Figure 6 ( 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 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 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 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 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 cells were infected with lentivirus expressing WT or T32A PAQR3 . Then PAQR3 expression levels were examined by IB . ( 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 .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6(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 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 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 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 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 cells were infected with lentivirus expressing WT or T32A PAQR3. Then PAQR3 expression levels were examined by IB.(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.


Figure 7 ( 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 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 . 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 , 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 ) and immunoblotting ( IB ) were performed using the antibodies as indicated . ( 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 assay ( n = 5 ; * * p < 0 . 01 ) .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O 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-PANEL_START 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 7(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 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. 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, 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) and immunoblotting (IB) were performed using the antibodies as indicated.(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 assay (n = 5; **p < 0.01).


Figure 1A . 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 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 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 .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1A. 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 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 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.


Figure 2A . 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 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 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 .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2A. 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 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 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.


Figure 3A . 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 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 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 .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3A. 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 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 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.


Figure 4A 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 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 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 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 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 the specificity of the GluA2 coIP . All error bars represent s . e . m .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4A 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 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 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 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 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 the specificity of the GluA2 coIP. All error bars represent s.e.m.


Figure 5A . 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 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 .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5A. 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 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.


Figure 6A . Individual traces of mEPSCs of both control and naspm treated neurons after TTX , TTX + DHPG , and TTX + DHPG + 2APB / dan treatment . 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 ( 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 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 ( 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 .

O B-PANEL_START O O O O O 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-PANEL_START O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6A. Individual traces of mEPSCs of both control and naspm treated neurons after TTX, TTX + DHPG, and TTX + DHPG + 2APB/dan treatment.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 (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 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 (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.


Figure 1Gata6 - 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 ) 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 ) 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 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 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 .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1Gata6-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) 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) 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 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 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.


Figure 2Normalised 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 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 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 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 , 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 and Gata6 - KOmye pMɸ stimulated with 100 ng . ml - 1 LPS for 3 h . Data shown are pooled from 3 independent experiments . 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 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 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 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 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 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 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 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 .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2Normalised 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 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 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 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, 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 and Gata6-KOmye pMɸ stimulated with 100 ng.ml-1 LPS for 3 h. Data shown are pooled from 3 independent experiments.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 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 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 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 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 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 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 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.


Figure 3IL - 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 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 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 analysis of Ptgis protein level of unstimulated pMФ from Gata6 - WT and Gata6 - KOmye mice . 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 .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3IL-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 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 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 analysis of Ptgis protein level of unstimulated pMФ from Gata6-WT and Gata6-KOmye mice.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.


Figure 4IL - 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 ) 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 ) 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 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 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 ) 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 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 .

O B-PANEL_START O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4IL-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) 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) 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 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 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) 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 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.


Figure 5Flow 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 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 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 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 .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5Flow 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 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 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 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.


Figure 6mRNA 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 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 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 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 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 .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6mRNA 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 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 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 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 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.


Figure 1CRH - 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 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 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 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 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 ) 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 .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1CRH-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 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 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 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 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) 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.


Figure 2CRH - 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 ) and typical NachBac - mediated APs showing lower threshold and long duration ( B , bottom , the inset highlighting the long duration ) in NachBac micethe 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 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 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 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 ) , 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 .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2CRH-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) and typical NachBac-mediated APs showing lower threshold and long duration (B, bottom, the inset highlighting the long duration) in NachBac micethe 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 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 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 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), 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.


Figure 3CRH - 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 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 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 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 ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3CRH-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 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 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 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).


Figure 4CRH - 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 ) 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 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 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 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 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 .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4CRH-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) 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 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 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 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 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.


Figure 5CRH - 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 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 . ( D - F ) 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 , * p = 0 . 0007 , unpaired Student ' s t test ) , and of the difference between day and night periods between the 2 groups ( F , p = 0 . 0791 , unpaired Student ' s t test ) . ( 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 , * * p = 0 . 0019 , unpaired Student ' s t test ) , and of the difference in food intake between day and night periods between the 2 groups ( I , * p = 0 . 0363 , unpaired Student ' s t test ) . n = 6 each . All data presented as mean + / - SEM .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5CRH-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 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.(D-F) 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, *p=0.0007, unpaired Student's t test), and of the difference between day and night periods between the 2 groups (F, p=0.0791, unpaired Student's t test).(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, **p=0.0019, unpaired Student's t test), and of the difference in food intake between day and night periods between the 2 groups (I, *p=0.0363, unpaired Student's t test). n=6 each. All data presented as mean +/- SEM.


Figure 1B . Size - exclusion chromatography ( SEC ) experiments of Mdm12 , tMmm1 , and the Mdm12 - tMmm1 complex comparing the molecular size of these proteins in solution . The proteins indicated were injected into a Superdex 200 column ( GE Healthcare ) with a buffer containing 25 mM Tris - HCl ( pH 7 . 5 ) , 150 mM NaCl , and 5 mM DTT . The standard molecular masses for the SEC experiments ( top ) are shown for relative molecular weight comparison ( blue dextran , void ; ferritin , 440 kDa ; aldolase , 158 kDa ; cobalbumin , 75 kDa ; ovalbumin , 44 kDa ; and carbonic anhydrase , 29 kDa ) . C . Graph indicating the molecular weights of Mdm12 , His6 - Mdm12 , and the Mdm12 - tMmm1 complex in solution as measured by analytical ultracentrifugation .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1B. Size-exclusion chromatography (SEC) experiments of Mdm12, tMmm1, and the Mdm12-tMmm1 complex comparing the molecular size of these proteins in solution. The proteins indicated were injected into a Superdex 200 column (GE Healthcare) with a buffer containing 25 mM Tris-HCl (pH 7.5), 150 mM NaCl, and 5 mM DTT. The standard molecular masses for the SEC experiments (top) are shown for relative molecular weight comparison (blue dextran, void; ferritin, 440 kDa; aldolase, 158 kDa; cobalbumin, 75 kDa; ovalbumin, 44 kDa; and carbonic anhydrase, 29 kDa).C. Graph indicating the molecular weights of Mdm12, His6-Mdm12, and the Mdm12-tMmm1 complex in solution as measured by analytical ultracentrifugation.


Figure 2A . Ribbon diagram of the yeastMdm12 dimer . The crystal structure of full - length Mdm12 was determined by SAD and refined with native data to 3 . 1 Å resolution . Lipids bound to Mdm12 are drawn with black stick models . B . Schematic diagram indicating the secondary structure elements and their organization in Mdm12 . Three structural elements of Mdm12 are highlighted in different colored boxes . C , D . Surface representations of the Mdm12 dimer are shown in different orientations .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O 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-PANEL_START O O O O O O O O O O O O O O O

Figure 2A. Ribbon diagram of the yeastMdm12 dimer. The crystal structure of full-length Mdm12 was determined by SAD and refined with native data to 3.1 Å resolution. Lipids bound to Mdm12 are drawn with black stick models.B. Schematic diagram indicating the secondary structure elements and their organization in Mdm12. Three structural elements of Mdm12 are highlighted in different colored boxes.C, D. Surface representations of the Mdm12 dimer are shown in different orientations.


Figure 3A . Ribbon diagram showing the 2 - fold dimerization interface of Mdm12 . Oxygen and nitrogen atoms are shown in red and blue , respectively . The orange dotted lines indicate intermolecular hydrogen bonds between two protomers of Mdm12 . The sequence alignment of yeastMdm12 orthologs is shown to highlight the sequence conservation in the N - terminus β1 - strand . Ten orthologs are aligned from residues 1 to 11 . Absolute and highly conserved residues are indicated in red and orange , respectively . B . The molecular weight of the Mdm12 ( I5P ) mutant was measured by size - exclusion chromatography ( below ) and ultracentrifugation ( top ) as in Figure 1B and C . C . Ribbon diagram showing the structures of the SMP domain in E - SYT2 , CETP , and BPI for the comparison of dimeric interfaces among SMP domains . Note that CETP and BPI are not dimers but monomers containing two tandem SMP domains .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3A. Ribbon diagram showing the 2-fold dimerization interface of Mdm12. Oxygen and nitrogen atoms are shown in red and blue, respectively. The orange dotted lines indicate intermolecular hydrogen bonds between two protomers of Mdm12. The sequence alignment of yeastMdm12 orthologs is shown to highlight the sequence conservation in the N-terminus β1-strand. Ten orthologs are aligned from residues 1 to 11. Absolute and highly conserved residues are indicated in red and orange, respectively.B. The molecular weight of the Mdm12 (I5P) mutant was measured by size-exclusion chromatography (below) and ultracentrifugation (top) as in Figure 1B and C.C. Ribbon diagram showing the structures of the SMP domain in E-SYT2, CETP, and BPI for the comparison of dimeric interfaces among SMP domains. Note that CETP and BPI are not dimers but monomers containing two tandem SMP domains.


Figure 4A . Simulated annealing omit map ( Fo - Fc , contoured at 1 . 5σ ) showing the molecule bound to Mdm12 ( left ) . The final model for the bound PE is shown as in stick representation . The electron density ( 2Fo - Fc ) calculated in the final model is shown with the stick model of PE in the right ( 3 . 1 Å resolution , contoured at 0 . 8σ ) . B . Surface representation of the Mdm12 dimer . Hydrophobic amino acids lining the Mdm12 channel are indicated by a blue mesh . Lipids built in Mdm12 are in space - filling representation . C . Ribbon diagram showing lipid coordination by Mdm12 . Mdm12 residues and lipid fatty acids are colored in green and yellow , respectively . D . Mdm12 binds NBD - PE . Wild - type and monomeric ( I5P mutant ) Mdm12 were incubated with NBD - PE and separated from free NBD - PE in native PAGE . Coomassie staining ( left ) and fluorescent ( right ) detection indicates that Mdm12 directly interacts with NBD - PEin vitro . E . Quantitative data showing binding affinities for NBD - PE by Mdm12 . The binding affinities of Mdm12 ( monomer / dimer shown in Native PAGE and I5P mutant ) for NBD - PE was measured with a NBD - PE concentration - dependent manner . All experiments were carried out three times and the means ± SD are given . F . Mdm12 mutants ( L256W , I262W , and L256W / I262W double mutant ) were incubated with NBD - PE , and subjected to native PAGE . Because wild - type Mdm12 separates as both monomer and dimer on native PAGE , the purely monomeric form ( I5P ) of Mdm12 was used as the wild type for clarity . The graph in the right indicates the quantities measured in the experiments . The bar shows the relative amounts of the band ratio ( fluorescence / Coomassie ) . Values represent the means and SD from three independent experiments .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O 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-PANEL_START 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4A. Simulated annealing omit map (Fo-Fc, contoured at 1.5σ) showing the molecule bound to Mdm12 (left). The final model for the bound PE is shown as in stick representation. The electron density (2Fo-Fc) calculated in the final model is shown with the stick model of PE in the right (3.1 Å resolution, contoured at 0.8σ).B. Surface representation of the Mdm12 dimer. Hydrophobic amino acids lining the Mdm12 channel are indicated by a blue mesh. Lipids built in Mdm12 are in space-filling representation.C. Ribbon diagram showing lipid coordination by Mdm12. Mdm12 residues and lipid fatty acids are colored in green and yellow, respectively.D. Mdm12 binds NBD-PE. Wild-type and monomeric (I5P mutant) Mdm12 were incubated with NBD-PE and separated from free NBD-PE in native PAGE. Coomassie staining (left) and fluorescent (right) detection indicates that Mdm12 directly interacts with NBD-PEin vitro.E. Quantitative data showing binding affinities for NBD-PE by Mdm12. The binding affinities of Mdm12 (monomer/dimer shown in Native PAGE and I5P mutant) for NBD-PE was measured with a NBD-PE concentration-dependent manner. All experiments were carried out three times and the means ± SD are given.F. Mdm12 mutants (L256W, I262W, and L256W/I262W double mutant) were incubated with NBD-PE, and subjected to native PAGE. Because wild-type Mdm12 separates as both monomer and dimer on native PAGE, the purely monomeric form (I5P) of Mdm12 was used as the wild type for clarity. The graph in the right indicates the quantities measured in the experiments. The bar shows the relative amounts of the band ratio (fluorescence/Coomassie). Values represent the means and SD from three independent experiments.


Figure 5A . Structural views suggesting that Mdm12 might have a preference for certain phospholipids . The surface representation of Mdm12 positioned around the binding site for the head group of PE is shown with charge distribution ( left ) and the sequence conservation ( right ) in the same orientation . Surface electrostatics and sequence conservation were calculated using an APBS program [ 31 ] with the nonlinear Poisson - Boltzmann equation and contoured at ± 3kT / e , and ConSurf website ( consurf . tau . ac . il ) [ 32 ] with 34 different yeast orthologs , respectively . The ribbon diagram shown in the middle indicates the overall orientation of Mdm12 . B . Lipid displacement experiments to identity the natural ligands of Mdm12in vitro ( see material and methods section for details ) . NBD - PE - preloaded Mdm12 - GST was mixed with a series of glycerophospholipids at different concentrations , and displacement of NBD - PE by non - labeled ligand was estimated from the decrease in fluorescence . Means ± SD are shown ( n = 3 independently performed experiments ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5A. Structural views suggesting that Mdm12 might have a preference for certain phospholipids. The surface representation of Mdm12 positioned around the binding site for the head group of PE is shown with charge distribution (left) and the sequence conservation (right) in the same orientation. Surface electrostatics and sequence conservation were calculated using an APBS program [31] with the nonlinear Poisson-Boltzmann equation and contoured at ±3kT/e, and ConSurf website (consurf.tau.ac.il) [32] with 34 different yeast orthologs, respectively. The ribbon diagram shown in the middle indicates the overall orientation of Mdm12.B. Lipid displacement experiments to identity the natural ligands of Mdm12in vitro (see material and methods section for details). NBD-PE-preloaded Mdm12-GST was mixed with a series of glycerophospholipids at different concentrations, and displacement of NBD-PE by non-labeled ligand was estimated from the decrease in fluorescence. Means ± SD are shown (n=3 independently performed experiments).


Figure 6A . Overall structure ( left ) and electron density ( right ) of Mdm12 in the asymmetric unit . Four molecules ( two Mdm12 dimers ) are organized with 2 - fold rotation symmetry . The 2 - fold axes are indicated with a black dotted line . B . Overall structure ( left ) and electron density ( right ) of ΔMdm12 in the asymmetric unit . Six ΔMdm12 molecules ( three Mdm12 dimers ) are arranged with 2 - fold rotation symmetry as shown above . C . Ribbon diagram showing a 2 - fold interface ( tail - to - tail ) in two crystal structures ( See text for details ) . D . Size - exclusion chromatography revealing that the N - terminus - truncated version of Mdm12 ( residues 11 - 271 ) retains the ability to interact with Mmm1 . The experiment was performed as in Figure 1B . Eluted fractions indicated by shading were subjected to SDS - PAGE followed by Coomassie Blue staining .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6A. Overall structure (left) and electron density (right) of Mdm12 in the asymmetric unit. Four molecules (two Mdm12 dimers) are organized with 2-fold rotation symmetry. The 2-fold axes are indicated with a black dotted line.B. Overall structure (left) and electron density (right) of ΔMdm12 in the asymmetric unit. Six ΔMdm12 molecules (three Mdm12 dimers) are arranged with 2-fold rotation symmetry as shown above.C. Ribbon diagram showing a 2-fold interface (tail-to-tail) in two crystal structures (See text for details).D. Size-exclusion chromatography revealing that the N-terminus-truncated version of Mdm12 (residues 11-271) retains the ability to interact with Mmm1. The experiment was performed as in Figure 1B. Eluted fractions indicated by shading were subjected to SDS-PAGE followed by Coomassie Blue staining.


Figure 1 ( C ) BN - PAGE and SDS - PAGE assays with inactive and activated Rx - NRC2 . C - terminally 6xHA tagged Rx and C - terminally 4xMyc - tagged NRC2EEE were co - expressed with either free GFP or C - terminally GFP - tagged CP . Free mCherry - 4xMyc and mCherry - 6xHA fusions were used as controls in the treatments without NRC2 and Rx , respectively . Total protein was extracted with a Tris - HCl - based buffer , as described in materials and methods . Extracts were run on native and denaturing PAGE assays in parallel and immunoblotted with the appropriate antisera labelled on the left . Approximate molecular weights ( kDa ) of the proteins are shown on the right . Red asterisks indicate bands corresponding to the activated NRC2 complex . Rubisco loading control was carried out using Ponceau stain ( PS ) . The experiment was repeated three times .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1(C) BN-PAGE and SDS-PAGE assays with inactive and activated Rx-NRC2. C-terminally 6xHA tagged Rx and C-terminally 4xMyc-tagged NRC2EEE were co-expressed with either free GFP or C-terminally GFP-tagged CP. Free mCherry-4xMyc and mCherry-6xHA fusions were used as controls in the treatments without NRC2 and Rx, respectively. Total protein was extracted with a Tris-HCl-based buffer, as described in materials and methods. Extracts were run on native and denaturing PAGE assays in parallel and immunoblotted with the appropriate antisera labelled on the left. Approximate molecular weights (kDa) of the proteins are shown on the right. Red asterisks indicate bands corresponding to the activated NRC2 complex. Rubisco loading control was carried out using Ponceau stain (PS). The experiment was repeated three times.