D Western blot analysis of ACE2 in HEK293T , 2fTGH , Caco - 2 and A549 cells treated with VitC at indicated concentrations for 24 hrs . Data are representative of three biological replicates .

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

D Western blot analysis of ACE2 in HEK293T, 2fTGH, Caco-2 and A549 cells treated with VitC at indicated concentrations for 24 hrs. Data are representative of three biological replicates.


G Western blot analysis of ACE2 , IRF3 and STAT1 in A549 cells treated with VitC at indicated concentrations for 24 hrs . Data are representative of three biological replicates .

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

G Western blot analysis of ACE2, IRF3 and STAT1 in A549 cells treated with VitC at indicated concentrations for 24 hrs. Data are representative of three biological replicates.


H Fluorescence microscopy of the SARS - CoV - 2 GFP / ΔN or VSV - GFP viruses in Caco - 2 - N cells pretreated with VitC ( 5 mM and 10 mM ) for 24 hrs , and then infected with SARS - CoV - 2 GFP / ΔN ( MOI = 0 . 1 ) or VSV - GFP ( MOI = 0 . 1 ) viruses for 24 hrs . Data are representative of three biological replicates . Scale bar : 100 µm .

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

H Fluorescence microscopy of the SARS-CoV-2 GFP/ΔN or VSV-GFP viruses in Caco-2-N cells pretreated with VitC (5 mM and 10 mM) for 24 hrs, and then infected with SARS-CoV-2 GFP/ΔN (MOI = 0.1) or VSV-GFP (MOI = 0.1) viruses for 24 hrs. Data are representative of three biological replicates. Scale bar: 100 µm.


A RT - qPCR analysis of Ace2 mRNA in 2fTGH cells treated with VitC ( 5 mM ) as indicated . Data are shown as mean and s . d . of three biological replicates ( n = 3 ) . N . S , not significant ( two - tailed unpaired Student ' s t - test ) .

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

A RT-qPCR analysis of Ace2 mRNA in 2fTGH cells treated with VitC (5 mM) as indicated. Data are shown as mean and s.d. of three biological replicates (n = 3). N.S, not significant (two-tailed unpaired Student's t-test).


F IP - IB analysis of ubiquitination types of Myc - ACE2 in HEK293T cells cotransfected with Myc - ACE2 and different types of HA - Ub , and then treated with VitC ( 5 mM ) for 12 hrs . The red dashed contour represents the group of K48 - linked ubiquitination we focused on studying here . Data are representative of three biological replicates .

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

F IP-IB analysis of ubiquitination types of Myc-ACE2 in HEK293T cells cotransfected with Myc-ACE2 and different types of HA-Ub, and then treated with VitC (5 mM) for 12 hrs. The red dashed contour represents the group of K48-linked ubiquitination we focused on studying here. Data are representative of three biological replicates.


H IP - IB analysis of K48 - Ub of endogenous ACE2 in 2fTGH cells treated with VitC ( 2 . 5 mM and 5 mM ) for 12 hrs . Data are representative of three biological replicates .

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

H IP-IB analysis of K48-Ub of endogenous ACE2 in 2fTGH cells treated with VitC (2.5 mM and 5 mM) for 12 hrs. Data are representative of three biological replicates.


C IP - IB analysis of the interaction between Flag - USP50 and Myc - ACE2 in HEK293T cells cotransfected with these two constructs . Data are representative of three biological replicates .

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

C IP-IB analysis of the interaction between Flag-USP50 and Myc-ACE2 in HEK293T cells cotransfected with these two constructs. Data are representative of three biological replicates.


G Western blot analysis of ACE2 in HEK293T cells transfected with Flag - USP50 and then treated with CHX ( 50 μM ) as indicated . Data are representative of three biological replicates .

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

G Western blot analysis of ACE2 in HEK293T cells transfected with Flag-USP50 and then treated with CHX (50 μM) as indicated. Data are representative of three biological replicates.


K Fluorescence microscopy of the VSV virus with a SARS - CoV - 2 - S gene and a GFP gene ( VSV - Spike ) in Usp50 + / + and Usp50 - / - HEK293T cells pretreated with or without VitC ( 5 mM ) for 12 hrs , followed by infection with the VSV - Spike virus ( MOI = 0 . 1 ) for 24 hrs . Scale bar : 100 µm . The average intensity each unit area was shown as mean and s . d . of three - unit areas ( n = 3 ) . N . S , not significant , * * * p < 0 . 001 ( two - tailed unpaired Student ' s t - test ) .

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

K Fluorescence microscopy of the VSV virus with a SARS-CoV-2-S gene and a GFP gene (VSV-Spike) in Usp50+/+ and Usp50-/- HEK293T cells pretreated with or without VitC (5 mM) for 12 hrs, followed by infection with the VSV-Spike virus (MOI = 0.1) for 24 hrs. Scale bar: 100 µm. The average intensity each unit area was shown as mean and s.d. of three-unit areas (n = 3). N.S, not significant, ***p < 0.001 (two-tailed unpaired Student's t-test).


D IP - IB analysis of ubiquitination types of Myc - ACE2 in HEK293T cells cotransfected with Myc - ACE2 , Flag - USP50 and different types of HA - Ub . The red dashed contour represents the group of K48 - linked ubiquitination we focused on studying here . Data are representative of three biological replicates .

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

D IP-IB analysis of ubiquitination types of Myc-ACE2 in HEK293T cells cotransfected with Myc-ACE2, Flag-USP50 and different types of HA-Ub. The red dashed contour represents the group of K48-linked ubiquitination we focused on studying here. Data are representative of three biological replicates.


E IP - IB analysis of K48 - Ub of endogenous ACE2 in HEK293T cells transfected with shCtrl ( - ) or shUSP50 ( # 1 , # 2 ) . Data are representative of three biological replicates .

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

E IP-IB analysis of K48-Ub of endogenous ACE2 in HEK293T cells transfected with shCtrl (-) or shUSP50 (#1, #2). Data are representative of three biological replicates.


B IP - IB analysis of Myc - ACE2 K48 - linked ubiquitination in HEK293T cells cotransfected with Myc - ACE2 ( WT or K788R ) and HA - K48 , together with shCtrl or shUSP50 . Data are representative of three biological replicates .

O O O O O O O O O O O O O O O O O O O O O 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 IP-IB analysis of Myc-ACE2 K48-linked ubiquitination in HEK293T cells cotransfected with Myc-ACE2 (WT or K788R) and HA-K48, together with shCtrl or shUSP50. Data are representative of three biological replicates.


C Western blot analysis of Myc - ACE2 in HEK293T cells transfected with Myc - ACE2 ( WT or K788R ) and then treated with CHX ( 50 μM ) as indicated . Data are representative of three biological replicates .

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

C Western blot analysis of Myc-ACE2 in HEK293T cells transfected with Myc-ACE2 (WT or K788R) and then treated with CHX (50 μM) as indicated. Data are representative of three biological replicates.


D IP - IB analysis of K48 - Ub of Myc - ACE2 in HEK293T cells transfected with Myc - ACE2 ( WT or K788R ) and then treated with VitC ( 5 mM ) for 12 hrs , by a specific anti - K48 - Ub antibody . Data are representative of three biological replicates .

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

D IP-IB analysis of K48-Ub of Myc-ACE2 in HEK293T cells transfected with Myc-ACE2 (WT or K788R) and then treated with VitC (5 mM) for 12 hrs, by a specific anti-K48-Ub antibody. Data are representative of three biological replicates.


G ACE2 - KO Caco - 2 cells transfected with ACE2 ( WT or K788R ) were treated with VitC ( 5 mM ) for 12 hrs , and then were infected with SARS - CoV - 2 GFP / ΔN ( MOI = 0 . 1 ) for 2 hrs . RT - qPCR was used to analyze SARS - CoV - 2 RNA levels . Data are shown as mean and s . d . of three biological replicates ( n = 3 ) . N . S , not significant , * * * p < 0 . 001 ( two - tailed unpaired Student ' s t - test ) .

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

G ACE2-KO Caco-2 cells transfected with ACE2 (WT or K788R) were treated with VitC (5 mM) for 12 hrs, and then were infected with SARS-CoV-2 GFP/ΔN (MOI = 0.1) for 2 hrs. RT-qPCR was used to analyze SARS-CoV-2 RNA levels. Data are shown as mean and s.d. of three biological replicates (n = 3). N.S, not significant, ***p < 0.001 (two-tailed unpaired Student's t-test).


A IP - IB analysis of the interaction between Flag - USP50 and Myc - ACE2 in HEK293T cells transfected with these two constructs and then treated with VitC ( 2 . 5 mM and 5 mM ) for 12 hrs . Data are representative of three biological replicates .

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

A IP-IB analysis of the interaction between Flag-USP50 and Myc-ACE2 in HEK293T cells transfected with these two constructs and then treated with VitC (2.5 mM and 5 mM) for 12 hrs. Data are representative of three biological replicates.


D Flag - USP50 proteins were obtained as ( C ) . Flag - USP50 and r - hACE2 proteins were mixed , together with increasing amounts of VitC or with VitC - Na + ( 20 mM ) . HCl is a pH control ( pH = 4 ) . After 2 hrs incubation , ACE2 proteins were analyzed by immunoblotting by a specific anti - ACE2 antibody . Data are representative of three biological replicates .

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

D Flag-USP50 proteins were obtained as (C). Flag-USP50 and r-hACE2 proteins were mixed, together with increasing amounts of VitC or with VitC-Na+ (20 mM). HCl is a pH control (pH = 4). After 2 hrs incubation, ACE2 proteins were analyzed by immunoblotting by a specific anti-ACE2 antibody. Data are representative of three biological replicates.


D Western blot analysis of hACE2 levels in lung tissues of hACE2 mice ( n = 6 ) administrated with VitC as ( B ) . Data are representative of three biological replicates .

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

D Western blot analysis of hACE2 levels in lung tissues of hACE2 mice (n = 6) administrated with VitC as (B). Data are representative of three biological replicates.


G RT - qPCR analysis of the SARS - CoV - 2 - Spike mRNA levels in lung , kidney , liver and spleen tissues of hACE2 mice treated with VitC and the VSV - Spike viruses as ( F ) . All graphs show the mean ± SEM for five individual mice ( n = 5 ) . * * * p < 0 . 001 ( two - tailed unpaired Student ' s t - test ) .

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

G RT-qPCR analysis of the SARS-CoV-2-Spike mRNA levels in lung, kidney, liver and spleen tissues of hACE2 mice treated with VitC and the VSV-Spike viruses as (F). All graphs show the mean ± SEM for five individual mice (n = 5). ***p < 0.001 (two-tailed unpaired Student's t-test).


( A - C ) qPCR and western blot analysis indicated an upregulation of hepatic ZBTB22 in diabetic db / db mice , obese ob / ob mice and high - fat diet for 12 weeks mice ( n = 6 mice ) . Data information : Data are means ± SEM . * * p < 0 . 01 , * * * p < 0 . 001 by Student ' s t test .

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

(A-C) qPCR and western blot analysis indicated an upregulation of hepatic ZBTB22 in diabetic db/db mice, obese ob/ob mice and high-fat diet for 12 weeks mice (n=6 mice). Data information: Data are means ± SEM. **p< 0.01, ***p< 0.001 by Student's t test.


( D ) qPCR and western blot analysis showed an increase of hepatic ZBTB22 in normal C57 mice after overnight fasting , and a recovery of ZBTB22 expression after refeeding ( n = 6 mice ) . Data information : Data are means ± SEM . * * p < 0 . 01 , * * * p < 0 . 001 by Student ' s t test .

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

(D) qPCR and western blot analysis showed an increase of hepatic ZBTB22 in normal C57 mice after overnight fasting, and a recovery of ZBTB22 expression after refeeding (n=6 mice). Data information: Data are means ± SEM. **p< 0.01, ***p< 0.001 by Student's t test.


( E ) qPCR and immunofluorescence analysis displayed an enhanced expression of ZBTB22 in MPHs after FSK treatment for 24h and a reduction of ZBTB22 in MPHs after insulin treatment for 15min ( n = 4 biological replicates ) , scale bars represent 10 μm . Data information : Data are means ± SEM . * * p < 0 . 01 , * * * p < 0 . 001 by Student ' s t test .

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

(E) qPCR and immunofluorescence analysis displayed an enhanced expression of ZBTB22 in MPHs after FSK treatment for 24h and a reduction of ZBTB22 in MPHs after insulin treatment for 15min (n=4 biological replicates), scale bars represent 10 μm. Data information: Data are means ± SEM. **p< 0.01, ***p< 0.001 by Student's t test.


( A - B ) qPCR analysis the change of genes involved in gluconeogenesis , lipogenesis and fatty acid oxidation in primary hepatocytes after ZBTB22 overexpression ( n = 4 biological replicates ) . Data information : Data are means ± SEM . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 by Student ' s t test .

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

(A-B) qPCR analysis the change of genes involved in gluconeogenesis, lipogenesis and fatty acid oxidation in primary hepatocytes after ZBTB22 overexpression (n=4 biological replicates). Data information: Data are means ± SEM. *p< 0.05, **p< 0.01, ***p< 0.001 by Student's t test.


( D - E ) ZBTB22 overexpression increase lipid deposition in primary hepatocytes , as shown by the enhanced lipid TOX probes and TG content ( n = 4 biological replicates ) , scale bars represent 10 μm . Data information : Data are means ± SEM . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 by Student ' s t test .

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

(D-E) ZBTB22 overexpression increase lipid deposition in primary hepatocytes, as shown by the enhanced lipid TOX probes and TG content (n=4 biological replicates), scale bars represent 10 μm. Data information: Data are means ± SEM. *p< 0.05, **p< 0.01, ***p< 0.001 by Student's t test.


( F - G ) qPCR analysis the change of genes involved in gluconeogenesis , lipogenesis , and fatty acid oxidation in primary hepatocytes after ZBTB22 knockdown ( n = 4 biological replicates ) . Data information : Data are means ± SEM . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 by Student ' s t test .

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

(F-G) qPCR analysis the change of genes involved in gluconeogenesis, lipogenesis, and fatty acid oxidation in primary hepatocytes after ZBTB22 knockdown (n=4 biological replicates). Data information: Data are means ± SEM. *p< 0.05, **p< 0.01, ***p< 0.001 by Student's t test.


( I - J ) ZBTB22 knockdown suppress lipid deposition in primary hepatocytes , as shown by the weakened lipid TOX probes and TG content ( n = 4 biological replicates ) , scale bars represent 10 μm . Data information : Data are means ± SEM . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 by Student ' s t test .

(I-J) ZBTB22 knockdown suppress lipid deposition in primary hepatocytes, as shown by the weakened lipid TOX probes and TG content (n=4 biological replicates), scale bars represent 10 μm. Data information: Data are means ± SEM. *p< 0.05, **p< 0.01, ***p< 0.001 by Student's t test.


( K ) qPCR analysis of mRNA levels of Pgc1α , PCK1 in primary hepatocytes infected with Ad - shCtrl or Ad - shZBTB22 with or without forskolin treatment ( n = 4 biological replicates ) . Data information : Data are means ± SEM . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 by Student ' s t test .

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

(K) qPCR analysis of mRNA levels of Pgc1α, PCK1 in primary hepatocytes infected with Ad-shCtrl or Ad-shZBTB22 with or without forskolin treatment (n=4 biological replicates). Data information: Data are means ± SEM. *p< 0.05, **p< 0.01, ***p< 0.001 by Student's t test.


ZBTB22 deletion effectively decrease the fat mass of mice fed on HFD ( n = 6 mice ) . Data information : Data are means ± SEM . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 by Student ' s t test .

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

ZBTB22 deletion effectively decrease the fat mass of mice fed on HFD (n=6 mice). Data information: Data are means ± SEM. *p< 0.05, **p< 0.01, ***p< 0.001 by Student's t test.


( D ) ZBTB22 deletion enhanced the expression of genes involved in thermogenesis and fatty acid oxidation in brown adipose tissue from mice fed on HFD ( n = 6 mice ) . Data information : Data are means ± SEM . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 by Student ' s t test .

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

(D) ZBTB22 deletion enhanced the expression of genes involved in thermogenesis and fatty acid oxidation in brown adipose tissue from mice fed on HFD (n=6 mice). Data information: Data are means ± SEM. *p< 0.05, **p< 0.01, ***p< 0.001 by Student's t test.


( E ) ZBTB22 deletion increased the heat production , CO2 generation and O2 consumption in mice fed on HFD ( n = 3 mice ) .

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

(E) ZBTB22 deletion increased the heat production, CO2 generation and O2 consumption in mice fed on HFD (n=3 mice).


ZBTB22 deletion effectively decrease the fasting blood glucose of mice fed on HFD ( n = 6 mice ) . Data information : Data are means ± SEM . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 by Student ' s t test .

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

ZBTB22 deletion effectively decrease the fasting blood glucose of mice fed on HFD (n=6 mice). Data information: Data are means ± SEM. *p< 0.05, **p< 0.01, ***p< 0.001 by Student's t test.


( D ) ZBTB22 deletion significantly changed hepatic expression of genes involved in gluconeogenesis , lipogenesis and fatty acid oxidation in mice fed on HFD ( n = 6 mice ) . Data information : Data are means ± SEM . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 by Student ' s t test .

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

(D) ZBTB22 deletion significantly changed hepatic expression of genes involved in gluconeogenesis, lipogenesis and fatty acid oxidation in mice fed on HFD (n=6 mice). Data information: Data are means ± SEM. *p< 0.05, **p< 0.01, ***p< 0.001 by Student's t test.


( E ) ZBTB22 deletion significantly improved the phosphorylation of AKT and GSK3β in the liver of mice fed on HFD .

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

(E) ZBTB22 deletion significantly improved the phosphorylation of AKT and GSK3β in the liver of mice fed on HFD.


( I ) Hepatic ZBTB22 overexpression induces glucose intolerance and insulin resistance in ZBTB22 deficiency mice fed on HFD ( n = 6 mice ) .

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

(I) Hepatic ZBTB22 overexpression induces glucose intolerance and insulin resistance in ZBTB22 deficiency mice fed on HFD (n=6 mice).


( J ) Hepatic ZBTB22 overexpression decreased the insulin - induced phosphorylation of AKT and GSK3β in the liver of ZBTB22 deficiency mice fed on HFD .

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

(J) Hepatic ZBTB22 overexpression decreased the insulin-induced phosphorylation of AKT and GSK3β in the liver of ZBTB22 deficiency mice fed on HFD.


( K ) qPCR analysis of hepatic genes involved in gluconeogenesis , lipogenesis and fatty acid oxidation in ZBTB22 deficiency mice fed on HFD ( n = 6 mice ) . Data information : Data are means ± SEM . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 by Student ' s t test .

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

(K) qPCR analysis of hepatic genes involved in gluconeogenesis, lipogenesis and fatty acid oxidation in ZBTB22 deficiency mice fed on HFD (n=6 mice). Data information: Data are means ± SEM. *p< 0.05, **p< 0.01, ***p< 0.001 by Student's t test.


( A - B ) Hepatic ZBTB22 silencing decreases the expression of gene involved in gluconeogenesis , lipogenesis , and fatty acid oxidation in db / db mice ( n = 6 mice ) . Data information : Data are means ± SEM . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 by Student ' s t test .

(A-B) Hepatic ZBTB22 silencing decreases the expression of gene involved in gluconeogenesis, lipogenesis, and fatty acid oxidation in db/db mice (n=6 mice). Data information: Data are means ± SEM. *p< 0.05, **p< 0.01, ***p< 0.001 by Student's t test.


( D ) Hepatic ZBTB22 silencing improved the glucose tolerance , insulin sensitivity and pyruvate tolerance in db / db mice ( n = 6 mice ) . Data information : Data are means ± SEM . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 by Student ' s t test .

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

(D) Hepatic ZBTB22 silencing improved the glucose tolerance, insulin sensitivity and pyruvate tolerance in db/db mice (n=6 mice). Data information: Data are means ± SEM. *p< 0.05, **p< 0.01, ***p< 0.001 by Student's t test.


( J - K ) Hepatic ZBTB22 silencing decreases serum inflammatory cytokines levels and hepatic inflammatory genes expression ( n = 6 mice ) . Data information : Data are means ± SEM . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 by Student ' s t test .

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

(J-K) Hepatic ZBTB22 silencing decreases serum inflammatory cytokines levels and hepatic inflammatory genes expression (n=6 mice). Data information: Data are means ± SEM. *p< 0.05, **p< 0.01, ***p< 0.001 by Student's t test.


Hepatic ZBTB22 overexpression increases the expression of genes involved in gluconeogenesis in normal C57 mice ( n = 6 mice ) . Data information : Data are means ± SEM . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 by Student ' s t test .

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

Hepatic ZBTB22 overexpression increases the expression of genes involved in gluconeogenesis in normal C57 mice (n=6 mice). Data information: Data are means ± SEM. *p< 0.05, **p< 0.01, ***p< 0.001 by Student's t test.


( C ) Hepatic ZBTB22 overexpression significantly induced glucose intolerance and insulin resistance in normal C57 mice ( n = 6 mice ) . Data information : Data are means ± SEM . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 by Student ' s t test .

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

(C) Hepatic ZBTB22 overexpression significantly induced glucose intolerance and insulin resistance in normal C57 mice (n=6 mice). Data information: Data are means ± SEM. *p< 0.05, **p< 0.01, ***p< 0.001 by Student's t test.


( D ) Hepatic ZBTB22 overexpression significantly decreased insulin - induced phosphorylation of hepatic AKT and GSK3β in normal C57 mice .

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

(D) Hepatic ZBTB22 overexpression significantly decreased insulin-induced phosphorylation of hepatic AKT and GSK3β in normal C57 mice.


Hepatic ZBTB22 overexpression increase serum inflammatory cytokines levels Data information : Data are means ± SEM . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 by Student ' s t test .

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

Hepatic ZBTB22 overexpression increase serum inflammatory cytokines levels Data information: Data are means ± SEM. *p< 0.05, **p< 0.01, ***p< 0.001 by Student's t test.


Hepatic ZBTB22 overexpression increase hepatic expression of inflammatory genes ( n = 6 mice ) . Data information : Data are means ± SEM . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 by Student ' s t test .

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

Hepatic ZBTB22 overexpression increase hepatic expression of inflammatory genes (n=6 mice). Data information: Data are means ± SEM. *p< 0.05, **p< 0.01, ***p< 0.001 by Student's t test.


Western Blot analysis showed that ZBTB22 - mediated induced the up - regulated expression of PEPCK1 in Ad - ZBTB22 infected mice primary hepatocytes and livers ( C )

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

Western Blot analysis showed that ZBTB22-mediated induced the up-regulated expression of PEPCK1 in Ad-ZBTB22 infected mice primary hepatocytes and livers (C)


Chip and Chip - qPCR displayed increase occupancy of ZBTB22 on the promoter region of PCK1 ( n = 3 biological replicates ) .

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

Chip and Chip-qPCR displayed increase occupancy of ZBTB22 on the promoter region of PCK1 (n=3 biological replicates).


PCK1 silencing , as well as the stimulatory effects of ZBTB22 on SREBP1C expression ( J ) ( n = 4 biological replicates ) and cellular glucose output ( K ) ( n = 4 biological replicates ) Data information : Data are means ± SEM . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 by Student ' s t test .

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

PCK1 silencing , as well as the stimulatory effects of ZBTB22 on SREBP1C expression (J) (n=4 biological replicates) and cellular glucose output (K) (n=4 biological replicates) Data information: Data are means ± SEM. *p< 0.05, **p< 0.01, ***p< 0.001 by Student's t test.


Hepatic ZBTB22 overexpression failed to change glucose tolerance , insulin sensitivity in PCK1 - silencing C57 mice ( n = 6 mice ) . Data information : Data are means ± SEM . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 by Student ' s t test .

Hepatic ZBTB22 overexpression failed to change glucose tolerance, insulin sensitivity in PCK1-silencing C57 mice (n=6 mice). Data information: Data are means ± SEM. *p < 0.05, **p< 0.01, ***p< 0.001 by Student's t test.


( G ) Hepatic ZBTB22 overexpression failed to change serum and hepatic TG and TC levels in PCK1 - silencing C57 mice ( n = 6 mice ) . Data information : Data are means ± SEM . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 by Student ' s t test .

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

(G) Hepatic ZBTB22 overexpression failed to change serum and hepatic TG and TC levels in PCK1-silencing C57 mice (n=6 mice). Data information: Data are means ± SEM. *p < 0.05, **p< 0.01, ***p< 0.001 by Student's t test.


B , ITC experiment shows the CBS - pair domain of CNNM3 binds PRL2 with a nanomolar Kd and one - to - one stoichiometry .

B, ITC experiment shows the CBS-pair domain of CNNM3 binds PRL2 with a nanomolar Kd and one-to-one stoichiometry.


D , Structure of CBS - pair domain of CNNM2 ( PDB 4IY0 ) with the residues DPFYE indicated .

D, Structure of CBS-pair domain of CNNM2 (PDB 4IY0) with the residues DPFYE indicated.


F , ITC data of CBS - pair domain mutants binding to PRL2 identifies aspartic acid 426 as essential for binding .

F, ITC data of CBS-pair domain mutants binding to PRL2 identifies aspartic acid 426 as essential for binding.


G , Kinetics of hydrolysis of a synthetic substrate by PRL2 shows burst kinetics . Addition of the wild - type CBS - pair domain but not the D426A mutant inhibits phosphatase activity .

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

G, Kinetics of hydrolysis of a synthetic substrate by PRL2 shows burst kinetics. Addition of the wild-type CBS-pair domain but not the D426A mutant inhibits phosphatase activity.


A , Treatment of PRL2 with a synthetic substrate leads to complete phosphorylation of the catalytic residue , cysteine 101 . Recombinant PRL2 was treated with 3 - O - methylfluorescein phosphate ( OMFP ) and analyzed by SDS - PAGE with the Phos - tag reagent . Phosphocysteine is hydrolyzed by boiling .

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

A, Treatment of PRL2 with a synthetic substrate leads to complete phosphorylation of the catalytic residue, cysteine 101. Recombinant PRL2 was treated with 3-O-methylfluorescein phosphate (OMFP) and analyzed by SDS-PAGE with the Phos-tag reagent. Phosphocysteine is hydrolyzed by boiling.


B , Detection of phosphorylated by electrospray mass spectrometry . OMFP - treatment increased the mass of the intact protein by 79 . 99 amu but had no effect on the C101A mutant .

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

B, Detection of phosphorylated by electrospray mass spectrometry. OMFP-treatment increased the mass of the intact protein by 79.99 amu but had no effect on the C101A mutant.


C , 31P NMR spectrum of the thioPO3 signal from phosphocysteine in PRL2 . The broad peak is TCEP in the buffer .

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

C, 31P NMR spectrum of the thioPO3 signal from phosphocysteine in PRL2. The broad peak is TCEP in the buffer.


D , Cysteine phosphorylated PRL2 does not bind to CNNM3 . Recombinant PRL2 was phosphorylated by treatment with OMFP and subjected to pull - down by the CBS - pair domain of CNNM3 as a GST fusion . Only unphosphorylated PRL2 was observed in the pull - down lanes while phosphorylated PRL2 was in the flow - through lanes .

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

D, Cysteine phosphorylated PRL2 does not bind to CNNM3. Recombinant PRL2 was phosphorylated by treatment with OMFP and subjected to pull-down by the CBS-pair domain of CNNM3 as a GST fusion. Only unphosphorylated PRL2 was observed in the pull-down lanes while phosphorylated PRL2 was in the flow-through lanes.


E , ITC measurement of the affinity of PRL2 for the CNNM3 GST - CBS - pair domain . Phosphorylation by OMFP completely blocked binding while oxidization of PRL2 by diamide decreased the affinity 200 - fold .

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

E, ITC measurement of the affinity of PRL2 for the CNNM3 GST-CBS-pair domain. Phosphorylation by OMFP completely blocked binding while oxidization of PRL2 by diamide decreased the affinity 200-fold.


F , The cysteine residues in the PRL2 active site are not required for binding the CNNM3 CBS - pair domain .

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

F, The cysteine residues in the PRL2 active site are not required for binding the CNNM3 CBS-pair domain.


A , Mutations in the CBS - pair loop of CNNM4 prevent binding to PRL3 . Lysates of COS7 cells transfected with the indicated constructs were subjected to immunoprecipitation ( IP ) and immunoblotting ( IB ) with the indicated antibodies .

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

A, Mutations in the CBS-pair loop of CNNM4 prevent binding to PRL3. Lysates of COS7 cells transfected with the indicated constructs were subjected to immunoprecipitation (IP) and immunoblotting (IB) with the indicated antibodies.


B C , Wild - type CNNM4 - dependent magnesium efflux is inhibited by co - transfection by PRL3 but the efflux by the CNNM4 D485A mutant , which is unable to bind PRL3 , is resistant to inhibition . HEK293 cells transfected with the indicated constructs were loaded with Magnesium Green and Mg2 + removed from the medium at the indicated time point ( arrowhead ) . The means of relative fluorescence intensities of 10 cells are graphed as a function of time in panel B and the mean and s . e . m . at 5 min plotted in panel C . P - values were calculated using one way ANOVA followed by Bonferroni ' s multiple comparison test . n . s . , not significant .

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

B C, Wild-type CNNM4-dependent magnesium efflux is inhibited by co-transfection by PRL3 but the efflux by the CNNM4 D485A mutant, which is unable to bind PRL3, is resistant to inhibition. HEK293 cells transfected with the indicated constructs were loaded with Magnesium Green and Mg2+ removed from the medium at the indicated time point (arrowhead). The means of relative fluorescence intensities of 10 cells are graphed as a function of time in panel B and the mean and s.e.m. at 5 min plotted in panel C. P-values were calculated using one way ANOVA followed by Bonferroni's multiple comparison test. n.s., not significant.


A , The CBS - pair domain ( yellow ) forms a dimer and binds two molecules of PRL2 via an extended loop . The positions of the PRL2 C - terminal CAAX prenylation motif [ 42 ] and CNNM3 N - terminus suggest that the membrane would sit above the complex .

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

A, The CBS-pair domain (yellow) forms a dimer and binds two molecules of PRL2 via an extended loop. The positions of the PRL2 C-terminal CAAX prenylation motif [42] and CNNM3 N-terminus suggest that the membrane would sit above the complex.


B , Contacts between the CNNM3 CBS2 loop and the PRL2 active site .

B, Contacts between the CNNM3 CBS2 loop and the PRL2 active site.


C , Stereoview of the 2FO - FC omit map of the PRL2 binding loop of CNNM3 contoured at 1 .

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

C, Stereoview of the 2FO-FC omit map of the PRL2 binding loop of CNNM3 contoured at 1 .


D , Detail of complex with reduced PRL2 , showing how CNNM3 Asp426 inserts into the pocket adjacent to the catalytic cysteine Cys101 .

D, Detail of complex with reduced PRL2, showing how CNNM3 Asp426 inserts into the pocket adjacent to the catalytic cysteine Cys101.


E , Model of phosphocysteine preventing CNNM binding . Crystal structure of PRL1 with a bound sulfate shows phosphocysteine would prevent Asp426 binding . Residues are numbered according to the PRL1 structure ( PDB 1XM2 ) .

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

E, Model of phosphocysteine preventing CNNM binding. Crystal structure of PRL1 with a bound sulfate shows phosphocysteine would prevent Asp426 binding. Residues are numbered according to the PRL1 structure (PDB 1XM2).


A , PRLs expressed in HEK293 cells are intrinsically phosphorylated on cysteine . Phos - tag analysis of cell lysates shows close to 50 % of transiently expressed FLAG - PRL1 , - 2 , and - 3 are phosphorylated . The phosphorylated PRL bands are marked by an asterisk .

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

A, PRLs expressed in HEK293 cells are intrinsically phosphorylated on cysteine. Phos-tag analysis of cell lysates shows close to 50% of transiently expressed FLAG-PRL1, -2, and -3 are phosphorylated. The phosphorylated PRL bands are marked by an asterisk.


B , Immunoblots of PRL1 expressed in HEK293 cells show phosphorylation requires the catalytic cysteine ( C104 ) but not the adjacent cysteine ( C49 ) in the active site .

O O O O O O O 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, Immunoblots of PRL1 expressed in HEK293 cells show phosphorylation requires the catalytic cysteine (C104) but not the adjacent cysteine (C49) in the active site.


C , Co - immunoprecipitation of Myc - PRL by FLAG - CNNMs shows only the unphosphorylated forms of PRL1 binds CNNM4 .

C, Co-immunoprecipitation of Myc-PRL by FLAG-CNNMs shows only the unphosphorylated forms of PRL1 binds CNNM4.


D , Endogenous PRLs in HeLa cells are cysteine phosphorylated .

D, Endogenous PRLs in HeLa cells are cysteine phosphorylated.


E , Levels of unphosphorylated PRL increase in cultured cells in response to removal of magnesium from the cultured medium .

E, Levels of unphosphorylated PRL increase in cultured cells in response to removal of magnesium from the cultured medium.


( A ) Third instar larval skeletal musculature stained with Phalloidin ( F - actin ) . In this and subsequent figures , we assayed the ventral longitudinal muscles ( highlighted in green ) .

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

(A) Third instar larval skeletal musculature stained with Phalloidin (F-actin). In this and subsequent figures, we assayed the ventral longitudinal muscles (highlighted in green).


( B - D ) GFP - Atg8 , overexpressed using the Dmef2 - Gal4 driver , labels autophagosomes . ( B ) , starved on low - nutrientfood for 6 h ( C ) , or starved on low - nutrientfood + 2 . 5 mg / ml CQ for 6 h ( D ) . GFP - Atg8 - labeled vesicles appeared only in the starved animals ( C - D ) , localizing around the nucleus and between myofibers . ( D ) CQ treatment caused accumulation of bloated GFP - Atg8 - labeled vesicles .

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

(B-D) GFP-Atg8, overexpressed using the Dmef2-Gal4 driver, labels autophagosomes. (B), starved on low-nutrientfood for 6 h (C), or starved on low-nutrientfood +2.5 mg/ml CQ for 6 h (D). GFP-Atg8-labeled vesicles appeared only in the starved animals (C-D), localizing around the nucleus and between myofibers. (D) CQ treatment caused accumulation of bloated GFP-Atg8-labeled vesicles.


( E - G ) Dmef2 - Gal4 , UAS - GFP - Atg8 / UAS - HRP - Lamp1 animals were assayed for Lamp1 and Atg8 localization ( anti - HRP , red ; GFP , green ; DAPI , blue ) . . ( E ) High - nutrient food suppressed formation of both GFP - Atg8 and HRP - Lamp1 - labeled vesicles . ( F ) Colocalization of GFP - Atg8 and HRP - Lamp in animals starved on low - nutrient food . The yellow arrowhead points to a vesicle positive for both Atg8 and Lamp . ( G ) Addition of CQ to the starvation diet resulted in accumulation of both GFP - Atg8 and HRP - Lamp - labeled vesicles , but they failed to colocalize .

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

(E-G) Dmef2-Gal4, UAS-GFP-Atg8/UAS-HRP-Lamp1 animals were assayed for Lamp1 and Atg8 localization (anti-HRP, red; GFP, green; DAPI, blue). . (E) High-nutrient food suppressed formation of both GFP-Atg8 and HRP-Lamp1-labeled vesicles. (F) Colocalization of GFP-Atg8 and HRP-Lamp in animals starved on low-nutrient food. The yellow arrowhead points to a vesicle positive for both Atg8 and Lamp. (G) Addition of CQ to the starvation diet resulted in accumulation of both GFP-Atg8 and HRP-Lamp-labeled vesicles, but they failed to colocalize.


( H ) Quantification of the number of GFP - Atg8 , HRP - Lamp1 , or GFP - Atg8 + HRP - Lamp1 vesicles in starved or starved + CQ muscles .

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

(H) Quantification of the number of GFP-Atg8, HRP-Lamp1, or GFP-Atg8+HRP-Lamp1 vesicles in starved or starved +CQ muscles.


( I - M ) The core Atg genes are required for starvation - induced autophagy in both wild - type and CQ - treated skeletal muscles . Dmef2 - Gal4 , UAS - GFP - Atg8 / UAS - Atg1 larvae were starved on low - nutrient food for 6 h ( I ) or starved on low - nutrient food + 2 . 5 mg / ml CQ for 6 h ( J ) . Note that Atg1 knockdown completely abolished the formation of GFP - Atg8 - labeled autophagosomes ( compare I - J to C - D ) . ( K - L ) Dmef2 - Gal4 , UAS - GFP - Atg8 , Atg1Δ3d larvae failed to form GFP - Atg8 vesicles when starved or starved and treated with CQ . ( M ) Quantification of autophagy changes due to Atg gene knockdown in Dmef2 - Gal4 , UAS - GFP - Atg8 larvae starved on low - nutrient food + 2 . 5 mg / ml CQ for 6 h . Each of the 10 UAS - Atg RNAi transgenes tested caused a highly significant decrease ( p < . 01 ) in the total area of GFP - Atg8vesicles . SEM is indicated , with n = 5 ventral longitudinal muscles from individual animals .

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

(I-M) The core Atg genes are required for starvation-induced autophagy in both wild-type and CQ-treated skeletal muscles. Dmef2-Gal4, UAS-GFP-Atg8/UAS-Atg1 larvae were starved on low-nutrient food for 6 h (I) or starved on low-nutrient food +2.5 mg/ml CQ for 6 h (J). Note that Atg1 knockdown completely abolished the formation of GFP-Atg8-labeled autophagosomes (compare I-J to C-D). (K-L) Dmef2-Gal4, UAS-GFP-Atg8, Atg1Δ3d larvae failed to form GFP-Atg8 vesicles when starved or starved and treated with CQ. (M) Quantification of autophagy changes due to Atg gene knockdown in Dmef2-Gal4, UAS-GFP-Atg8 larvae starved on low-nutrient food +2.5 mg/ml CQ for 6 h. Each of the 10 UAS-Atg RNAi transgenes tested caused a highly significant decrease (p<.01) in the total area of GFP-Atg8vesicles. SEM is indicated, with n = 5 ventral longitudinal muscles from individual animals.


. ( N - O ) EM of muscles from Dmef2 - Gal4 , UAS - whitei larvae . Animals starved on low - nutrient food + 2 . 5 mg / ml CQ ( O ) accumulated vesicles in the intermyofibril spaces ( red asterisk ) , disrupting the integrity of the sarcomere compared to non - CQ - treated control muscles ( N ) .

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

. (N-O) EM of muscles from Dmef2-Gal4, UAS-whitei larvae. Animals starved on low-nutrient food +2.5 mg/ml CQ (O) accumulated vesicles in the intermyofibril spaces (red asterisk), disrupting the integrity of the sarcomere compared to non-CQ-treated control muscles (N).


( P ) CQ treatment increased the larval crawling time of Dmef2 - Gal4 , UAS - whitei larvae in starved animals , and weakly in fed animals . ( Q ) CQ treatment increased the larval righting time of Dmef2 - Gal4 , UAS - whitei larvae in starved but not fed animals . For both locomotor assays , SEM is indicated for n = 10 larvae ( * p < . 05 , * * p < . 01 ) .

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

(P) CQ treatment increased the larval crawling time of Dmef2-Gal4, UAS-whitei larvae in starved animals, and weakly in fed animals. (Q) CQ treatment increased the larval righting time of Dmef2-Gal4, UAS-whitei larvae in starved but not fed animals. For both locomotor assays, SEM is indicated for n = 10 larvae (*p<.05, **p<.01).


( A ) Sectioned third instar OreR larva stained with Periodic acid - Schiff ( PAS ) . The muscles , but not the fat body , are stained purple , indicating high levels of glycogen ( m , muscle ; bw , body wall ; fb , fat body ) .

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

(A) Sectioned third instar OreR larva stained with Periodic acid-Schiff (PAS). The muscles, but not the fat body, are stained purple, indicating high levels of glycogen (m, muscle; bw, body wall; fb, fat body).


( B ) Glycogen was also detected in muscle from Dmef2 - Gal4 , UAS - GFP - Atg8 larvae , immunostained with an antiglycogen monoclonal antibody .

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

(B) Glycogen was also detected in muscle from Dmef2-Gal4, UAS-GFP-Atg8 larvae, immunostained with an antiglycogen monoclonal antibody.


( C ) GFP - Atg8 vesicles colocalized with glycogen in Dmef2 - Gal4 , UAS - GFP - Atg8 larvae starved on low - nutrient food + 2 . 5 mg / ml CQ for 6 h ( GFP , green ; antiglycogen , red ) .

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

(C) GFP-Atg8 vesicles colocalized with glycogen in Dmef2-Gal4, UAS-GFP-Atg8 larvae starved on low-nutrient food +2.5 mg/ml CQ for 6 h (GFP, green; antiglycogen, red).


D ) HRP - Lamp1 vesicles show less colocalization with glycogen in UAS - HRP - Lamp1 ; Dmef2 - Gal4 larvae starved and treated with CQ .

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

D) HRP-Lamp1 vesicles show less colocalization with glycogen in UAS-HRP-Lamp1;Dmef2-Gal4 larvae starved and treated with CQ.


( E ) Quantification of GFP - Atg8 or HRP - Lamp1 vesicles with glycogen .

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

(E) Quantification of GFP-Atg8 or HRP-Lamp1 vesicles with glycogen.


( F - G ) EM from Dmef2 - Gal4 , UAS - whitei larvae starved on low - nutrient food + 2 . 5 mg / ml CQ for 6 h . ( F ) Double - and single - membrane vesicles containing glycogen granules accumulated between myofibers ( s , sarcomere ; m , mitochondrion ; AVs , autophagic vesicles ) . ( G ) Higher magnification view of region outlined in ( E ) . ( H ) CQ treatment is not required for glycogen autophagy as seen in an EM from a Dmef2 - Gal4 , UAS - whitei larva starved on low - nutrient food for 6 h . Arrow points to double membrane .

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

(F-G) EM from Dmef2-Gal4, UAS-whitei larvae starved on low-nutrient food +2.5 mg/ml CQ for 6 h. (F) Double- and single-membrane vesicles containing glycogen granules accumulated between myofibers (s, sarcomere; m, mitochondrion; AVs, autophagic vesicles). (G) Higher magnification view of region outlined in (E). (H) CQ treatment is not required for glycogen autophagy as seen in an EM from a Dmef2-Gal4, UAS-whitei larva starved on low-nutrient food for 6 h. Arrow points to double membrane.


( A - D ) Time course of autophagy induction in Dmef2 - Gal4 , UAS - GFP - Atg8 muscles , accompanied by quantification of GFP - Atg8 and glycogen colocalization . Animals were fed for 18 h in high - nutrient food + 2 . 5 mg / ml CQ , then starved on low - nutrient food + 2 . 5 mg / ml CQ for 0 - 8 h ( antiglycogen , red ; GFP , green ; DAPI , blue ) . ( A ) At time point 0 , following 18 h in high - nutrient food + CQ , the muscles contained large amounts of glycogen with no apparent autophagy . ( B ) At 3 h of starvation , glycogen stores were still high , and GFP - Atg8 - labeled vesicles began to appear . ( C - D ) At 6 and 8 h of starvation , the majority of GFP - Atg8 - labeled vesicles colocalized with glycogen .

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

(A-D) Time course of autophagy induction in Dmef2-Gal4, UAS-GFP-Atg8 muscles, accompanied by quantification of GFP-Atg8 and glycogen colocalization. Animals were fed for 18 h in high-nutrient food +2.5 mg/ml CQ, then starved on low-nutrient food +2.5 mg/ml CQ for 0-8 h (antiglycogen, red; GFP, green; DAPI, blue). (A) At time point 0, following 18 h in high-nutrient food +CQ, the muscles contained large amounts of glycogen with no apparent autophagy. (B) At 3 h of starvation, glycogen stores were still high, and GFP-Atg8-labeled vesicles began to appear. (C-D) At 6 and 8 h of starvation, the majority of GFP-Atg8-labeled vesicles colocalized with glycogen.


( E ) Time course of glycogen levels in Dmef2 - Gal4 carcasses ( muscle + body wall ) . Animals were fed for 24 h in high - nutrient food , then starved on low - nutrient food + / − 2 . 5 mg / ml CQ for 0 - 24 h . Starvation caused reduction of glycogen levels in both untreated and CQ - treated larvae over time . However , after 6 h of starvation , CQ treatment significantly increased glycogen levels compared to controls . SEM is indicated for n = 5 - 8 samples ( * p < . 05 , * * p < . 01 ) .

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

(E) Time course of glycogen levels in Dmef2-Gal4 carcasses (muscle+body wall). Animals were fed for 24 h in high-nutrient food, then starved on low-nutrient food +/− 2.5 mg/ml CQ for 0-24 h. Starvation caused reduction of glycogen levels in both untreated and CQ-treated larvae over time. However, after 6 h of starvation, CQ treatment significantly increased glycogen levels compared to controls. SEM is indicated for n = 5-8 samples (*p<.05, **p<.01).


( F - G ) activation of the Tor pathway blocked autophagy in the muscles from larvae starved on low - nutrient food + 2 . 5 mg / ml CQ for 6 h . ( F ) Autophagy levels were high in control Dmef2 - Gal4 / UAS - whitei larvae . Muscles from ( G ) UAS - Rheb / + ; Dmef2 - Gal4 / + , ( H ) Dmef2 - Gal4 / UAS - Tsc1i , and ( I ) Dmef2 - Gal4 / UAS - gigi all failed to induce autophagy .

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

(F-G) activation of the Tor pathway blocked autophagy in the muscles from larvae starved on low-nutrient food +2.5 mg/ml CQ for 6 h. (F) Autophagy levels were high in control Dmef2-Gal4/UAS-whitei larvae. Muscles from (G) UAS-Rheb/+; Dmef2-Gal4/+, (H) Dmef2-Gal4/UAS-Tsc1i, and (I) Dmef2-Gal4/UAS-gigi all failed to induce autophagy.


( A - B ) Glycogen phosphorylase is not required for glycogen autophagy ( antiglycogen , red ; GFP , green ; DAPI , blue ) . ( A ) UAS - GlyPi / + ; Dmef2 - Gal4 , UAS - GFP - Atg8 / + larvae starved on low - nutrient food + 2 . 5 mg / ml CQ for 6 h exhibited high levels of colocalization between GFP - Atg8 and glycogen . ( B ) Higher magnification of region outlined in ( A )

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

(A-B) Glycogen phosphorylase is not required for glycogen autophagy (antiglycogen, red; GFP, green; DAPI, blue). (A) UAS-GlyPi/+; Dmef2-Gal4, UAS-GFP-Atg8/+ larvae starved on low-nutrient food +2.5 mg/ml CQ for 6 h exhibited high levels of colocalization between GFP-Atg8 and glycogen.(B) Higher magnification of region outlined in (A)


C - F ) Dmef2 - Gal4 , UAS - GFP - Atg8 larvae with GlyP and / or Atg1 knockdown were fed on high - nutrient food for 18 h before being starved on low - nutrient food ( antiglycogen , red ; GFP , green ; DAPI , blue ) . ( C ) UAS - GlyPi / + ; Dmef2 - Gal4 , UAS - GFP - Atg8 / + larval muscle contained high levels of glycogen prior to starvation , indicating no defect in glycogen synthesis . ( D ) Following 24 h starvation UAS - GlyPi / + ; Dmef2 - Gal4 , UAS - GFP - Atg8 / + muscles contained no glycogen detectable by antibody staining . ( E ) Following 24 h of starvation Dmef2 - Gal4 , UAS - GFP - Atg8 / UAS - Atg1i muscles contained no glycogen . ( F ) Double - mutant larvae UAS - GlyPi / + ; Dmef2 - Gal4 , UAS - GFP - Atg8 / UAS - Atg1i larval muscles contained high levels of glycogen after 24 h of starvation , indicating an inability to break down glycogen .

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

C-F) Dmef2-Gal4, UAS-GFP-Atg8 larvae with GlyP and/or Atg1 knockdown were fed on high-nutrient food for 18 h before being starved on low-nutrient food (antiglycogen, red; GFP, green; DAPI, blue). (C) UAS-GlyPi/+; Dmef2-Gal4, UAS-GFP-Atg8/+ larval muscle contained high levels of glycogen prior to starvation, indicating no defect in glycogen synthesis. (D) Following 24 h starvation UAS-GlyPi/+; Dmef2-Gal4, UAS-GFP-Atg8/+ muscles contained no glycogen detectable by antibody staining. (E) Following 24 h of starvation Dmef2-Gal4, UAS-GFP-Atg8/UAS-Atg1i muscles contained no glycogen. (F) Double-mutant larvae UAS-GlyPi/+; Dmef2-Gal4, UAS-GFP-Atg8/UAS-Atg1i larval muscles contained high levels of glycogen after 24 h of starvation, indicating an inability to break down glycogen.


( G ) Time course of glycogen levels in Dmef2 - Gal4 carcasses ( muscle + body wall ) with expression of UAS - RNAi transgenes targeting white , GlyP , Atg1 , or GlyP + Atg1 . Simultaneous knockdown of GlyP and Atg1 , but not either gene alone , significantly reduced glycogen degradation compared to the white control after 24 h of starvation , consistent with immunostaining ( C - F ) . Between 6 and 12 h of starvation , individual knockdown of GlyP or Atg1 caused a significant increase in glycogen levels , indicating a reduced rate of glycogen degradation . SEM is indicated for n = 5 - 8 samples . The p values were calculated relative to white RNAi control at each time point ( * p < . 05 , * * p < . 01 ) .

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

(G) Time course of glycogen levels in Dmef2-Gal4 carcasses (muscle+body wall) with expression of UAS-RNAi transgenes targeting white, GlyP, Atg1, or GlyP+Atg1. Simultaneous knockdown of GlyP and Atg1, but not either gene alone, significantly reduced glycogen degradation compared to the white control after 24 h of starvation, consistent with immunostaining (C-F). Between 6 and 12 h of starvation, individual knockdown of GlyP or Atg1 caused a significant increase in glycogen levels, indicating a reduced rate of glycogen degradation. SEM is indicated for n = 5-8 samples. The p values were calculated relative to white RNAi control at each time point (*p<.05, **p<.01).


( A - D ) Glycogen synthase ( GlyS ) is required for glycogen synthesis in D . melanogaster muscles . PAS staining for glycogen was absent in Dmef2 - Gal4 / UAS - GlySi muscles ( B ) compared to control Dmef2 - Gal4 / UAS - whitei muscles ( A ) . Antiglycogen immunostaining for glycogen was absent in Dmef2 - Gal4 / UAS - GlySi muscles ( D ) compared to Dmef2 - Gal4 / UAS - whitei control muscles ( C ) .

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

(A-D) Glycogen synthase (GlyS) is required for glycogen synthesis in D. melanogaster muscles. PAS staining for glycogen was absent in Dmef2-Gal4/UAS-GlySi muscles (B) compared to control Dmef2-Gal4/UAS-whitei muscles (A). Antiglycogen immunostaining for glycogen was absent in Dmef2-Gal4/UAS-GlySi muscles (D) compared to Dmef2-Gal4/UAS-whitei control muscles (C).


( C ) . ( E - K ) GlyS is required for the formation of large CQ - induced autophagosomes . Vesicles are much smaller in Dmef2 - Gal4 , UAS - GFP - Atg8 / UAS - GlySi larval muscle starved 6 h in low - nutrient food + 2 . 5 mg / ml CQ ( G ) than in control Dmef2 - Gal4 , UAS - GFP - Atg8 / UAS - whitei larval muscle ( E ) . ( F , H ) The difference in autophagosome size is clearly evident at high magnification .

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

(C). (E-K) GlyS is required for the formation of large CQ-induced autophagosomes. Vesicles are much smaller in Dmef2-Gal4, UAS-GFP-Atg8/UAS-GlySi larval muscle starved 6 h in low-nutrient food +2.5 mg/ml CQ (G) than in control Dmef2-Gal4, UAS-GFP-Atg8/UAS-whitei larval muscle (E). (F, H) The difference in autophagosome size is clearly evident at high magnification.


( I - K ) Quantification of autophagy changes due to GlyS gene knockdown in Dmef2 - Gal4 , UAS - GFP - Atg8 larvae starved on low - nutrient food + 2 . 5 mg / ml CQ for 6 h . SEM is indicated , with n = 5 ( I ) or n = 10 ( J - K ) ventral longitudinal muscles from individual animals ( * p < . 05 , * * p < . 01 ) . ( I ) Each of the four UAS - GlyS RNAi transgenes tested caused a significant decrease in the total area of GFP - Atg8vesicles in the muscle compared to the UAS - whitei control . ( J ) Vesicle number was unchanged by GlyS knockdown . ( K ) UAS - GlyS RNAi caused a highly significant decrease in the mean vesicle size ( area ) compared to the control .

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

(I-K) Quantification of autophagy changes due to GlyS gene knockdown in Dmef2-Gal4, UAS-GFP-Atg8 larvae starved on low-nutrient food +2.5 mg/ml CQ for 6 h. SEM is indicated, with n = 5 (I) or n = 10 (J-K) ventral longitudinal muscles from individual animals (*p<.05, **p<.01). (I) Each of the four UAS-GlyS RNAi transgenes tested caused a significant decrease in the total area of GFP-Atg8vesicles in the muscle compared to the UAS-whitei control. (J) Vesicle number was unchanged by GlyS knockdown. (K) UAS-GlyS RNAi caused a highly significant decrease in the mean vesicle size (area) compared to the control.


( L - M ) GlyS gene expression , monitored using a MiMIC transposon insertion ( MI01490 ) , showed expression in the larval muscle ( L ) but not the fat body ( M ) ( green , GFP ; blue , DAPI ) .

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

(L-M) GlyS gene expression, monitored using a MiMIC transposon insertion (MI01490), showed expression in the larval muscle (L) but not the fat body (M) (green, GFP; blue, DAPI).


N - O ) Larvae were starved on low - nutrient food for 6 h prior to dissection of the fat bodies . Autophagy in Cg - Gal4 / + ; UAS - GFP - Atg8 / UAS - GlySi ( O ) was not substantially different from autophagy in Cg - Gal4 / + ; UAS - GFP - Atg8 / UAS - whitei control fat bodies ( GFP , green ; DAPI , blue ) .

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

N-O) Larvae were starved on low-nutrient food for 6 h prior to dissection of the fat bodies. Autophagy in Cg-Gal4/+; UAS-GFP-Atg8/UAS-GlySi (O) was not substantially different from autophagy in Cg-Gal4/+; UAS-GFP-Atg8/UAS-whitei control fat bodies (GFP, green; DAPI, blue).


. ( P ) EM of muscle from Dmef2 - Gal4 / UAS - GlySi animal starved on low - nutrient food + CQ . Note that the intermyofibril spaces ( red asterisk ) and sarcomere structure are not distorted .

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

. (P) EM of muscle from Dmef2-Gal4/UAS-GlySi animal starved on low-nutrient food +CQ. Note that the intermyofibril spaces (red asterisk) and sarcomere structure are not distorted.


Q ) GlyS or Atg1 knockdown significantly improved the crawling time of larvae treated with CQ and starved for 6 h . SEM is indicated for n = 10 larvae . The p values were calculated relative to white RNAi control larvae ( * p < . 05 , * * p < . 01 ) .

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

Q) GlyS or Atg1 knockdown significantly improved the crawling time of larvae treated with CQ and starved for 6 h. SEM is indicated for n = 10 larvae. The p values were calculated relative to white RNAi control larvae (*p<.05, **p<.01).


( A ) Protein sequence alignment of the C - terminal region of D . melanogaster , human , and yeast Glycogen synthases . Identical residues are blue ; all other residues are red . Conserved in all three species , R593 , W609 , and S651 are underlined .

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

(A) Protein sequence alignment of the C-terminal region of D. melanogaster, human, and yeast Glycogen synthases. Identical residues are blue; all other residues are red. Conserved in all three species, R593, W609, and S651 are underlined.


( B ) Western blot / Co - immunoprecipitation ( co - IP ) showing that Flag - Atg8 binds to a Venus - GlyS or Venus - GlyS ( S651A ) protein complex in response to starvation . Flag - Atg8 is unable to co - IP with either Venus - GlyS ( R593A ) or Venus - GlyS ( W609A ) . Venus - GlyS and Venus - GlyS mutants were co - IP ' d from muscle lysate from Dmef2 - Gal4 / UAS - Flag - Atg8 or UAS - Venus - GlyS ( WT or mutant ) / + ; Dmef2 - Gal4 / UAS - Flag - Atg8third instar larvae . These were fed on high - nutrientfood for 18 h , and then transferred to fresh high - nutrientfood or low - nutrientfood for 6 h .

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

(B) Western blot/Co-immunoprecipitation (co-IP) showing that Flag-Atg8 binds to a Venus-GlyS or Venus-GlyS (S651A) protein complex in response to starvation. Flag-Atg8 is unable to co-IP with either Venus-GlyS (R593A) or Venus-GlyS (W609A). Venus-GlyS and Venus-GlyS mutants were co-IP'd from muscle lysate from Dmef2-Gal4/UAS-Flag-Atg8 or UAS-Venus-GlyS(WT or mutant)/+;Dmef2-Gal4/UAS-Flag-Atg8third instar larvae. These were fed on high-nutrientfood for 18 h, and then transferred to fresh high-nutrientfood or low-nutrientfood for 6 h.


( C ) Venus - GlyS was localized predominantly to the Flag - labeled autophagosomes , with weak staining in the rest of the cytoplasm . ( D ) Venus - GlyS ( R593A ) was found throughout the cytoplasm and did not colocalize with autophagosomes . ( E ) Venus - GlyS ( S651A ) was localized to the autophagosomes . ( F ) Venus - GlyS ( W609A ) did not colocalize with the Flag - labeled autophagosomes .

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

(C) Venus-GlyS was localized predominantly to the Flag-labeled autophagosomes, with weak staining in the rest of the cytoplasm. (D) Venus-GlyS (R593A) was found throughout the cytoplasm and did not colocalize with autophagosomes. (E) Venus-GlyS (S651A) was localized to the autophagosomes. (F) Venus-GlyS (W609A) did not colocalize with the Flag-labeled autophagosomes.


( B ) Lineage tracing of the anagen matrix from β - actin - CreER : Confetti , 7 days after induction with either a high dose of tamoxifen ( TM ) ( top ) or low dose ( bottom ) for single cell clonal lineages .

O O O O O O O O O O O O O O O O O O O O O O O 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) Lineage tracing of the anagen matrix from β-actin-CreER:Confetti, 7 days after induction with either a high dose of tamoxifen (TM) (top) or low dose (bottom) for single cell clonal lineages.


( C ) Images of skin sections at various stages of the hair cycle , immunostained for Gata6 ( red ) , CD34 or K14 ( green ) and DNA ( blue ) .

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

(C) Images of skin sections at various stages of the hair cycle, immunostained for Gata6 (red), CD34 or K14 (green) and DNA (blue).


( D ) images ( D ' ) for Gata6 inducible knockout ( iKO ) and wild type ( WT ) mice over time , showing the loss of Gata6 ( red ) from the hair follicle . Scale bars : 30 m .

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

(D) images (D') for Gata6 inducible knockout (iKO) and wild type (WT) mice over time, showing the loss of Gata6 (red) from the hair follicle. Scale bars: 30 m.


( B ) Representative images of hematoxylin staining of skin sections from the telogen induced mice sacrificed at 2 , 4 , and 10 days ( d ) post TM induction . ( C ) Images of hematoxylin staining of skin section from the anagen induced mice . Quantification shown in Figure EV2D . Scale bars : 30 m .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Representative images of hematoxylin staining of skin sections from the telogen induced mice sacrificed at 2, 4, and 10 days (d) post TM induction. (C) Images of hematoxylin staining of skin section from the anagen induced mice. Quantification shown in Figure EV2D. Scale bars: 30 m.


( B - G ) Gata6 WT and iKO skin after 3 days after TM induction and 12 hours after BrdU labeling or 5 days after TM induction and 2 days after BrdU labeling stained with markers as indicated in corresponding color .

O O O O O O O O O O O O O O O O O O O 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-G) Gata6 WT and iKO skin after 3 days after TM induction and 12 hours after BrdU labeling or 5 days after TM induction and 2 days after BrdU labeling stained with markers as indicated in corresponding color.


( A ) Hair follicles exhibit immunostaining signal for DNA damage marker H2A . X ( red ) and apoptosis marker Caspase3 ( green ) as early as one day after Gata6 iKO induction . Top panel shows a section of UV irradiated epidermis as a positive control for DNA damage . Note that Caspase 3 is not expressed in the H2A . X cells of the epidermis but is found in some iKO hair follicle matrix cells . Right panels show single color images as indicated at top ( B ) Quantification ( average SD ) of the percentage of apoptotic cells per matrix as determined by counting of Caspase3 + cells ( n = 50 follicles from 3 mice per group ) ( C ) Quantification ( average SD ) of the percentage of DNA damaged cells per matrix as determined by counting of H2A . X + Caspase3 - cells ( n = 50 follicles from 3 mice per group ) .

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

(A) Hair follicles exhibit immunostaining signal for DNA damage marker H2A.X (red) and apoptosis marker Caspase3 (green) as early as one day after Gata6 iKO induction. Top panel shows a section of UV irradiated epidermis as a positive control for DNA damage. Note that Caspase 3 is not expressed in the H2A.X cells of the epidermis but is found in some iKO hair follicle matrix cells. Right panels show single color images as indicated at top (B) Quantification (average SD) of the percentage of apoptotic cells per matrix as determined by counting of Caspase3+ cells (n=50 follicles from 3 mice per group) (C) Quantification (average SD) of the percentage of DNA damaged cells per matrix as determined by counting of H2A.X+Caspase3- cells (n=50 follicles from 3 mice per group).


( D ) Regional distribution of apoptosis was analyzed by dividing the matrix into four compartments as shown in the legend ( left ) and counting the fraction of Caspase3 + cells per each matrix compartments ( right ; n = 50 follicles from 3 mice per group ) .

(D) Regional distribution of apoptosis was analyzed by dividing the matrix into four compartments as shown in the legend (left) and counting the fraction of Caspase3+ cells per each matrix compartments (right; n=50 follicles from 3 mice per group).


( E ) Proliferation in the matrix compartments shown in ( D ) was assessed by BrdU labeling for 1 hour , followed by staining , and quantification ( F ) ( interior cells labeled with solid bars , exterior cells labeled with striped bars ) . Unpaired t - test : * P - value < 0 . 05 . n = 20 follicles from 2 mice per group .

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

(E) Proliferation in the matrix compartments shown in (D) was assessed by BrdU labeling for 1 hour, followed by staining, and quantification (F) (interior cells labeled with solid bars, exterior cells labeled with striped bars). Unpaired t-test: *P-value < 0.05. n=20 follicles from 2 mice per group.


( A ) Colony formation of WT and TM induced Gata6 iKO keratinocytes stained with hematoxylin . Primary keratinocytes were isolated from induced iKO newborn mice , plated on feeder cells , and stained with hematoxylin at day 7 after plating ( top ) . Phase contrast images of cells are shown below . Scale bars : 30 m . ( B ) Quantification of colony formation from ( A ) , average SD from n = 3 mice in each genotype .

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

(A) Colony formation of WT and TM induced Gata6 iKO keratinocytes stained with hematoxylin. Primary keratinocytes were isolated from induced iKO newborn mice, plated on feeder cells, and stained with hematoxylin at day 7 after plating (top). Phase contrast images of cells are shown below. Scale bars: 30 m. (B) Quantification of colony formation from (A), average SD from n=3 mice in each genotype.


( C ) Western blot of WT and iKO cultured keratinocytes after 0 or 6 hours of tamoxifen induction , blotted with actin and Gata6 antibodies , show a decrease in Gata6 protein levels after 6 hours .

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

(C) Western blot of WT and iKO cultured keratinocytes after 0 or 6 hours of tamoxifen induction, blotted with actin and Gata6 antibodies, show a decrease in Gata6 protein levels after 6 hours.


( D ) Phase contrast images of WT and Gata6 iKO keratinocytes 3 days after induction with TM ( n > 3 per genotype ) .

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

(D) Phase contrast images of WT and Gata6 iKO keratinocytes 3 days after induction with TM (n > 3 per genotype).


( E - H ) Images and quantification of DNA damage in cultured keratinocytes based on staining for DNA damage marker H2A . X ( E and G ) and comet assay ( F and H ) . Arrowheads in ( F ) indicate DNA damaged cell nuclei with comet shape . Quantification of H2A . X ( G ) represents average SD from at least 300 cells per condition from 3 experiments using different cell lines . * Unpaired t - test P - values : 6h = 0 . 01 and 24h = 0 . 004 . ( H ) Quantification of comet assay represents average SD ( n = 3 ) .

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

(E-H) Images and quantification of DNA damage in cultured keratinocytes based on staining for DNA damage marker H2A.X (E and G) and comet assay (F and H). Arrowheads in (F) indicate DNA damaged cell nuclei with comet shape. Quantification of H2A.X (G) represents average SD from at least 300 cells per condition from 3 experiments using different cell lines . *Unpaired t-test P-values: 6h=0.01 and 24h=0.004. (H) Quantification of comet assay represents average SD (n=3).


( I - J ) FACS cell cycle profiles and quantification show that Gata6 iKO keratinocytes show decreased proliferation and G2 - M arrest .

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

(I-J) FACS cell cycle profiles and quantification show that Gata6 iKO keratinocytes show decreased proliferation and G2-M arrest.


( I - J ) FACS cell cycle profiles and quantification show that Gata6 iKO keratinocytes show decreased proliferation and G2 - M arrest . ( J ) Quantification of FACS cell cycle analysis represent average SD ( n = 3 per genotype ) . * Unpaired t - test P - values : G1 = 0 . 03 , S = 5x10 - 4 , G2 - M = 7x10 - 4 .

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

(I-J) FACS cell cycle profiles and quantification show that Gata6 iKO keratinocytes show decreased proliferation and G2-M arrest. (J) Quantification of FACS cell cycle analysis represent average SD (n=3 per genotype). *Unpaired t-test P-values: G1 = 0.03, S = 5x10-4, G2-M = 7x10-4.


( K ) Quantification of the frequency of H2A . X + staining in keratinocytes within S phase ( EdU + ) or another cell cycle stage ( EdU - ) following a 20 minute EdU pulse labeling ( average SD , n = 5 ) . * Unpaired t - test P - values WT = 0 . 1 , iKO = 0 . 05 . Mann - Whitney U test P - values : WT = 0 . 09 , iKO = 0 . 03 .

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

(K) Quantification of the frequency of H2A.X+ staining in keratinocytes within S phase (EdU+) or another cell cycle stage (EdU-) following a 20 minute EdU pulse labeling (average SD, n=5). *Unpaired t-test P-values WT=0.1, iKO= 0.05. Mann-Whitney U test P-values: WT=0.09, iKO=0.03.


( L - M ) Cultured keratinocytes were made quiescent by gradual serum depletion . ( L ) Quantification of EdU + cells in proliferative and serum depleted quiescent cells ( average SD , n = 4 ) . ( M ) Quantification of H2A . X + keratinocytes during proliferative and quiescent conditions ( average SD , n = 4 ) . * Unpaired t - test P - value = 0 . 04 . Mann - Whitney U test P - value = 0 . 06 .

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

(L-M) Cultured keratinocytes were made quiescent by gradual serum depletion. (L) Quantification of EdU+ cells in proliferative and serum depleted quiescent cells (average SD, n=4). (M) Quantification of H2A.X+ keratinocytes during proliferative and quiescent conditions (average SD, n=4). *Unpaired t-test P-value = 0.04. Mann-Whitney U test P-value=0.06.


( A ) Heatmap of differentially expressed genes ( adjusted P - value < 0 . 05 and fold change > 1 . 5 ) from RNA - seq ( n = 3 different WT and iKO cell lines ) .

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

(A) Heatmap of differentially expressed genes (adjusted P-value < 0.05 and fold change > 1.5) from RNA-seq (n=3 different WT and iKO cell lines).


( B ) qRT - PCR validation of candidate genes selected from RNA - seq analysis using RNA from cultured cells ( average SD , n = 4 ) . * Unpaired t - test P - values : Edaradd = 0 . 03 , Apcdd1 = 0 . 005 , Gls2 = 7x10 - 5 , Ppargc1b = 3x10 - 4 , Rab17 = 2x10 - 5 , Foxn1 = 0 . 03 . Mann - Whitney U test P - values : Edaradd = 0 . 06 , Apcdd1 = 0 . 03 , Gls2 = 0 . 03 , Ppargc1b = 0 . 03 , Rab17 = 0 . 03 , Foxn1 = 0 . 03 . ( C ) Table summarizing selection of candidate genes from ( B ) . Genes were selected based on known function or skin phenotype related to Gata6 iKO phenotype .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) qRT-PCR validation of candidate genes selected from RNA-seq analysis using RNA from cultured cells (average SD, n=4). *Unpaired t-test P-values: Edaradd=0.03, Apcdd1=0.005, Gls2=7x10-5, Ppargc1b=3x10-4, Rab17=2x10-5, Foxn1=0.03. Mann-Whitney U test P-values: Edaradd=0.06, Apcdd1=0.03, Gls2=0.03, Ppargc1b=0.03, Rab17=0.03, Foxn1=0.03. (C) Table summarizing selection of candidate genes from (B). Genes were selected based on known function or skin phenotype related to Gata6 iKO phenotype.


( D ) Staining of ROS by DHE . LY83583 treated cells served as positive control with elevated ROS . ( E ) Quantification of fold change in ROS at 6 and 24 hours after TM compared to WT , based on fluorescence intensity of DHE ( average SD , n = 2 ) .

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

(D) Staining of ROS by DHE. LY83583 treated cells served as positive control with elevated ROS. (E) Quantification of fold change in ROS at 6 and 24 hours after TM compared to WT, based on fluorescence intensity of DHE (average SD, n=2).


( F ) Analysis of transcription factor binding site overrepresentation ( oPOSSUM ) in differentially expressed genes from RNA - seq . Points represent transcription factors plotted as Z . score of the frequency of transcription factor binding site occurrences in the promoters of downregulated genes compared to the background rate of occurrences in all gene promoters and Fisher P - value of the proportion of downregulated gene promoters containing each transcription factor binding site compared to the frequency in all gene promoters .

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

(F) Analysis of transcription factor binding site overrepresentation (oPOSSUM) in differentially expressed genes from RNA-seq. Points represent transcription factors plotted as Z.score of the frequency of transcription factor binding site occurrences in the promoters of downregulated genes compared to the background rate of occurrences in all gene promoters and Fisher P-value of the proportion of downregulated gene promoters containing each transcription factor binding site compared to the frequency in all gene promoters.


( H ) qRT - PCR of selected genes found to be overlapping between Gata6 iKO and Eda transgenic analyzed in Gata6 WT and iKO keratinocytes ( average SD , n = 4 ) . * Unpaired t - test P - values : Mcm3 = 0 . 001 , Mcm6 = 0 . 02 , Mcm10 = 0 . 04 , Exo1 = 0 . 02 , Pold1 = 0 . 02 . Mann - Whitney U test P - values : Mcm3 = 0 . 03 , Mcm6 = 0 . 03 , Mcm10 = 0 . 03 , Exo1 = 0 . 03 , Pold1 = 0 . 06 .

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

(H) qRT-PCR of selected genes found to be overlapping between Gata6 iKO and Eda transgenic analyzed in Gata6 WT and iKO keratinocytes (average SD, n=4). *Unpaired t-test P-values: Mcm3=0.001, Mcm6=0.02, Mcm10=0.04, Exo1=0.02, Pold1=0.02. Mann-Whitney U test P-values: Mcm3=0.03, Mcm6=0.03, Mcm10=0.03, Exo1=0.03, Pold1=0.06.


( B ) qRT - PCR confirmation of the change in Edaradd mRNA expression in - vivo from mouse whole skin ( average SD , 1d n = 3 , 2d n = 4 ) . * Unpaired t - test P - values : 1d = 0 . 02 , 2d = 0 . 05 . Mann - Whitney U test P - values : 1d = 0 . 06 , 2d = 0 . 03 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) qRT-PCR confirmation of the change in Edaradd mRNA expression in-vivo from mouse whole skin (average SD, 1d n=3, 2d n=4). *Unpaired t-test P-values: 1d = 0.02, 2d = 0.05. Mann-Whitney U test P-values: 1d=0.06, 2d=0.03.


( C ) ChIP - qPCR for Gata6 at three conserved binding sites within the Edaradd promoter . GAPDH served as a negative control . Binding site 1 , 2 , and 3 are located 50 bp , 1 kb , and 1 . 5 kb upstream of the transcription start site , respectively ( average SD , n = 4 ) . * Unpaired t - test P - values GAPDH = 0 . 4 , 1 = 0 . 002 , 2 = 0 . 8 , 3 = 0 . 2 . Mann - Whitney U test P - values : GAPDH = 0 . 3 , 1 = 0 . 06 , 2 = 0 . 9 , 3 = 0 . 2 .

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

(C) ChIP-qPCR for Gata6 at three conserved binding sites within the Edaradd promoter. GAPDH served as a negative control. Binding site 1, 2, and 3 are located 50 bp, 1 kb, and 1.5 kb upstream of the transcription start site, respectively (average SD, n=4). *Unpaired t-test P-values GAPDH=0.4, 1=0.002, 2=0.8, 3=0.2. Mann-Whitney U test P-values: GAPDH=0.3, 1=0.06, 2=0.9, 3=0.2.


( D ) Western blot from whole skin lysate . Note decrease of Edaradd and activated pNF - B in the iKO .

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

(D) Western blot from whole skin lysate. Note decrease of Edaradd and activated pNF-B in the iKO.


( E - F ) Staining for Edaradd and quantification show decrease in Edaradd protein levels in the matrix of Gata6 iKO . * Unpaired t - test P - value = 3 . 5x10 - 14 . n = 100 follicles from 3 mice per genotype .

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

(E-F) Staining for Edaradd and quantification show decrease in Edaradd protein levels in the matrix of Gata6 iKO. *Unpaired t-test P-value = 3.5x10-14. n=100 follicles from 3 mice per genotype.


( G - H ) Staining for pNF - B and quantification show decrease of active NF - B in the Gata6 iKO matrix . * Unpaired t - test P - value = 2 . 2x10 - 16 . n = 50 follicles from 3 mice .

(G-H) Staining for pNF-B and quantification show decrease of active NF-B in the Gata6 iKO matrix. *Unpaired t-test P-value = 2.2x10-16. n=50 follicles from 3 mice.


( I ) Rescue of Gata6 iKO keratinocyte growth by transfection with pGata6 or pEdaradd . Cell plates of keratinocytes are stained with Rhodanile Blue ( n = 3 cell lines each ) .

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

(I) Rescue of Gata6 iKO keratinocyte growth by transfection with pGata6 or pEdaradd. Cell plates of keratinocytes are stained with Rhodanile Blue (n=3 cell lines each).


( J ) Quantification of H2A . X in cells stably transfected with pMock , pGata6 , or pEdaradd plasmids , followed by induction with TM for 6 hours ( average SD , n = 4 with > 500 cells counted per condition ) . * Unpaired t - test P - values = 2x10 - 4 ( iKO pGata6 vs pMock ) , 2x10 - 4 ( iKO pEdaradd vs pMock ) .

(J) Quantification of H2A.X in cells stably transfected with pMock, pGata6, or pEdaradd plasmids, followed by induction with TM for 6 hours (average SD, n=4 with > 500 cells counted per condition). *Unpaired t-test P-values = 2x10-4 (iKO pGata6 vs pMock), 2x10-4 (iKO pEdaradd vs pMock).


( K ) qRT - PCR analysis of Mcm10 expression in pMock , pGata6 , and pEdaradd WT and iKO expressing cells ( average SD , n = 4 ) . * Unpaired t - test P - values : 8x10 - 4 ( iKO pGata6 vs pMock ) , 0 . 03 ( iKO pEdaradd vs pMock ) . Mann - Whitney U test P - values : 0 . 03 ( iKO pGata6 vs pMock ) , 0 . 03 ( iKO pEdaradd vs pMock ) .

(K) qRT-PCR analysis of Mcm10 expression in pMock, pGata6, and pEdaradd WT and iKO expressing cells (average SD, n=4). *Unpaired t-test P-values: 8x10-4 (iKO pGata6 vs pMock), 0.03 (iKO pEdaradd vs pMock). Mann-Whitney U test P-values: 0.03 (iKO pGata6 vs pMock), 0.03 (iKO pEdaradd vs pMock).


( A ) OCR of wt and gas - 1 mutant nematodes ( normalized to nematode size as given by the COPAS Biosort ( Time of Flight - TOF ) ) . One representative curve shown ( mean ± SEM ; n = 3 ) * * * * p value < 0 . 0001 ; Kruskal - Wallis Test , Dunn ' s post hoc correction .

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

(A) OCR of wt and gas-1 mutant nematodes (normalized to nematode size as given by the COPAS Biosort (Time of Flight -TOF)). One representative curve shown (mean ± SEM; n=3) ****p value< 0.0001; Kruskal-Wallis Test, Dunn's post hoc correction.


Representative curves show the lifespan of wt and gas - 1 ( fc21 ) nematodes compared to ( B ) age - 1 and age - 1 ; gas - 1 mutants animals . Average median lifespans ± SEM from all replicates indicated beneath representative curves .

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

Representative curves show the lifespan of wt and gas-1(fc21) nematodes compared to (B) age-1 and age-1; gas-1 mutants animals. Average median lifespans ± SEM from all replicates indicated beneath representative curves.


Representative curves show the lifespan of wt and gas - 1 ( fc21 ) nematodes compared to ( C ) daf - 16 and daf - 16 ; age - 1 ; gas - 1 mutant animals . Average median lifespans ± SEM from all replicates indicated beneath representative curves .

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

Representative curves show the lifespan of wt and gas-1(fc21) nematodes compared to (C) daf-16 and daf-16; age-1; gas-1 mutant animals. Average median lifespans ± SEM from all replicates indicated beneath representative curves.


( D ) Volcano plot of differentially phosphorylated proteins in gas - 1 ( fc21 ) mutants compared to wt . Cutoffs : 20 % FDR and | log2 ( foldchange ) | > 0 . 345 ( in red ) .

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

(D) Volcano plot of differentially phosphorylated proteins in gas-1(fc21) mutants compared to wt. Cutoffs: 20% FDR and |log2(foldchange)| >0.345 (in red).


( F ) Lifespan analysis of mev - 1 ( kn1 ) nematodes compared to wt , age - 1 ( hx546 ) and age - 1 ( hx546 ) ; mev - 1 ( kn1 ) animals . ( G )

(F) Lifespan analysis of mev-1(kn1) nematodes compared to wt, age-1(hx546) and age-1(hx546); mev-1(kn1) animals. (G)


( G ) ATP measurements in wt , age - 1 , gas - 1 and age - 1 ; gas - 1 mutant nematodes ( normalized to wt ) , n = 4 . Error bars : Mean ± SEM , * p value < 0 . 05 , * * * p value = 0 . 001 , Kruskal - Wallis Test , Dunn ' s post hoc correction .

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

(G) ATP measurements in wt, age-1, gas-1 and age-1; gas-1 mutant nematodes (normalized to wt), n=4. Error bars: Mean ± SEM, *p value <0.05, ***p value =0.001, Kruskal-Wallis Test, Dunn's post hoc correction.


( H ) OCR of gas - 1 and age - 1 ; gas - 1 mutant nematodes ( normalized to TOF ) . Left panel : One representative curve ( Mean ± SEM ) . Right panel : Basal respiration in wt , gas - 1 and age - 1 ; gas - 1 animals , pooled from 3 biological replicates , * * * * p value < 0 . 0001 , ns = non - significant , Kruskal - Wallis Test , Dunn ' s post hoc correction .

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

(H) OCR of gas-1 and age-1; gas-1 mutant nematodes (normalized to TOF). Left panel: One representative curve (Mean ± SEM). Right panel: Basal respiration in wt, gas-1 and age-1; gas-1 animals, pooled from 3 biological replicates, ****p value <0.0001, ns= non-significant, Kruskal-Wallis Test, Dunn's post hoc correction.


( I ) STED images of representative mitochondrial morphology in muscle cells of L4 mutant worms . Scale bar : 2 μm . ( J ) Left panel : Percentage of FRAP . Each point : mean recovery of ~ 30 bleached regions ( 2 regions per animal , 15 animals per condition ) ± SEM . Right panel : values of the maximum ( total ) recovery per strain ± SEM , * * * p value < 0 . 001 , * * * * p value < 0 . 0001 , one - way ANOVA , Tukey ' s post - hoc correction .

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

(I) STED images of representative mitochondrial morphology in muscle cells of L4 mutant worms. Scale bar: 2 μm. (J) Left panel: Percentage of FRAP. Each point: mean recovery of ~30 bleached regions (2 regions per animal, 15 animals per condition) ± SEM. Right panel: values of the maximum (total) recovery per strain ± SEM, ***p value <0.001, **** p value <0.0001, one-way ANOVA, Tukey's post-hoc correction.


( K ) Representative lifespan of age - 1 ; gas - 1 nematodes fed with empty vector ( EV ) and fzo - 1 RNAi bacteria . Average median lifespans ± SEM from all replicates indicated beneath representative curves . For all panels : wt = wild type , a = age - 1 , g = gas - 1 , a ; g = age - 1 ; gas - 1 .

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

(K) Representative lifespan of age-1; gas-1 nematodes fed with empty vector (EV) and fzo-1 RNAi bacteria. Average median lifespans ± SEM from all replicates indicated beneath representative curves. For all panels: wt= wild type, a= age-1, g= gas-1, a; g= age-1; gas-1.


( A ) Left panel : Venn Diagram of the number of genes significantly dysregulated in age - 1 ; gas - 1 versus wt ( green ) and gas - 1 versus wt ( red ) . Right panel : Scatter - plots of the expression of genes significantly downregulated ( left ) or upregulated ( right ) in gas - 1 and age - 1 ; gas - 1 mutants compared to wt . In black : expression levels of genes that are significantly up - or down - regulated in gas - 1 mutants compared to wt . In red : expression levels of the same genes in age - 1 ; gas - 1 mutants compared to wt .

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

(A) Left panel: Venn Diagram of the number of genes significantly dysregulated in age-1; gas-1 versus wt (green) and gas-1 versus wt (red). Right panel: Scatter-plots of the expression of genes significantly downregulated (left) or upregulated (right) in gas-1 and age-1; gas-1 mutants compared to wt. In black: expression levels of genes that are significantly up- or down-regulated in gas-1 mutants compared to wt. In red: expression levels of the same genes in age-1; gas-1 mutants compared to wt.


( C ) Representative lifespans of the indicated strains with average median lifespans ± SEM from all replicates indicated beneath representative

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

(C) Representative lifespans of the indicated strains with average median lifespans ± SEM from all replicates indicated beneath representative curves


( D ) Volcano plot of TMT / iTRAQ - based quantitative proteomic analysis . In red , proteins significantly dysregulated in age - 1 ; gas - 1 compared to gas - 1 ( FDR < 20 % ; | log2 ( fold change ) | > 0 . 47 ) . Dashed lines indicate cutoffs . Proteins involved in carbohydrate ( green ) and lipid ( blue ) metabolism are labeled .

(D) Volcano plot of TMT/iTRAQ-based quantitative proteomic analysis. In red, proteins significantly dysregulated in age-1; gas-1 compared to gas-1 (FDR< 20%; |log2 (fold change)| >0.47). Dashed lines indicate cutoffs. Proteins involved in carbohydrate (green) and lipid (blue) metabolism are labeled.


( H ) Left panel : Glucose quantification in wt , gas - 1 and age - 1 ; gas - 1 nematode lysates with NMR , n = 7 , ns = non - significant , Mann Whitney U test . Right panel : Quantification of total carbohydrates in the indicated strains with phenol - sulfuric assay . Error bars : mean ± SEM , 2 biological replicates ( 5 technical replicates each ) , normalized to wt and number of animals used . * * p value < 0 . 01 , ns = non - significant , Mann - Whitney U test .

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

(H) Left panel: Glucose quantification in wt, gas-1 and age-1; gas-1 nematode lysates with NMR, n=7, ns= non-significant, Mann Whitney U test. Right panel: Quantification of total carbohydrates in the indicated strains with phenol-sulfuric assay. Error bars: mean ± SEM, 2 biological replicates (5 technical replicates each), normalized to wt and number of animals used. **p value < 0.01, ns= non-significant, Mann-Whitney U test.


( C ) Immunoblot analysis of phospho - AAK - 2 in wt , age - 1 , gas - 1 and age - 1 ; gas - 1 mutant nematodes ( actin loading control ) . Numbers correspond to band densitometries across 3 biological replicates .

(C) Immunoblot analysis of phospho-AAK-2 in wt, age-1, gas-1 and age-1; gas-1 mutant nematodes (actin loading control). Numbers correspond to band densitometries across 3 biological replicates.


( D ) Representative lifespan of age - 1 ; gas - 1 double mutant and age - 1 ; aak - 2 ; gas - 1 triple mutant nematodes with average median lifespans ± SEM from all replicates indicated beneath representative curves .

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

(D) Representative lifespan of age-1; gas-1 double mutant and age-1; aak-2; gas-1 triple mutant nematodes with average median lifespans ± SEM from all replicates indicated beneath representative curves.


( E ) Lifespan assay of gas - 1 mutant animals compared to gas - 1 ; AAK - 2 overexpressing ( O / E ) nematodes and their wt counterparts . Inset : immunoblot analysis of phospho - AAK - 2 in gas - 1 ( g ) and gas - 1 ; AAK - 2 O / E ( g ; a O / E ) animals ( actin loading control ) , showing the overexpressed protein ( lower band ) and the endogenous one ( upper band ) .

(E) Lifespan assay of gas-1 mutant animals compared to gas-1; AAK-2 overexpressing (O/E) nematodes and their wt counterparts. Inset: immunoblot analysis of phospho-AAK-2 in gas-1 (g) and gas-1; AAK-2 O/E (g; a O/E) animals (actin loading control), showing the overexpressed protein (lower band) and the endogenous one (upper band).