMɸ unstimulated (-) or stimulated with 100 ng.ml-1 LPS, beraprost (1 and 10 µM), cicaprost (1 and 10 µM), Iloprost (1 or 10 nM) or vehicle control (Vh, DMSO). Data shown are representative of at least 3 independent experiments.


IL - 10 ( D ) IL - 1β ( E ) ELISA of supernatants of Gata6 - WT Gata6 - KOmye pMɸ unstimulated ( - ) or stimulated with 100 ng . ml - 1 LPS , 10 µM beraprost , 10 µM PGE2 , 10 µM U46619 , 100 µM picotamide or vehicle control ( Vh , methyl acetate ) . Data shown are representative of at least 3 independent experiments .

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IL-10 (D) and IL-1β (E) ELISA of supernatants of Gata6-WT and Gata6-KOmye pMɸ unstimulated (-) or stimulated with 100 ng.ml-1 LPS, 10 µM beraprost, 10 µM PGE2, 10 µM U46619, 100 µM picotamide or vehicle control (Vh, methyl acetate). Data shown are representative of at least 3 independent experiments.


IL - 1β ELISA of supernatants of Gata6 - WT Gata6 - KOmye pMɸ unstimulated ( - ) or stimulated with 100 ng . ml - 1 LPS , 10 ng . ml - 1 IL - 10 , 10 µM beraprost or vehicle control ( Vh ) . Data shown are representative of at least 3 independent experiments .

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IL-1β ELISA of supernatants of Gata6-WT and Gata6-KOmye pMɸ unstimulated (-) or stimulated with 100 ng.ml-1 LPS, 10 ng.ml-1 IL-10, 10 µM beraprost or vehicle control (Vh). Data shown are representative of at least 3 independent experiments.


IL - 1β ELISA of supernatants of Gata6 - WT Gata6 - KOmye pMɸ unstimulated ( - ) or stimulated with 100 ng . ml - 1 LPS , 10 µM beraprost , 5 µg . ml - 1 αIL - 10R or isotype antibody . Data shown are representative of at least 3 independent experiments .

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IL-1β ELISA of supernatants of Gata6-WT and Gata6-KOmye pMɸ unstimulated (-) or stimulated with 100 ng.ml-1 LPS, 10 µM beraprost, 5 µg.ml-1 αIL-10R or isotype antibody. Data shown are representative of at least 3 independent experiments.


IL - 1β ( H ) IL - 10 ( I ) ELISA of supernatants of Gata6 - WT Gata6 - KOmye pMɸ unstimulated ( - ) or stimulated with 100 ng . ml - 1 LPS , 10 µM MCC950 , 5 µg . ml - 1 αIL - 10R or isotype antibody stimulations were performed for 16 h . Data shown are representative of at least 3 independent experiments .

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IL-1β (H) and IL-10 (I) ELISA of supernatants of Gata6-WT and Gata6-KOmye pMɸ unstimulated (-) or stimulated with 100 ng.ml-1 LPS, 10 µM MCC950, 5 µg.ml-1 αIL-10R or isotype antibody All stimulations were performed for 16 h. Data shown are representative of at least 3 independent experiments.


IL - 1β ELISA of supernatants of Gata6 - WT Gata6 - KOmye pMɸ unstimulated ( - ) or stimulated with 100 ng . ml - 1 LPS , 10 ng . ml - 1 IL - 10 5 mM ATP . Data shown are a pool of 3 independent experiments .

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IL-1β ELISA of supernatants of Gata6-WT and Gata6-KOmye pMɸ unstimulated (-) or stimulated with 100 ng.ml-1 LPS, 10 ng.ml-1 IL-10 and 5 mM ATP. Data shown are a pool of 3 independent experiments.


Flow cytometry of Mitotracker ( MT ) Green Red integration in Gata6 - WT Gata6 - KOmye naïve pMɸ ( gated on F4 / 80hi / + Tim4 + ) in vivo , 30 min after intraperitoneal injection ( i . p . ) of 1 µM of Mitotracker Green Red . Data shown are representative of 3 to 5 independent mice of each genotype are expressed as mean + / - . Student ' s t was performed .

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Flow cytometry analysis of Mitotracker (MT) Green and Red integration in Gata6-WT and Gata6-KOmye naïve pMɸ (gated on F4/80hi/+ Tim4+) in vivo, 30 min after intraperitoneal injection (i.p.) of 1 µM of Mitotracker Green and Red. Data shown are representative of 3 to 5 independent mice of each genotype and are expressed as mean +/- SEM. Student's t test analysis was performed.


Flow cytometry of Mitotracker green red in Gata6 - WT Gata6 - KOmye pMɸ ( gated on F4 / 80hi / + Tim4 + ) unstimulated ( - ) or after LPS ( 100 ng . ml - 1 ) , IL - 10 ( 10 ng . ml - 1 ) , αIL - 10R ( 5 µg . ml - 1 ) or isotype ( 5 µg . ml - 1 ) stimulation for 16 h in vitro . Data shown are pooled from 3 independent experiments normalized to WT unstimulated .

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Flow cytometry analysis of Mitotracker green and red staining in Gata6-WT and Gata6-KOmye pMɸ (gated on F4/80hi/+ Tim4+) unstimulated (-) or after LPS (100 ng.ml-1), IL-10 (10 ng.ml-1), αIL-10R (5 µg.ml-1) or isotype (5 µg.ml-1) stimulation for 16 h in vitro. Data shown are pooled from 3 independent experiments and normalized to WT unstimulated.


Flow cytometry of MitoSOX in Gata6 - WT Gata6 - KOmye pMɸ ( gated on F4 / 80hi / + Tim4 + ) unstimulated ( - ) or after LPS ( 100 ng . ml - 1 ) , IL - 10 ( 10 ng . ml - 1 ) αIL - 10R ( 5 µg . ml - 1 ) stimulation for 16 h in vitro . Data shown are pooled from 2 independent experiments normalized to WT unstimulated .

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Flow cytometry analysis of MitoSOX staining in Gata6-WT and Gata6-KOmye pMɸ (gated on F4/80hi/+ Tim4+) unstimulated (-) or after LPS (100 ng.ml-1), IL-10 (10 ng.ml-1) and αIL-10R (5 µg.ml-1) stimulation for 16 h in vitro. Data shown are pooled from 2 independent experiments and normalized to WT unstimulated.


IL - 1β ELISA of supernatants of Gata6 - WT Gata6 - KOmye pMɸ unstimulated ( - ) or stimulated with 100 ng . ml - 1 LPS , 500 µM MitoTempo , 10 mM NAC , 0 . 5 µM MitoQ for 16 h . Data are representative of at least 3 experiments .

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IL-1β ELISA of supernatants of Gata6-WT and Gata6-KOmye pMɸ unstimulated (-) or stimulated with 100 ng.ml-1 LPS, 500 µM MitoTempo, 10 mM NAC, 0.5 µM MitoQ for 16 h. Data are representative of at least 3 experiments.


mRNA expression of Il1b , Nlrp3 Caspase1 ( Casp1 ) of Gata6 - WT Gata6 - KOmye pMФ stimulated for 3 h with 100 ng . ml - 1 LPS , 10 µM beraprost , 10 ng . ml - 1 IL - 10 , 5 µg . ml - 1 αIL - 10R or 5 µg . ml - 1 isotype . Data are representative of at least 3 independent experiments .

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

mRNA expression analysis of Il1b, Nlrp3 and Caspase1 (Casp1) of Gata6-WT and Gata6-KOmye pMФ stimulated for 3 h with 100 ng.ml-1 LPS, 10 µM beraprost, 10 ng.ml-1 IL-10, 5 µg.ml-1 αIL-10R or 5 µg.ml-1 isotype. Data are representative of at least 3 independent experiments.


Western blot ( left ) ( right ) of Gata6 - WT Gata6 - KOmye pMФ stimulated for 6 h with 100 ng . ml - 1 LPS , 10 µM beraprost , 10 ng . ml - 1 IL - 10 or 5 µg . ml - 1 αIL - 10R . Data are representative of 3 independent experiments .

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

Western blot analysis (left) and quantification (right) of Gata6-WT and Gata6-KOmye pMФ stimulated for 6 h with 100 ng.ml-1 LPS, 10 µM beraprost, 10 ng.ml-1 IL-10 or 5 µg.ml-1 αIL-10R. Data are representative of 3 independent experiments.


Caspase1 activity of Gata6 - WT Gata6 - KOmye pMФ stimulated for 16 h with 100 ng . ml - 1 LPS , 10 µM beraprost , 10 ng . ml - 1 IL - 10 , 5 µg . ml - 1 αIL - 10R or 5 µg . ml - 1 isotype . n = 5 to 8 mice .

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

Caspase1 activity analysis of Gata6-WT and Gata6-KOmye pMФ stimulated for 16 h with 100 ng.ml-1 LPS, 10 µM beraprost, 10 ng.ml-1 IL-10, 5 µg.ml-1 αIL-10R or 5 µg.ml-1 isotype. n = 5 to 8 individual mice.


Mean ( MFI ) of pro - IL - 1β of Gata6 - WT Gata6 - KOmye pMФ stimulated for 3 h with 100 ng . ml - 1 LPS , 10 µM beraprost , 10 ng . ml - 1 IL - 10 , 5 µg . ml - 1 αIL - 10R or 5 µg . ml - 1 isotype for 16 h or freshly isolated . n = 6 to 13 mice . Data were log transformed before performing statistical .

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

Mean fluorescence intensity (MFI) of pro-IL-1β of Gata6-WT and Gata6-KOmye pMФ stimulated for 3 h with 100 ng.ml-1 LPS, 10 µM beraprost, 10 ng.ml-1 IL-10, 5 µg.ml-1 αIL-10R or 5 µg.ml-1 isotype for 16 h or freshly isolated. n = 6 to 13 individual mice. Data were log transformed before performing statistical analysis.


mRNA expression of Il10 Tnf of Gata6 - WT Gata6 - KOmye pMФ stimulated for 3 h with 100 ng . ml - 1 LPS , 10 µM beraprost , 10 ng . ml - 1 IL - 10 or 5 µg . ml - 1 αIL - 10R . Data are representative of at least 3 independent experiments .

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

mRNA expression analysis of Il10 and Tnf of Gata6-WT and Gata6-KOmye pMФ stimulated for 3 h with 100 ng.ml-1 LPS, 10 µM beraprost, 10 ng.ml-1 IL-10 or 5 µg.ml-1 αIL-10R. Data are representative of at least 3 independent experiments.


CRH - Cre : : Ai9 reporter mice fed on chow or 4 on HFD were used to examine c - Fos expression in PVH in 24hr fasting or refed after 24hr fasting . A ) Representative showing CRH neurons ( red in first colomn ) , c - Fos expression ( green in second colomn ) , merged of red green ( third colomn ) amplied pictures for boxed areas in merged ( forth colomn ) . experimental conditions for each group ( fasting verus refed , chow versus HFD ) are labelled on left . Scale bar = 200µm .

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

CRH-Cre::Ai9 reporter male mice fed on chow or 4 weeks on HFD were used to examine c-Fos expression in the PVH in 24hr fasting or refed after 24hr fasting. A) Representative images showing CRH neurons (red in the first colomn), c-Fos expression (green in the second colomn), merged of red and green (the third colomn) and the amplied pictures for the boxed areas in the merged images (the forth colomn). The experimental conditions for each group (fasting verus refed, chow versus HFD) are labelled on the left. Scale bar=200µm.


B - C ) Comparison of average neuron numbers from counting on total number of c - Fos positive neurons in PVH ( B , * * p = 0 . 0032 : chow fasting versus chow refed * p = 0 . 023 , HFD fasting versus HFD refed ) total number of c - Fos positive CRH neurons ( C , * * p < 0 . 0001 : chow fasting versus chow refed p = 0 . 7158 : HFD fasting versus HFD refed ) , n = 3 - 4 each , data presented as mean + / - , two way ANOVA .

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 O O O O 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 I-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 O O O O O O O O O O O O O O O O O O O O

B-C) Comparison of average neuron numbers from cell counting on total number of c-Fos positive neurons in the PVH (B, **p=0.0032: chow fasting versus chow refed and *p=0.023, HFD fasting versus HFD refed) and total number of c-Fos positive CRH neurons (C, **p<0.0001: chow fasting versus chow refed and p=0.7158: HFD fasting versus HFD refed), n=3-4 each, data presented as mean +/-SEM, two way ANOVA tests.


Effects of HFD feeding on diurnal rhythmicity in feeding ( D E ) measurement was collected with mice fed chow or after 4 HFD feeding , n = 6 each , data presented as mean + / - , * * p = 0 . 0066 in E * * p = 0 . 0 . 0007 in G , unpaired Student ' s t .

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

Effects of HFD feeding on diurnal rhythmicity in feeding (D and E) The measurement was collected with male mice fed chow or after 4 weeks HFD feeding, n=6 each, data presented as mean +/- SEM, **p=0.0066 in E and **p=0.0.0007 in G, unpaired Student's t tests.


Effects of HFD feeding on diurnal rhythmicity in energy expenditure ( F G ) . measurement was collected with mice fed chow or after 4 HFD feeding , n = 6 each , data presented as mean + / - , * * p = 0 . 0066 in E * * p = 0 . 0 . 0007 in G , unpaired Student ' s t .

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

Effects of HFD feeding on diurnal rhythmicity in energy expenditure (F and G). The measurement was collected with male mice fed chow or after 4 weeks HFD feeding, n=6 each, data presented as mean +/- SEM, **p=0.0066 in E and **p=0.0.0007 in G, unpaired Student's t tests.


CRH - Cre mice received delivery of AAV - Flex - GCaMP6m viral vectors to bilateral PVH optic fiber implantation targeting PVH neurons , were used for in vivo live recording . ( H - I ) Representative traces showing recordings of activity changes in CRH neuron in response to stressor ( water spray ) in chow - fed controls ( H ) , 4 after HFD ( I ) , ( J ) Comparison of average responses in two conditions , n = 4 each , * * p = 0 . 0006 , paired Student ' s t .

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

CRH-Cre mice received delivery of AAV-Flex-GCaMP6m viral vectors to bilateral PVH and optic fiber implantation targeting PVH neurons, and were used for in vivo live recording. (H-I) Representative traces showing recordings of activity changes in CRH neuron in response to stressor (water spray) in chow-fed controls (H), and 4 weeks after HFD (I), (J) Comparison of average responses in the two conditions, n=4 each, **p=0.0006, paired Student's t test.


K - L ) Representative traces showing recordings of activity changes in CRH neurons in response to Dexamethasone ( Dex ) injections in chow - fed controls ( K ) after 4 on HFD ( L ) . M ) Comparison of average responses in two conditions , n = 4 each , data presented as mean + / - , * * P = 0 . 0012 , paired Student ' s t .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-L) Representative traces showing recordings of activity changes in CRH neurons in response to Dexamethasone (Dex) injections in chow-fed controls (K) and after 4 weeks on HFD (L). M) Comparison of average responses in the two conditions, n=4 each, data presented as mean +/- SEM, **P=0.0012, paired Student's t tests.


CRH - Cre mice received stereotaxic delivery of AAV - EF1a - Flex - EGFP - P2A - mNachBac vectors at 7 - 8 of recordings were made at least 4 after viral delivery on neurons with NachBac ( one side ) or control neurons ( side ) of same ( A ) ;

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

CRH-Cre male mice received stereotaxic delivery of AAV-EF1a-Flex-EGFP-P2A-mNachBac vectors at 7-8 weeks of age and recordings were made at least 4 weeks after viral delivery on neurons with NachBac (one side) or control neurons (contralateral side) of the same animal (A);


typical action potentials ( APs ) in controls ( B , top panel ) typical NachBac - mediated APs showing lower threshold long duration ( B , bottom , inset highlighting long duration ) in NachBac

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

typical action potentials (APs) in controls (B, top panel) and typical NachBac-mediated APs showing lower threshold and long duration (B, bottom, the inset highlighting the long duration) in NachBac mice


distribution of potential firing in two groups ( C , ratios in bracket denoting neuron number out of total number recorded with indicated behavior ) .

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

the distribution of the membrane potential and firing cells in the two groups (C, ratios in the bracket denoting neuron number out of total number recorded with the indicated behavior).


CRH - Cre mice were injected with either AAV - Flex - GFP or AAV - EF1a - Flex - EGFP - P2A - mNachBac to examine c - Fos expression in PVH in 24hr fasting or refed after fasting . ( D ) Representative showing CRH neurons expressing GFP or NachBac ( GFP in top 2 rows NachBac in bottom 2 rows of first colomn ) , c - Fos expression ( red in second colomn ) , merged of red green ( third colomn ) amplied pictures for boxed areas in merged ( forth colomn ) . experimental conditions for each group ( fasting verus refed ) are labelled on left .

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

CRH-Cre male mice were injected with either AAV-Flex-GFP or AAV-EF1a-Flex-EGFP-P2A-mNachBac to examine c-Fos expression in the PVH in 24hr fasting or refed after fasting. (D) Representative images showing CRH neurons expressing GFP or NachBac (GFP in the top 2 rows and NachBac in the bottom 2 rows of the first colomn), c-Fos expression (red in the second colomn), merged of red and green (the third colomn) and the amplied pictures for the boxed areas in the merged images (the forth colomn). The experimental conditions for each group (fasting verus refed) are labelled on the left.


( E - F ) Comparison of average neuron numbers from counting on total number of c - Fos positive neurons in PVH ( E , * * p < 0 . 0001 : fasting versus refed ; * * p = 0 . 0026 : fasting versus fasting / NachBac ; * * p < 0 . 0001 : fasting / NachBac versus refed / NachBac ) total number of c - Fos positive CRH neurons ( F , * * p < 0 . 0001 : fasting versus refed ; * * p < 0 . 0001 : fasting versus fasting / NachBac ; p = 0 . 3423 : fasting / NachBac versus refed / NachBac ) , n = 3 - 4 each , data presented as mean + / - , two way ANOVA .

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

(E-F) Comparison of average neuron numbers from cell counting on total number of c-Fos positive neurons in the PVH (E, **p<0.0001: fasting versus refed; **p=0.0026: fasting versus fasting/NachBac; **p<0.0001: fasting/NachBac versus refed/NachBac) and total number of c-Fos positive CRH neurons (F, **p<0.0001: fasting versus refed; **p<0.0001: fasting versus fasting/NachBac; p=0.3423: fasting/NachBac versus refed/NachBac), n=3-4 each, data presented as mean +/- SEM, two way ANOVA tests.


CRH - Cre mice received stereotaxic delivery of a mixture of AAV - Flex - GCaMP6m AAV - EF1a - Flex - EGFP - P2A - mNachBac vectors at 7 - 8 of with optic fiber implantation targeting PVH neurons . ( G - H ) Representative traces showing typical responses of PVH CRH neurons to water spay ( arrows ) in NachBac mice ( G ) , comparison of responsiveness to water spray between NachBac control mice ( H ) .

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

CRH-Cre male mice received stereotaxic delivery of a mixture of AAV-Flex-GCaMP6m and AAV-EF1a-Flex-EGFP-P2A-mNachBac vectors at 7-8 weeks of age with optic fiber implantation targeting PVH neurons. (G-H) Representative traces showing typical responses of PVH CRH neurons to water spay (arrows) in NachBac mice (G), and comparison of responsiveness to water spray between NachBac and control mice (H).


( I - J ) Representative traces showing typical responses of PVH CRH neurons to water spay ( arrows ) in NachBac mice ( I ) , comparison of responsiveness to water spray between NachBac control mice ( J ) . Gray traces showed Ca2 + independent signal for system stability red ones showed Ca2 + dependent signals for neuron activity , N = 5 - 7 each , , data presented as mean + / - , * * p < 0 . 0001 in both ( H J ) , unpaired Student ' s t .

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

(I-J) Representative traces showing typical responses of PVH CRH neurons to water spay (arrows) in NachBac mice (I), and comparison of responsiveness to water spray between NachBac and control mice (J).Gray traces showed Ca2+ independent signal for system stability and red ones showed Ca2+ dependent signals for neuron activity, N=5-7 each, males, data presented as mean +/-SEM, **p<0.0001 in both (H and J), unpaired Student's t tests.


CRH - Cre mice received stereotaxic delivery of AAV - EF1a - Flex - EGFP - P2A - mNachBac or control GFP vectors at 7 - 8 of were maintained on chow diet for 7 before switching to HFD . Weekly gain after viral delivery ( A ) at 11 after viral delivery .

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

CRH-Cre male mice received stereotaxic delivery of AAV-EF1a-Flex-EGFP-P2A-mNachBac or control GFP vectors at 7-8 weeks of age and were maintained on chow diet for 7 weeks before switching to HFD. Weekly body weight gain after viral delivery (A) at 11 weeks after viral delivery.


CRH - Cre mice received stereotaxic delivery of AAV - EF1a - Flex - EGFP - P2A - mNachBac or control GFP vectors at 7 - 8 of were maintained on chow diet for 7 before switching to HFD . measurements of fat mass ( B ) lean mass ( C ) at 11 after viral delivery .

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

CRH-Cre male mice received stereotaxic delivery of AAV-EF1a-Flex-EGFP-P2A-mNachBac or control GFP vectors at 7-8 weeks of age and were maintained on chow diet for 7 weeks before switching to HFD. measurements of fat mass (B) and lean mass (C) at 11 weeks after viral delivery.


Measurements of energy expenditure during 7th week after viral delivery . Comparison of real time O2 consumption traces during 4 - day measurement period ( D ) , of average O2 consumption between 2 groups ( E ) , of difference between day night periods between 2 groups ( F ) .

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

Measurements of energy expenditure during the 7th week after viral delivery. Comparison of real time O2 consumption traces during the 4-day measurement period (D), of average O2 consumption between the 2 groups (E), and of the difference between day and night periods between the 2 groups (F).


( G - I ) Measurements of feeding during 7th week after viral delivery . Comparison of real time feeding c traces during 4 - day measurement period ( G ) , of average food intake between 2 groups ( H ) , of difference in food intake between day night periods between 2 groups ( I ) .

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

(G-I) Measurements of feeding during the 7th week after viral delivery. Comparison of real time feeding c traces during the 4-day measurement period (G), of average food intake between the 2 groups (H), and of the difference in food intake between day and night periods between the 2 groups (I).


CRH - Cre mice received stereotaxic delivery of AAV - EF1a - DIO - Kir2 . 1 - P2A - dTomato vectors AAV - Flex - GFP at 7 - 8 of recording was made at least 4 after viral delivery on neurons with Kir2 . 1 ( one side ) or control GFP neurons ( side ) of same ( A )

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

CRH-Cre male mice received stereotaxic delivery of AAV-EF1a-DIO-Kir2.1-P2A-dTomato vectors and AAV-Flex-GFP at 7-8 weeks of age and recording was made at least 4 weeks after viral delivery on neurons with Kir2.1 (one side) or control GFP neurons (contralateral side) of the same animal (A)


typical spontaneous neuron activity recorded in GFP controls ( B , top panel ) Kir2 . 1 neurons ( B , bottom ) ; comparison in resting potentials between two groups ( C , * * p < 0 . 0001 , unpaired Student ' s t ) .

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 O O O O O O O O O O

typical spontaneous neuron activity recorded in GFP controls (B, top panel) and Kir2.1 neurons (B, bottom); and comparison in resting membrane potentials between the two groups (C, **p<0.0001, unpaired Student's t test).


CRH - Cre mice were injected with either AAV - Flex - mCherry or AAV - EF1a - DIO - Kir2 . 1 - P2A - dTomato to examine c - Fos expression in PVH in 24hr fasting or refed after fasting . ( D ) Representative showing CRH neurons expressing mCherry or Kir2 . 1 ( mCherry in top 2 rows Kir2 . 1 in bottom 2 rows of second colomn ) , c - Fos expression ( green in first colomn ) , merged of red green ( third colomn ) amplied pictures for boxed areas in merged ( forth colomn ) . experimental conditions for each group ( fasting verus refed ) are labelled on left .

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

CRH-Cre male mice were injected with either AAV-Flex-mCherry or AAV-EF1a-DIO-Kir2.1-P2A-dTomato to examine c-Fos expression in the PVH in 24hr fasting or refed after fasting. (D) Representative images showing CRH neurons expressing mCherry or Kir2.1 (mCherry in the top 2 rows and Kir2.1 in the bottom 2 rows of the second colomn), c-Fos expression (green in the first colomn), merged of red and green (the third colomn) and the amplied pictures for the boxed areas in the merged images (the forth colomn). The experimental conditions for each group (fasting verus refed) are labelled on the left.


CRH - Cre mice were injected with either AAV - Flex - mCherry or AAV - EF1a - DIO - Kir2 . 1 - P2A - dTomato to examine c - Fos expression in PVH in 24hr fasting or refed after fasting . ( E - F ) Comparison of average neuron numbers from counting on total number of c - Fos positive neurons in PVH ( E , * * p = 0 . 0001 : fasting versus refed ; * p = 0 . 0497 : refed versus refed / Kir2 . 1 ; * * p = 0 . 0025 : fasting / Kir2 . 1 versus refed Kir2 . 1 ) total number of c - Fos positive CRH neurons ( F , * * p < 0 . 0001 : fasting versus refed ; p = 0 . 9586 : fasting / Kir2 . 1 versus refed / Kir2 . 1 ) , n = 3 - 4 each , two way ANOVA . data presented as mean + / - .

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

CRH-Cre male mice were injected with either AAV-Flex-mCherry or AAV-EF1a-DIO-Kir2.1-P2A-dTomato to examine c-Fos expression in the PVH in 24hr fasting or refed after fasting. (E-F) Comparison of average neuron numbers from cell counting on total number of c-Fos positive neurons in the PVH (E, **p=0.0001: fasting versus refed; *p=0.0497: refed versus refed/Kir2.1; **p=0.0025: fasting/Kir2.1 versus refed Kir2.1) and total number of c-Fos positive CRH neurons (F, **p<0.0001: fasting versus refed; p=0.9586: fasting/Kir2.1 versus refed/Kir2.1), n=3-4 each, two way ANOVA tests. All data presented as mean +/- SEM.


CRH - Cre mice received stereotaxic delivery of a mixture of AAV - Flex - GCaMP6m AAV - EF1a - DIO - Kir2 . 1 - P2A - dTomato vectors at 7 - 8 of with optic fiber implantation targeting PVH neurons . ( G - H ) Representative traces showing typical responses of PVH CRH neurons to water spay ( arrows ) in Kir2 . 1 mice ( G ) , comparison of responsiveness to water spray between Kir2 . 1 control mice ( H ) .

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

CRH-Cre male mice received stereotaxic delivery of a mixture of AAV-Flex-GCaMP6m and AAV-EF1a-DIO-Kir2.1-P2A-dTomato vectors at 7-8 weeks of age with optic fiber implantation targeting PVH neurons. (G-H) Representative traces showing typical responses of PVH CRH neurons to water spay (arrows) in Kir2.1 mice (G), and comparison of responsiveness to water spray between Kir2.1 and control mice (H).


CRH - Cre mice received stereotaxic delivery of a mixture of AAV - Flex - GCaMP6m AAV - EF1a - DIO - Kir2 . 1 - P2A - dTomato vectors at 7 - 8 of with optic fiber implantation targeting PVH neurons .

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 O O O

CRH-Cre male mice received stereotaxic delivery of a mixture of AAV-Flex-GCaMP6m and AAV-EF1a-DIO-Kir2.1-P2A-dTomato vectors at 7-8 weeks of age with optic fiber implantation targeting PVH neurons.


CRH - Cre mice received stereotaxic delivery of AAV - EF1a - DIO - Kir2 . 1 - P2A - dTomato or control AAV - Flex - mCherry vectors at 7 - 8 of were maintained on chow diet for 7 before switching to HFD . Weekly gain after viral delivery ( A , * p < 0 . 0001 , unpaired Student ' s t ) , at 15 after viral delivery .

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

CRH-Cre male mice received stereotaxic delivery of AAV-EF1a-DIO-Kir2.1-P2A-dTomato or control AAV-Flex-mCherry vectors at 7-8 weeks of age and were maintained on chow diet for 7 weeks before switching to HFD. Weekly body weight gain after viral delivery (A, *p<0.0001, unpaired Student's t test), at 15 weeks after viral delivery.


CRH - Cre mice received stereotaxic delivery of AAV - EF1a - DIO - Kir2 . 1 - P2A - dTomato or control AAV - Flex - mCherry vectors at 7 - 8 of were maintained on chow diet for 7 before switching to HFD . measurements of fat mass ( B , * * p = 0 . 0006 , unpaired Student ' s t ) lean mass ( C , p = 0 . 3728 , unpaired Student ' s t ) at 15 after viral delivery .

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

CRH-Cre male mice received stereotaxic delivery of AAV-EF1a-DIO-Kir2.1-P2A-dTomato or control AAV-Flex-mCherry vectors at 7-8 weeks of age and were maintained on chow diet for 7 weeks before switching to HFD. measurements of fat mass (B, **p=0.0006, unpaired Student's t test) and lean mass (C, p=0.3728, unpaired Student's t test) at 15 weeks after viral delivery.