( D ) Left panel , representative high - content microscopy of RIPosomes in control ATG5 siRNA transfected THP - 1 infected with S . flexneri ( 8 h ) . Right panel , graph depicts average number of RIPosomes / . About 50000 were plated per well RIPosomes were screened in 35 fields per well . Mean ± SD , n = 3 ( biological replicates ) , * * * p < 0 . 0005 , Student ' s unpaired t - .

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

(D) Left panel, representative high-content microscopy images of RIPosomes in control and ATG5 siRNA transfected THP-1 cells infected with S. flexneri (8 h). Right panel, the graph depicts average number of RIPosomes/cell. About 50000 cells were plated per well and RIPosomes were screened in 35 fields per well. Mean ± SD, n=3 (biological replicates), ***p < 0.0005, Student's unpaired t-test.


( E ) soluble insoluble fractions of S . flexneri infected ( MOI 1 : 25 , 8 h ) control ATG5 siRNA knockdown THP - 1 were subjected to Western blot with indicated antibodies .

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

(E) The soluble and insoluble fractions of S. flexneri infected (MOI 1:25, 8 h) control and ATG5 siRNA knockdown THP-1 cells were subjected to Western blot analysis with indicated antibodies.


( F ) Representative confocal of HEK293T transfected with GFP - RIPK2 immunostained with anti - FK2 antibodies . profile : co - localization using profiles . Scale bar , 3 µm .

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

(F) Representative confocal images of HEK293T cells transfected with GFP-RIPK2 and immunostained with anti-FK2 antibodies. Line profile: co-localization analysis using line intensity profiles. Scale bar, 3 µm.


( G ) Autophagy receptor screen using co - immunoprecipitation ( Co - IP ) to analyze interaction between GFP - RIPK2 Flag - p62 or Flag - NBRI or Flag - NDP52 or Flag - TAX1BP1 or Flag - OPTINEURIN in HEK293T lysate .

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

(G) Autophagy receptor screen using co-immunoprecipitation (Co-IP) assay to analyze the interaction between GFP-RIPK2 and Flag-p62 or Flag-NBRI or Flag-NDP52 or Flag-TAX1BP1 or Flag-OPTINEURIN in HEK293T cell lysate.


( H ) Western blot of soluble insoluble fractions of control p62 siRNA transfected S . flexneri infected THP - 1 ( MOI 1 : 25 , 8 h ) .

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(H) Western blot analysis of soluble and insoluble fractions of control and p62 siRNA transfected and S. flexneri infected THP-1 cells (MOI 1:25, 8 h).


( I ) Representative confocal of HEK293T expressing GFP - RIPK2 ( 6 h ) immunostained with p62 LC3B antibodies . Zoom panels are digital magnifications .

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(I) Representative confocal images of HEK293T cells expressing GFP-RIPK2 (6 h) immunostained with p62 and LC3B antibodies. Zoom panels are digital magnifications.


( J ) GST pull - down using purified GST or GST - RIPK2 with in - vitro translated 35S radiolabeled myc - ULK1 or myc - ATG16L or myc - BECLIN1 .

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(J) GST pull-down assay using purified GST or GST-RIPK2 proteins with in-vitro translated 35S radiolabeled myc-ULK1 or myc-ATG16L or myc-BECLIN1.


Representative confocal of HEK293T transfected with GFP - RIPK2 ( 9 h ) immunofluorescence performed with antibodies specific to ( K ) ULK1 profile : co - localization using profiles . Scale bar , 5 µm or 3 µm as indicated . Zoom panels are digital magnifications .

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

Representative confocal images of HEK293T cells transfected with GFP-RIPK2 (9 h) and immunofluorescence assay performed with antibodies specific to (K) ULK1 Line profile: co-localization analysis using line intensity profiles. Scale bar, 5 µm or 3 µm as indicated. Zoom panels are digital magnifications.


Representative confocal of HEK293T transfected with GFP - RIPK2 or mcherry - RIPK2 ( 9 h ) immunofluorescence performed with antibodies specific to ( L ) ATG16L ( M ) BECLIN1 . profile : co - localization using profiles . Scale bar , 5 µm or 3 µm as indicated . Zoom panels are digital magnifications .

O O 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 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

Representative confocal images of HEK293T cells transfected with GFP-RIPK2 or mcherry-RIPK2 (9 h) and immunofluorescence assay performed with antibodies specific to (L) ATG16L (M) BECLIN1. Line profile: co-localization analysis using line intensity profiles. Scale bar, 5 µm or 3 µm as indicated. Zoom panels are digital magnifications.


( N ) Luciferase assays were performed with ATG5 or p62 knockdown HEK293T transfected with NF - κB luciferase reporter vector pGL4 . 32 NFκB - RE , GFP - RIPK2 , Flag - NOD1 plasmids . Mean ± SD , n = 3 ( biological replicates ) , * * * * p < 0 . 00005 , ordinary one - way ANOVA ( Tukey ' s multiple comparisons ) .

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

(N) Luciferase assays were performed with ATG5 or p62 knockdown HEK293T cells transfected with NF-κB luciferase reporter vector pGL4.32 NFκB-RE, GFP-RIPK2, and Flag-NOD1 plasmids. Mean ± SD, n=3 (biological replicates), ****p < 0.00005, ordinary one-way ANOVA (Tukey's multiple comparisons test).


( A - B ) THP - 1 lysates were subjected to immunoprecipitation ( A ) untreated treated with iE - DAP ( 40 μg / ml , 6 h ) , ( B ) untreated treated with MDP ( 40 μg / ml , 6 h ) . IP was performed with isotype control IgG or IRGM antibody Western blotting was performed with indicated antibodies . IgG ( H ) ; IgG heavy chain .

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

(A-B) The THP-1 cell lysates were subjected to immunoprecipitation analysis (A) untreated and treated with iE-DAP (40 μg/ml, 6 h), (B) untreated and treated with MDP (40 μg/ml, 6 h). IP was performed with isotype control IgG or IRGM antibody and Western blotting was performed with indicated antibodies. IgG (H); IgG heavy chain.


( C - D ) Co - IP of interactions between ( C ) GFP - RIPK2 Flag - IRGM or ( D ) Flag - NOD1 GFP - IRGM in HEK293T lysates .

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

(C-D) Co-IP analysis of the interactions between (C) GFP-RIPK2 and Flag-IRGM or (D) Flag-NOD1 and GFP-IRGM in HEK293T cell lysates.


( E ) GST pull - down using purified GST GST - IRGM in - vitro translated 35S radiolabeled myc - NOD2 , myc - NOD1 , myc - RIPK2 .

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

(E) GST pull-down assay using purified GST and GST-IRGM and in-vitro translated 35S radiolabeled myc-NOD2, myc-NOD1, and myc-RIPK2.


( F ) Representative confocal of HEK293T expressing GFP - RIPK2 mCherry or mCherry - IRGM . profile : co - localization using profiles . Scale bar , 5 µm .

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

(F) Representative confocal images of HEK293T cells expressing GFP-RIPK2 and mCherry or mCherry-IRGM. Line profile: co-localization analysis using line intensity profiles. Scale bar, 5 µm.


Representative confocal of THP - 1 infected with RFP expressing S . flexneri , ( MOI 1 : 25 , 8 h ) immunostained with , ( H ) IRGM RIPK2 antibody . profile : co - localization using profiles . Scale bar , 5 µm . Zoom panels are digital magnifications . In image H , for better contrast , RFP expressing Shigella is pseudocolored to 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 I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Representative confocal images of THP-1 cells infected with RFP expressing S. flexneri, (MOI 1:25, 8 h) and immunostained with, (H) IRGM and RIPK2 antibody. Line profile: co-localization analysis using line intensity profiles. Scale bar, 5 µm. Zoom panels are digital magnifications. In image H, for better contrast, RFP expressing Shigella is pseudocolored to blue.


( J ) Representative confocal of HEK293T expressing GFP - RIPK2 , Flag - NOD1 , mCherry - IRGM . profile : co - localization using profiles . Scale bar , 3 µm . Zoom panels are digital magnifications .

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

(J) Representative confocal images of HEK293T cells expressing GFP-RIPK2, Flag-NOD1, and mCherry-IRGM. Line profile: co-localization analysis using line intensity profiles. Scale bar, 3 µm. Zoom panels are digital magnifications.


( M ) A co - IP is performed with HEK293T lysates expressing various domains of RIPK2 IRGM to map domain / s of RIPK2 interacting with IRGM . Asterisk indicate main band of overexpressed .

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

(M) A co-IP analysis is performed with HEK293T cell lysates expressing various domains of RIPK2 and IRGM to map the domain/s of RIPK2 interacting with IRGM. Asterisk indicate the main band of overexpressed protein.


( O ) A co - IP is performed with HEK293T lysates expressing various domains of NOD1 IRGM to map domain / s of NOD1 interacting with IRGM . Asterisk indicate main band of overexpressed

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

(O) A co-IP analysis is performed with HEK293T cell lysates expressing various domains of NOD1 and IRGM to map the domain/s of NOD1 interacting with IRGM. Asterisk indicate the main band of overexpressed protein


( A ) Western blot with lysates of control CRISPR CRISPR - Cas9 mediated IRGM partial knockout ( IRGM + / - ) HT - 29 ( 2 clones were tested ) . Densitometric was performed using Image J software . FC , fold change .

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

(A) Western blot analysis with the cell lysates of control CRISPR cells and CRISPR-Cas9 mediated IRGM partial knockout (IRGM+/-) HT-29 cells (2 clones were tested). Densitometric analysis was performed using Image J software. FC, fold change.


( B ) Left panel , soluble insoluble fractions of S . flexneri ( MOI 1 : 25 , 8 h ) infected mouse BMDMs from Irgm1 + / + Irgm1 - / - mice were subjected to immunoblot with antibodies as indicated . Right panel , Western blot with colon lysates from Irgm1 + / + Irgm1 - / - mice with indicated antibodies . graph indicate of band ( measured using ImageJ ) actin ( n = 5 , Mean ± SD , * p < 0 . 05 , * * p < 0 . 005 , * * * * p < 0 . 00005 , Student ' s unpaired t - )

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

(B) Left panel, soluble and insoluble fractions of S. flexneri (MOI 1:25, 8 h) infected mouse BMDMs from Irgm1+/+ and Irgm1-/- mice were subjected to immunoblot analysis with antibodies as indicated. Right panel, Western blot analysis with the colon lysates from Irgm1+/+ and Irgm1-/- mice with indicated antibodies. The graph indicate ratio of band intensity (measured using ImageJ) and actin (n=5, Mean ± SD, *p < 0.05, **p < 0.005, and ****p < 0.00005, Student's unpaired t-test)


( C ) Doxycycline - inducible stable GFP - RIPK2 HeLa were transfected with control siRNA or IRGM siRNA followed by infection with S . flexneri ( MOI 1 : 25 , 4h ) . were fixed subjected to high - content microscopy to quantitate number of RIPosomes formed . graph depicts an average number of RIPosome / . About 10000 were plated per well RIPosomes were screened in 35 fields per well . Mean ± SD , n = 4 ( biological replicates ) , * * * * p < 0 . 00005 , Student ' s unpaired t - .

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

(C) Doxycycline-inducible stable GFP-RIPK2 HeLa cells were transfected with control siRNA or IRGM siRNA followed by infection with S. flexneri (MOI 1:25, 4h). The cells were fixed and subjected to high-content microscopy to quantitate the number of RIPosomes formed. The graph depicts an average number of RIPosome/cell. About 10000 cells were plated per well and RIPosomes were screened in 35 fields per well. Mean ± SD, n=4 (biological replicates), ****p < 0.00005, Student's unpaired t-test.


Western blot with lysate of ( D ) HT - 29 clones stably expressing Flag - vector control or Flag - IRGM Densitometric was performed using Image J software . FC , fold change .

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

Western blot analysis with the cell lysate of (D) HT-29 clones stably expressing Flag-vector control or Flag-IRGM Densitometric analysis was performed using Image J software. FC, fold change.


Western blot with lysate of ( E ) THP - 1 transiently transfected with GFP or GFP - IRGM for 6 h . Densitometric was performed using Image J software . FC , fold change .

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

Western blot analysis with the cell lysate of (E) THP-1 cells transiently transfected with GFP or GFP-IRGM for 6 h. Densitometric analysis was performed using Image J software. FC, fold change.


( F ) Left panel , representative high - content microscopy of NODo - RIPosomes in HEK293T transfected with GFP - RIPK2 myc - NOD2 ( upper panel ) or GFP - RIPK2 , myc - NOD2 , Flag - IRGM ( lower panel ) . Right panel , graph depicts average number of NODo - RIPosome / . About 15000 plated per well RIPosomes were screened in 35 fields per well . Mean ± SD , n = 5 ( biological replicates ) , * * * * p < 0 . 00005 , Student ' s unpaired t - .

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(F) Left panel, representative high-content microscopy images of NODo-RIPosomes in HEK293T cells transfected with GFP-RIPK2 and myc-NOD2 (upper panel) or GFP-RIPK2, myc-NOD2, and Flag-IRGM (lower panel). Right panel, the graph depicts average number of NODo-RIPosome/cell. About 15000 cells plated per well and RIPosomes were screened in 35 fields per well. Mean ± SD, n=5 (biological replicates), ****p < 0.00005, Student's unpaired t-test.


( G ) soluble insoluble fractions of HEK293T transfected with indicated plasmids were subjected to immunoblot with indicated antibodies .

(G) The soluble and insoluble fractions of HEK293T cells transfected with indicated plasmids were subjected to immunoblot analysis with indicated antibodies.


( H ) Representative confocal of HEK293T transfected with mCherry GFP - RIPK2CARD or mCherry - IRGM GFP - RIPK2CARD for 9 h . profile : co - localization using profiles . Scale bar , 5 µm .

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) Representative confocal images of HEK293T cells transfected with mCherry and GFP-RIPK2CARD or mCherry-IRGM and GFP-RIPK2CARD for 9 h. Line profile: co-localization analysis using line intensity profiles. Scale bar, 5 µm.


( I - K ) Western blot with lysates of HEK293T transfected with indicated plasmids probe with Actin , Flag , Myc , , GFP antibodies as indicated . Densitometric was performed using Image J software . FC , fold change .

O O O O O O O O O O O O O O O O O O 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) Western blot analysis with the cell lysates of HEK293T transfected with indicated plasmids and probe with Actin, Flag, Myc, and, GFP antibodies as indicated. Densitometric analysis was performed using Image J software. FC, fold change.


( L ) Left panels , representative high - content microscopy of HEK293T transfected with GFP - RIPK2 or GFP - RIPK2 Flag - IRGM or GFP - RIPK2 Flag - IRGM ( S47N ) . graph depicts average number of RIPosome / . Mean ± SD , n = 4 ( biological replicates ) , * * * * p < 0 . 00005 , ordinary one - way ANOVA ( Tukey ' s multiple comparisons ) .

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 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

(L) Left panels, representative high-content microscopy images of HEK293T cells transfected with GFP-RIPK2 or GFP-RIPK2 and Flag-IRGM or GFP-RIPK2 and Flag-IRGM (S47N). The graph depicts the average number of RIPosome/cell. Mean ± SD, n=4 (biological replicates), ****p < 0.00005, ordinary one-way ANOVA (Tukey's multiple comparisons test).


( C ) Left panel , representative pictures of colons of Irgm1 + / + Irgm - / - mice untreated or treated with GSK583 infected with S . flexneri . Right panel , graph depicts average colon lengths of mice groups . Mean ± SD , n = 6 , * * p < 0 . 005 , * * * p < 0 . 0005 , Student ' s unpaired t - .

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(C) Left panel, representative pictures of colons of Irgm1+/+ and Irgm-/- mice untreated or treated with GSK583 infected with S. flexneri. Right panel, the graph depicts the average colon lengths of the mice groups. Mean ± SD, n=6, **p < 0.005, ***p < 0.0005, Student's unpaired t-test.


( D ) Representative microscopic of H & E of colon tissues of Irgm1 + / + Irgm - / - mice untreated or treated with GSK583 administrated with DSS . graph depicts combined histological scores . Mean ± SD , n = 3 ( DSS ) , * * p < 0 . 005 , Student ' s unpaired t - . Scale bar , 200

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(D) Representative microscopic images of H&E staining of colon tissues of Irgm1+/+ and Irgm-/- mice untreated or treated with GSK583 administrated with DSS. The graph depicts the combined histological scores. Mean ± SD, n=3 (DSS), **p < 0.005, Student's unpaired t-test. Scale bar, 200 µm


( E ) soluble insoluble fractionations of lysates from colon tissues of Shigella - infected or DSS - treated Irgm1 + / + Irgm - / - mice treated with GSK583 as indicated , were subjected to immunoblot with indicated antibodies .

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(E) The soluble and insoluble fractionations of lysates from colon tissues of Shigella-infected or DSS-treated Irgm1+/+ and Irgm-/- mice treated with GSK583 as indicated, were subjected to immunoblot analysis with indicated antibodies.


( H - I ) Western blot with colon tissue lysates of DSS - treated or S . flexneri infected mice groups as indicated .

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(H-I) Western blot analysis with the colon tissue lysates of DSS-treated or S. flexneri infected mice groups as indicated.


A . Arginine methylase inhibitor decreases SIRT7 methylation . HEK293T expressing Flag - tagged SIRT7 were treated with increasing doses of AdOx as indicated for 24 hrs . After immunoprecipitated with Flag - beads , SIRT7 was analyzed by western blot detected by an antibody against mono - methylarginine ( α - me1 ) . Relative methylation ( ) was calculated by normalizing arginine methylation level against Flag - SIRT7 . WCL , whole

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

A. Arginine methylase inhibitor decreases SIRT7 methylation. HEK293T cells expressing Flag-tagged SIRT7 were treated with increasing doses of AdOx as indicated for 24 hrs. After immunoprecipitated with Flag-beads, SIRT7 was analyzed by western blot and detected by an antibody against mono-methylarginine (α-me1). Relative methylation ratio (Ratio) was calculated by normalizing arginine methylation level against the Flag-SIRT7 protein. WCL, whole cell lysate


C . PRMT inhibitor reduces arginine methylation of wildtype SIRT7 , but not its RK / RF mutants . HEK293T expressing wildtype SIRT7 or its RK / RF mutants were treated with or without AdOx for 24 hrs . Arginine methylation of immunopurified SIRT7 was determined by western

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

C. PRMT inhibitor reduces arginine methylation of wildtype SIRT7, but not its RK/RF mutants. HEK293T cells expressing wildtype SIRT7 or its RK/RF mutants were treated with or without AdOx for 24 hrs. Arginine methylation of immunopurified SIRT7 was determined by western blot


D . SIRT7 is mono - asymmetrically di - methylated at R388 . Flag - tagged SIRT7 its RK / RF mutants were expressed in HEK293T . Immunopurified SIRT7 was blotted with antibodies against mono - methylarginine ( α - me1 ) , asymmetric di - methylarginine ( α - me2a ) , symmetric di - methylarginine ( α - me2s ) ,

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

D. SIRT7 is mono- and asymmetrically di-methylated at R388. Flag-tagged SIRT7 and its RK/RF mutants were expressed in HEK293T cells. Immunopurified SIRT7 was blotted with antibodies against mono-methylarginine (α-me1), asymmetric di-methylarginine (α-me2a), and symmetric di-methylarginine (α-me2s), respectively


E . R388 site - specific methylation antibody recognizes R388 - methylated peptide , but not unmodified peptide . unmodified peptide corresponding to R388 , R388 - monomethylated ( R388me1 ) peptide , R388 - asymmetrically di - methylated ( R388me2a ) peptide were blotted with α - meSIRT7 ( R388 )

O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. R388 site-specific methylation antibody recognizes R388-methylated peptide, but not the unmodified peptide. The unmodified peptide corresponding to R388, R388-monomethylated (R388me1) peptide, R388-asymmetrically di-methylated (R388me2a) peptide were blotted with α-meSIRT7(R388) antibody


F . R388 - methylated peptide , but not unmodified peptide , blocks α - meSIRT7 ( R388 ) antibody . After incubation with unmodified peptide or R388 - methylated peptide ( R388me2a ) , α - meSIRT7 ( R388 ) antibody was used to detect immunopurified

O O O O O O O O O O O O O O O O O O O O O O O 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. R388-methylated peptide, but not the unmodified peptide, blocks α-meSIRT7(R388) antibody. After incubation with unmodified peptide or R388-methylated peptide (R388me2a), α-meSIRT7(R388) antibody was used to detect immunopurified SIRT7


G . RK RF mutations disrupt SIRT7 methylation . Flag - tagged wildtype SIRT7 its RK / RF mutants were immunoprecipitated blotted with α - meSIRT7 ( R388 )

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

G. RK and RF mutations disrupt SIRT7 methylation. Flag-tagged wildtype SIRT7 and its RK/RF mutants were immunoprecipitated and blotted with α-meSIRT7(R388) antibody


H . SIRT7 is methylated at R388 . HEK293T ectopically expressing Flag - tagged SIRT7 were treated with increasing concentrations of AdOx for 24 hrs as indicated . SIRT7 was pulled down by Flag - beads . Arginine methylation of SIRT7 was detected with a site - specific antibody against R388 methylation [ α - meSIRT7 ( R388 ) ]

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

H. SIRT7 is methylated at R388. HEK293T cells ectopically expressing Flag-tagged SIRT7 were treated with increasing concentrations of AdOx for 24 hrs as indicated. SIRT7 protein was pulled down by Flag-beads. Arginine methylation of SIRT7 was detected with a site-specific antibody against R388 methylation [α-meSIRT7(R388)]


I - J . AdOx treatment decreases R388 methylation of SIRT7 . MEF L02 were treated with AdOx for 24 hrs . Methylation of immunoprecipitated endogenous SIRT7 was determined by western blot ( I ) . band of western blots was measured to quantify relative R388 methylation ( J ) ( mean ± SD , n = 3 experimental replicates , * * * p < 0 . 001 , unpaired two - t - )

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I-J. AdOx treatment decreases R388 methylation of SIRT7. MEF and L02 cells were treated with AdOx for 24 hrs. Methylation of immunoprecipitated endogenous SIRT7 was determined by western blot (I). The band intensity of western blots was measured to quantify relative R388 methylation ratio (J) (mean ± SD, n=3 experimental replicates, ***p < 0.001, unpaired two-tailed t-test)


K - L . of R388 - methylated endogenous SIRT7 in HEK293T L02 . R388 - methylated endogenous SIRT7 was captured from HEK293T L02 by using R388 site - specific methylation antibody ( K ) . Input , output , immunoprecipitate were analyzed by western blot to quantify of R388 - methylated ( L ) ( mean ± SD , n = 3 experimental replicates )

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K-L. Quantification of R388-methylated endogenous SIRT7 in HEK293T and L02 cells. R388-methylated endogenous SIRT7 was captured from HEK293T and L02 cells by using R388 site-specific methylation antibody (K). Input, output, and immunoprecipitate were analyzed by western blot to quantify the percentage of R388-methylated protein (L) (mean±SD, n=3 experimental replicates)


A . SIRT7 binds to PRMT1 , PRMT3 , PRMT6 . GFP - tagged PRMTs ( PRMT1 - 9 ) were expressed in HEK293T . PRMT was immunoprecipitated with GFP antibody analyzed by western

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A. SIRT7 binds to PRMT1, PRMT3, and PRMT6. GFP-tagged PRMTs (PRMT1-9) were expressed in HEK293T cells. PRMT protein was immunoprecipitated with GFP antibody and analyzed by western blot


B . Co - expression of PRMT6 , but not PRMT1 or PRMT3 , increases R388 methylation of SIRT7 . Flag - tagged SIRT7 GFP - tagged PRMT1 , PRMT3 , or PRMT6 were co - expressed in HEK293T . R388 methylation of immunopurified SIRT7 was analyzed by western

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B. Co-expression of PRMT6, but not PRMT1 or PRMT3, increases R388 methylation of SIRT7. Flag-tagged SIRT7 and GFP-tagged PRMT1, PRMT3, or PRMT6 were co-expressed in HEK293T cells. R388 methylation of immunopurified SIRT7 was analyzed by western blot


C . Wildtype PRMT6 , but not its catalytic - deficient mutant , upregulates SIRT7 R388 methylation . Wildtype PRMT6 or its V86K / D88A mutant ( DM ) was co - expressed with Flag - tagged SIRT7 in HEK293T . R388 methylation of immunopurified SIRT7 was detected by western

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C. Wildtype PRMT6, but not its catalytic-deficient mutant, upregulates SIRT7 R388 methylation. Wildtype PRMT6 or its V86K/D88A mutant (DM) was co-expressed with Flag-tagged SIRT7 in HEK293T cells. R388 methylation of immunopurified SIRT7 was detected by western blot


D - E . PRMT6 physically interacts with endogenous SIRT7 . Endogenous SIRT7 ( D ) or PRMT6 ( E ) was pulled down using corresponding antibody from HEK293T , MEF , mouse hepatocytes , respectively . Input immunoprecipitates were analyzed by western

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D-E. PRMT6 physically interacts with endogenous SIRT7. Endogenous SIRT7 (D) or PRMT6 (E) was pulled down using corresponding antibody from HEK293T, MEF, and primary mouse hepatocytes, respectively. Input and immunoprecipitates were analyzed by western blot


F . SIRT7 associates with PRMT6 in vitro . Recombinant GST - PRMT6 were incubated with HEK293T lysates . After GST pulldown , interaction of recombinant with endogenous SIRT7 was analyzed by western blot . arrows indicate GST GST - PRMT6 ,

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F. SIRT7 associates with PRMT6 in vitro. Recombinant GST-PRMT6 were incubated with HEK293T lysates. After GST pulldown, the interaction of recombinant protein with endogenous SIRT7 was analyzed by western blot. The arrows indicate GST and GST-PRMT6 protein, respectively


G . PRMT6 inhibitor reduces arginine methylation of wildtype SIRT7 , but not RK / RF mutants . Wildtype SIRT7 or its RK / RF mutants were stably expressed in MEF . After treating with or without PRMT6 - specific chemical inhibitor ( PRMT6i ) for 24 hrs , SIRT7 was immunoprecipitated blotted with R388 - specific methylation

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G. PRMT6 inhibitor reduces arginine methylation of wildtype SIRT7, but not RK/RF mutants. Wildtype SIRT7 or its RK/RF mutants were stably expressed in MEF cells. After treating cells with or without PRMT6-specific chemical inhibitor (PRMT6i) for 24 hrs, SIRT7 was immunoprecipitated and blotted with R388-specific methylation antibody


H . Prmt6 depletion decreases R388 methylation of endogenous Sirt7 . Scramble control ( scr ) or MEF stably expressing two different shRNAs ( # 1 # 2 ) against Prmt6 was established . R388 methylation of immunopurified endogenous Sirt7 was determined by western

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H. Prmt6 depletion decreases R388 methylation of endogenous Sirt7. Scramble control cells (scr) or MEF cells stably expressing two different shRNAs (#1 and #2) against Prmt6 was established. R388 methylation of immunopurified endogenous Sirt7 was determined by western blot


I . Treatment with PRMT6 inhibitor reduces R388 methylation of endogenous SIRT7 in a dose - dependent manner . MEF HEK293T were treated with increasing doses of PRMT6i for 24 hrs as indicated . R388 methylation of immunoprecipitated endogenous SIRT7 was determined by western

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I. Treatment with PRMT6 inhibitor reduces R388 methylation of endogenous SIRT7 in a dose-dependent manner. MEF and HEK293T cells were treated with increasing doses of PRMT6i for 24 hrs as indicated. R388 methylation of immunoprecipitated endogenous SIRT7 was determined by western blot


J - K . Prmt6 depletion reduces of R388 - methylated endogenous SIRT7 in MEF . R388 - methylated endogenous SIRT7 was captured from scramble control ( scr ) Prmt6 - knockdown MEF by using R388 site - specific methylation antibody ( J ) . Input , output , immunoprecipitate were analyzed by western blot to quantify of R388 - methylated ( K ) ( mean ± SD , n = 3 experimental replicates )

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J-K. Prmt6 depletion reduces the percentage of R388-methylated endogenous SIRT7 in MEF cells. R388-methylated endogenous SIRT7 was captured from scramble control cells (scr) and Prmt6-knockdown MEF cells by using R388 site-specific methylation antibody (J). Input, output, and immunoprecipitate were analyzed by western blot to quantify the percentage of R388-methylated protein (K) (mean±SD, n=3 experimental replicates)


L . Recombinant PRMT6 directly methylates SIRT7 at R388 in vitro . Bacterially expressed PRMT6 SIRT7 were incubated with SAM or PRMT6i as indicated . Heat - inactivated PRMT6 was included as negative control R388 methylation of recombinant SIRT7 was determined by western

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L. Recombinant PRMT6 directly methylates SIRT7 at R388 in vitro. Bacterially expressed PRMT6 and SIRT7 proteins were incubated with SAM or PRMT6i as indicated. Heat-inactivated PRMT6 was included as negative control R388 methylation of recombinant SIRT7 protein was determined by western blot


A . RF , but not RK mutant , suppresses H3K18 deacetylase activity of SIRT7 . Flag - SIRT7 its RK / RF mutants were expressed in HEK293T MEF , respectively . lysates were subjected to western blot to detect histone H3K18 acetylation . H3K27ac H3K56ac were included as negative control . level of each histone acetylation marker was normalized against histone H3 ( )

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A. RF, but not RK mutant, suppresses H3K18 deacetylase activity of SIRT7. Flag-SIRT7 and its RK/RF mutants were expressed in HEK293T and MEF cells, respectively. Cell lysates were subjected to western blot to detect histone H3K18 acetylation. H3K27ac and H3K56ac were included as negative control. The level of each histone acetylation marker was normalized against histone H3 (Ratio)


B . R388 methylation of SIRT7 decreases its H3K18 deacetylase activity . HEK293T expressing wildtype SIRT7 or its RK / RF mutants were treated with or without AdOx for 24 hrs . SIRT7 enzyme was immunopurified with Flag - beads further incubated with extracted in vitro . R388 methylation of immunopurified SIRT7 H3K18ac of in vitro deacetylated were determined by western

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B. R388 methylation of SIRT7 decreases its H3K18 deacetylase activity. HEK293T cells expressing wildtype SIRT7 or its RK/RF mutants were treated with or without AdOx for 24 hrs. The SIRT7 enzyme was immunopurified with Flag-beads and further incubated with extracted chromatin in vitro. R388 methylation of immunopurified SIRT7 and H3K18ac of in vitro deacetylated chromatin were determined by western blot


C . RF , but not RK mutant , shows reduced H3K18 deacetylase activity in vitro . was extracted from HEK293T incubated with recombinant GST - tagged SIRT7 ( wildtype RK / RF mutants ) in vitro for 2 hrs . H3K18ac was determined by western

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C. RF, but not RK mutant, shows reduced H3K18 deacetylase activity in vitro. Chromatin was extracted from HEK293T cells and incubated with recombinant GST-tagged SIRT7 (wildtype and RK/RF mutants) in vitro for 2 hrs. H3K18ac was determined by western blot


D . Wildtype PRMT6 , but not its methylase - dead mutant , decreases H3K18 deacetylase activity of SIRT7 . Flag - tagged SIRT7 was co - expressed with wildtype PRMT6 or its catalytic - inactive mutant ( DM ) in HEK293T . H3K18 deacetylase activity of immunopurified SIRT7 was assayed in

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D. Wildtype PRMT6, but not its methylase-dead mutant, decreases the H3K18 deacetylase activity of SIRT7. Flag-tagged SIRT7 was co-expressed with wildtype PRMT6 or its catalytic-inactive mutant (DM) in HEK293T cells. The H3K18 deacetylase activity of immunopurified SIRT7 was assayed in vitro


E . PRMT6 deficiency promotes H3K18 deacetylase activity of SIRT7 . Control sgRNA or two independent sgRNAs ( # 1 # 2 ) against PRMT6 was introduced into HEK293T . Flag - tagged SIRT7 was ectopically expressed in these . histone deacetylase activity of immunoprecipitated SIRT7 was assayed in vitro using as a substrate . R388 methylation of immunopurified SIRT7 H3K18 acetylation level of in vitro deacetylated was determined by western

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E. PRMT6 deficiency promotes the H3K18 deacetylase activity of SIRT7. Control sgRNA or two independent sgRNAs (#1 and #2) against PRMT6 was introduced into HEK293T cells. Flag-tagged SIRT7 was ectopically expressed in these cells. The histone deacetylase activity of immunoprecipitated SIRT7 was assayed in vitro using chromatin as a substrate. R388 methylation of immunopurified SIRT7 and H3K18 acetylation level of in vitro deacetylated chromatin was determined by western blot


F - G . Methylated recombinant SIRT7 shows reduced H3K18 deacetylase activity . In vitro methylated SIRT7 from Fig 2L was subjected to in vitro deacetylation . H3K18 acetylation level of in vitro deacetylated was determined by western blot ( F ) . Relative H3K18ac level was quantified from three independent experiments ( G ) ( mean ± SD , n = 3 experimental replicates , * * p < 0 . 01 , n . s . = not significant , unpaired two - t )

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F-G. Methylated recombinant SIRT7 shows reduced H3K18 deacetylase activity. In vitro methylated SIRT7 from Fig 2L was subjected to in vitro deacetylation assay. H3K18 acetylation level of in vitro deacetylated chromatin was determined by western blot (F). Relative H3K18ac level was quantified from three independent experiments (G) (mean±SD, n=3 experimental replicates, **p<0.01, n.s.=not significant, unpaired two-tailed t test)


Wildtype SIRT7 or its RF mutant was re - expressed in Sirt7 - knockdown MEF ( A )

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Wildtype SIRT7 or its RF mutant was re-expressed in Sirt7-knockdown MEF cells (A)


Wildtype SIRT7 , but not its RF mutant , restores mitochondria mas in Sirt7 - deficient MEF Mitochondria mass was determined by Mito Tracker Red ( B ) ( mean ± SD , n = 3 experimental replicates , * p < 0 . 05 ; * * p < 0 . 01 ; n . s . = not significant , unpaired two - t )

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Wildtype SIRT7, but not its RF mutant, restores mitochondria mas in Sirt7-deficient MEF cells Mitochondria mass was determined by Mito Tracker Red staining (B) (mean ± SD, n=3 experimental replicates, *p<0.05; **p<0.01; n.s.=not significant, unpaired two-tailed t test)


Wildtype SIRT7 , but not its RF mutant , restore ATP leve in Sirt7 - deficient MEF ATP was quantified normalized to number ( C ) ( mean ± SD , n = 3 experimental replicates , * p < 0 . 05 ; * * p < 0 . 01 ; n . s . = not significant , unpaired two - t )

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

Wildtype SIRT7, but not its RF mutant, restore ATP leve in Sirt7-deficient MEF cells Cellular ATP was quantified and normalized to cell number (C) (mean ± SD, n=3 experimental replicates, *p<0.05; **p<0.01; n.s.=not significant, unpaired two-tailed t test)


Wildtype SIRT7 , but not its RF mutant , restore respiration in Sirt7 - deficient MEF Respiration flux was determined as indicated ( D ) . ( mean ± SD , n = 3 experimental replicates , * p < 0 . 05 ; * * p < 0 . 01 ; n . s . = not significant unpaired two - t )

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Wildtype SIRT7, but not its RF mutant, restore cellular respiration in Sirt7-deficient MEF cells Respiration flux was determined as indicated (D). (mean ± SD, n=3 experimental replicates, *p<0.05; **p<0.01; n.s.=not significant unpaired two-tailed t test)


Wildtype SIRT7 , but not its RF mutant , downregulates SIRT7 target genes involved in mitochondria biogenesis . Wildtype SIRT7 its RF mutant were stably expressed in MEF ( E )

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

Wildtype SIRT7, but not its RF mutant, downregulates SIRT7 target genes involved in mitochondria biogenesis. Wildtype SIRT7 and its RF mutant were stably expressed in MEF cells (E)


Wildtype SIRT7 , but not its RF mutant , downregulates SIRT7 target genes involved in mitochondria biogenesis SIRT7 target gene expression was determined presented as a on a log2 scale ( F )

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Wildtype SIRT7, but not its RF mutant, downregulates SIRT7 target genes involved in mitochondria biogenesis SIRT7 target gene expression was determined and presented as a heatmap on a log2 scale (F)


Control stable Sirt7 - knockdown MEF were treated with or without 5 µM PRMT6i for 24 hrs . H3K18ac level at promoter of SIRT7 target genes ( Gfm2 , Mrps31 , Mrps33 , Mrpl40 ) was determined by ChIP - qPCR , Rabbit IgG was used as a negative control ( G ) ( Data shown are mean ± SD , n = 3 experimental replicates , * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 ; n . s . = not significant , unpaired two - t

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

Control cells and stable Sirt7-knockdown MEF cells were treated with or without 5 µM PRMT6i for 24 hrs. The H3K18ac level at the promoter of SIRT7 target genes (Gfm2, Mrps31, Mrps33, and Mrpl40) was determined by ChIP-qPCR, Rabbit IgG was used as a negative control (G) ( Data shown are mean±SD, n=3 experimental replicates, *p<0.05; **p<0.01; ***p < 0.001; n.s.=not significant, unpaired two-tailed t test


Inhibition of Prmt6 decreases mitochondria mas of control , but not Sirt7 - knockdown MEF Control stable Sirt7 - knockdown MEF were treated with or without 5 µM PRMT6i for 24 hrs Mitochondria mass was determined by Mito Tracker Red ( H ) ( Data shown are mean ± SD , n = 3 experimental replicates , * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 ; n . s . = not significant , unpaired two - t

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Inhibition of Prmt6 decreases mitochondria mas of control cells, but not Sirt7-knockdown MEF cells Control cells and stable Sirt7-knockdown MEF cells were treated with or without 5 µM PRMT6i for 24 hrs Mitochondria mass was determined by Mito Tracker Red staining (H) ( Data shown are mean±SD, n=3 experimental replicates, *p<0.05; **p<0.01; ***p < 0.001; n.s.=not significant, unpaired two-tailed t test


Inhibition of Prmt6 decrease ATP leve of control , but not Sirt7 - knockdown MEF . Control stable Sirt7 - knockdown MEF were treated with or without 5 µM PRMT6i for 24 hrs ATP was quantified normalized to number ( I ) ( Data shown are mean ± SD , n = 3 experimental replicates , * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 ; n . s . = not significant , unpaired two - t

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Inhibition of Prmt6 decrease ATP leve of control cells, but not Sirt7-knockdown MEF cells. Control cells and stable Sirt7-knockdown MEF cells were treated with or without 5 µM PRMT6i for 24 hrs Cellular ATP was quantified and normalized to cell number (I) ( Data shown are mean±SD, n=3 experimental replicates, *p<0.05; **p<0.01; ***p < 0.001; n.s.=not significant, unpaired two-tailed t test


Inhibition of Prmt6 decrease respiration flux of control , but not Sirt7 - knockdown MEF . Control stable Sirt7 - knockdown MEF were treated with or without 5 µM PRMT6i for 24 hrs Respiration flux was determined as indicated ( J ) . ( Data shown are mean ± SD , n = 3 experimental replicates , * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 ; n . s . = not significant , unpaired two - t

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

Inhibition of Prmt6 decrease the respiration flux of control cells, but not Sirt7-knockdown MEF cells. Control cells and stable Sirt7-knockdown MEF cells were treated with or without 5 µM PRMT6i for 24 hrs Respiration flux was determined as indicated (J). ( Data shown are mean±SD, n=3 experimental replicates, *p<0.05; **p<0.01; ***p < 0.001; n.s.=not significant, unpaired two-tailed t test


A . Glucose depletion reduces SIRT7 methylation . MEF stably expressing Flag - SIRT7 were cultured in medium depleted with glucose ( 12 hrs ) , FBS ( 12 hrs ) or glutamine ( 24 hrs ) . R388 methylation of immunopurified SIRT7 Ampk phosphorylation were determined by western blot . R388 methylation level was normalized to Flag - SIRT7 ( )

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A. Glucose depletion reduces SIRT7 methylation. MEF cells stably expressing Flag-SIRT7 were cultured in medium depleted with glucose (12 hrs), FBS (12 hrs) or glutamine (24 hrs). R388 methylation of immunopurified SIRT7 and Ampk phosphorylation were determined by western blot. R388 methylation level was normalized to Flag-SIRT7 (Ratio)


B . Glucose starvation decreases R388 methylation of SIRT7 in a time - dependent manner . MEF stably expressing Flag - SIRT7 were cultured in glucose - free media as indicated . R388 methylation level of immunopurified SIRT7 was determined by western

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B. Glucose starvation decreases R388 methylation of SIRT7 in a time-dependent manner. MEF cells stably expressing Flag-SIRT7 were cultured in glucose-free media as indicated. R388 methylation level of immunopurified SIRT7 was determined by western blot


C . Glucose dose - dependently upregulates R388 methylation of SIRT7 . MEF stably expressing Flag - SIRT7 were cultured with increasing concentrations of glucose as indicated for 12 hrs . R388 methylation of SIRT7 Ampk phosphorylation were determined by western

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C. Glucose dose-dependently upregulates R388 methylation of SIRT7. MEF cells stably expressing Flag-SIRT7 were cultured with increasing concentrations of glucose as indicated for 12 hrs. R388 methylation of SIRT7 and Ampk phosphorylation were determined by western blot


D . AICAR treatment decreases R388 methylation of SIRT7 . MEF stably expressing Flag - SIRT7 were treated with or without AICAR for 12 hrs . R388 methylation of immunoprecipitated SIRT7 was determined by western

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D. AICAR treatment decreases R388 methylation of SIRT7. MEF cells stably expressing Flag-SIRT7 were treated with or without AICAR for 12 hrs. R388 methylation of immunoprecipitated SIRT7 was determined by western blot


E - F . AICAR reduces of R388 - methylated endogenous SIRT7 in MEF . R388 - methylated endogenous SIRT7 was captured from control AICAR - treated MEF by using R388 site - specific methylation antibody ( E ) . Input , output , immunoprecipitate were analyzed by western blot to quantify of R388 - methylated ( F ) ( mean ± SD , n = 3 experimental replicates )

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E-F. AICAR reduces the percentage of R388-methylated endogenous SIRT7 in MEF cells. R388-methylated endogenous SIRT7 was captured from control and AICAR-treated MEF cells by using R388 site-specific methylation antibody (E). Input, output, and immunoprecipitate were analyzed by western blot to quantify the percentage of R388-methylated protein (F) (mean±SD, n=3 experimental replicates)


G . AMPK deletion increases SIRT7 R388 methylation . Flag - tagged SIRT7 was stably expressed in Ampk wildtype ( WT ) Ampk α1 / α2 double - knockout ( DKO ) MEF 293A . SIRT7 was immunopurified with Flag - beads , R388 methylation was determined by western

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G. AMPK deletion increases SIRT7 R388 methylation. Flag-tagged SIRT7 was stably expressed in Ampk wildtype (WT) and Ampk α1/α2 double-knockout (DKO) MEF cells and 293A cells. SIRT7 protein was immunopurified with Flag-beads, R388 methylation was determined by western blot


H . AMPK is required for glucose starvation - induced R388 hypomethylation of SIRT7 . Ampk wildtype ( WT ) Ampk α1 / α2 double - knockout ( DKO ) MEF stably expressing Flag - SIRT7 were cultured with or without glucose for 12 hrs . SIRT7 was purified with Flag - beads R388 methylation was determined by western

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

H. AMPK is required for glucose starvation-induced R388 hypomethylation of SIRT7. Ampk wildtype (WT) and Ampk α1/α2 double-knockout (DKO) MEF cells stably expressing Flag-SIRT7 were cultured with or without glucose for 12 hrs. SIRT7 was purified with Flag-beads and R388 methylation was determined by western blot


I . PRMT6 is necessary for AMPK - induced SIRT7 hypomethylation . Flag - tagged SIRT7 was stably expressed in scramble control MEF or expressing two independent shRNAs targeting Prmt6 ( # 1 # 2 ) . were cultured with or without glucose for 12 hrs . R388 methylation of immunopurified SIRT7 was determined by western

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

I. PRMT6 is necessary for AMPK-induced SIRT7 hypomethylation. Flag-tagged SIRT7 was stably expressed in scramble control MEF cells or cells expressing two independent shRNAs targeting Prmt6 (#1 and #2). Cells were cultured with or without glucose for 12 hrs. R388 methylation of immunopurified SIRT7 was determined by western blot


J . PRMT6 is necessary for AMPK - induced SIRT7 hypomethylation . Wildtype MEFs Ampk DKO were treated with PRMT6i for 24 hrs . SIRT7 was purified by Flag - beads , R388 methylation of SIRT7 was determined by western

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

J. PRMT6 is necessary for AMPK-induced SIRT7 hypomethylation. Wildtype MEFs and Ampk DKO cells were treated with PRMT6i for 24 hrs. SIRT7 was purified by Flag-beads, and R388 methylation of SIRT7 was determined by western blot


K . Both AICAR treatment glucose depletion impair association between Prmt6 Sirt7 . MEF were cultured with or without glucose treated with AICAR as indicated . Co - immunoprecipitation was performed using SIRT7 antibody . Input immunoprecipitates were analyzed by western

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K. Both AICAR treatment and glucose depletion impair the association between Prmt6 and Sirt7. MEF cells were cultured with or without glucose and treated with AICAR as indicated. Co-immunoprecipitation was performed using SIRT7 antibody. Input and immunoprecipitates were analyzed by western blot


L . R388 methylation of SIRT7 does not modulate its phosphorylation . MEF stably expressing Flag - tagged wildtype SIRT7 or RK mutant were treated with PRMT6i ( 24 hrs ) cultured with or without glucose ( 12 hrs ) as indicated . SIRT7 was immunopurified with Flag - beads , R388 methylation phosphorylation of SIRT7 were determined by western

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L. R388 methylation of SIRT7 does not modulate its phosphorylation. MEF cells stably expressing Flag-tagged wildtype SIRT7 or RK mutant were treated with PRMT6i (24 hrs) and cultured with or without glucose (12 hrs) as indicated. SIRT7 protein was immunopurified with Flag-beads, R388 methylation and phosphorylation of SIRT7 were determined by western blot


Glucose administration leads to R388 hypermethylation of Sirt7 in mouse hepatocyte . After fasting for 16 hrs , mice were injected with glucose . Hepatic tissue was harvested in a time - dependent manner as indicated ( n = 3 for each time point ) . SIRT7 was immunoprecipitated from whole liver extracts . R388 methylation of Sirt7 was determined by western blot ( A Scale bar in upper panel , 500 µm ; scale bar in lower panel , 100

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

Glucose administration leads to R388 hypermethylation of Sirt7 in mouse hepatocyte. After fasting for 16 hrs, mice were injected with glucose. Hepatic tissue was harvested in a time-dependent manner as indicated (n=3 for each time point). SIRT7 protein was immunoprecipitated from whole liver extracts. R388 methylation of Sirt7 was determined by western blot (A Scale bar in the upper panel, 500 µm; scale bar in the lower panel, 100 µm


Glucose administration leads to R388 hypermethylation of Sirt7 in mouse hepatocyte . After fasting for 16 hrs , mice were injected with glucose . Hepatic tissue was harvested in a time - dependent manner as indicated ( n = 3 for each time point ) . SIRT7 was immunoprecipitated from whole liver extracts . R388 methylation of Sirt7 was determined b immunohistochemical ( B ) . Scale bar in upper panel , 500 µm ; scale bar in lower panel , 100

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

Glucose administration leads to R388 hypermethylation of Sirt7 in mouse hepatocyte. After fasting for 16 hrs, mice were injected with glucose. Hepatic tissue was harvested in a time-dependent manner as indicated (n=3 for each time point). SIRT7 protein was immunoprecipitated from whole liver extracts. R388 methylation of Sirt7 was determined b immunohistochemical staining (B). Scale bar in the upper panel, 500 µm; scale bar in the lower panel, 100 µm


AICAR PRMT6 inhibitor decrease R388 methylation epigenetically downregulate SIRT7 - target genes . MEF stably expressing Flag - SIRT7 were incubated with or without PRMT6i for 12 hrs , followed by treatment with 1 mM AICAR for 12 hrs . R388 methylation of SIRT7 was determined by western blot ( C ) ( Data shown are mean ± SD , n = 3 experimental replicates , * * p < 0 . 01 ; * * * p < 0 . 001 , unpaired two - t

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AICAR and PRMT6 inhibitor decrease R388 methylation and epigenetically downregulate SIRT7-target genes. MEF cells stably expressing Flag-SIRT7 were incubated with or without PRMT6i for 12 hrs, followed by treatment with 1 mM AICAR for 12 hrs. R388 methylation of SIRT7 was determined by western blot (C) (Data shown are mean±SD, n=3 experimental replicates, **p<0.01; ***p < 0.001, unpaired two-tailed t test


AICAR PRMT6 inhibitor decrease R388 methylation epigenetically downregulate SIRT7 - target genes . MEF stably expressing Flag - SIRT7 were incubated with or without PRMT6i for 12 hrs , followed by treatment with 1 mM AICAR for 12 hrs H3K18ac level at SIRT7 - target gene promoters were determined by ChIP - qPCR . Normal rabbit IgG was used as a negative control ( D ) ( Data shown are mean ± SD , n = 3 experimental replicates , * * p < 0 . 01 ; * * * p < 0 . 001 , unpaired two - t

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-CONTROLLED_VAR 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

AICAR and PRMT6 inhibitor decrease R388 methylation and epigenetically downregulate SIRT7-target genes. MEF cells stably expressing Flag-SIRT7 were incubated with or without PRMT6i for 12 hrs, followed by treatment with 1 mM AICAR for 12 hrs H3K18ac level at SIRT7-target gene promoters were determined by ChIP-qPCR. Normal rabbit IgG was used as a negative control (D) (Data shown are mean±SD, n=3 experimental replicates, **p<0.01; ***p < 0.001, unpaired two-tailed t test


AICAR PRMT6 inhibitor decrease R388 methylation epigenetically downregulate SIRT7 - target genes . MEF stably expressing Flag - SIRT7 were incubated with or without PRMT6i for 12 hrs , followed by treatment with 1 mM AICAR for 12 hrs mRNA expression was determined by qPCR ( E ) ( Data shown are mean ± SD , n = 3 experimental replicates , * * p < 0 . 01 ; * * * p < 0 . 001 , unpaired two - t

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-CONTROLLED_VAR 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

AICAR and PRMT6 inhibitor decrease R388 methylation and epigenetically downregulate SIRT7-target genes. MEF cells stably expressing Flag-SIRT7 were incubated with or without PRMT6i for 12 hrs, followed by treatment with 1 mM AICAR for 12 hrs mRNA expression was determined by qPCR analysis (E) (Data shown are mean±SD, n=3 experimental replicates, **p<0.01; ***p < 0.001, unpaired two-tailed t test


AICAR PRMT6 inhibitor decrease R388 methylation epigenetically downregulate SIRT7 - target genes . MEF stably expressing Flag - SIRT7 were incubated with or without PRMT6i for 12 hrs , followed by treatment with 1 mM AICAR for 12 hrs Mitochondria mass was determined by Mito Tracker Red ( F ) . ( Data shown are mean ± SD , n = 3 experimental replicates , * * p < 0 . 01 ; * * * p < 0 . 001 , unpaired two - t

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-CONTROLLED_VAR 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

AICAR and PRMT6 inhibitor decrease R388 methylation and epigenetically downregulate SIRT7-target genes. MEF cells stably expressing Flag-SIRT7 were incubated with or without PRMT6i for 12 hrs, followed by treatment with 1 mM AICAR for 12 hrs Mitochondria mass was determined by Mito Tracker Red staining (F). (Data shown are mean±SD, n=3 experimental replicates, **p<0.01; ***p < 0.001, unpaired two-tailed t test


B . Normalized fitness functions of E . coli BW25113 in absence of tet ( B ) their corresponding model fits in presence of varying concentrations of TET , DOX , MCN in media ( [ S ] M ) . Normalized quadruplicate data is shown for each antibiotic .

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B. Normalized fitness functions of E. coli BW25113 in the absence of tet(B) and their corresponding model fits in the presence of varying concentrations of antibiotics TET, DOX, and MCN in the media ([S]M). Normalized quadruplicate data is shown for each antibiotic.


B . Normalized fitness functions of expressing wild type tet ( B ) their corresponding model fits in presence of TET , DOX MCN . Triplicate data is shown for each

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

B. Normalized fitness functions of cells expressing wild type tet(B) and their corresponding model fits in the presence of TET, DOX and MCN. Triplicate data is shown for each antibiotic


B . Model fits for E . coli BW25113 strains expressing a chromosomal copy of tet ( B ) WT seven variants in TET , DOX , MCN . Our model fit triplicate fitness function data for strains ( Figure S2 ) . At low drug concentrations for , tet ( B ) G366C exhibits much slower growth rates than wild - type . Compared to wild - type , strains expressing tet ( B ) L9F exhibit similar growth rates at low high drug concentrations but have faster growth rates at intermediate drug concentrations .

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

B. Model fits for E. coli BW25113 strains expressing a chromosomal copy of tet(B)WT and seven variants in TET, DOX, and MCN. Our model fit triplicate fitness function data for all strains (Figure S2). At low drug concentrations for all antibiotics, tet(B)G366C exhibits much slower growth rates than wild-type. Compared to wild-type, strains expressing tet(B)L9F exhibit similar growth rates at low and high drug concentrations but have faster growth rates at intermediate drug concentrations.


C . Fitness landscapes with mutants overlaid for TET = 35 µg / mL , DOX = 10 µg / mL , MCN = 2 . 5 µg / mL corresponding to dashed lines in 3B .

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C. Fitness landscapes with mutants overlaid for TET=35 µg/mL, DOX=10 µg/mL, and MCN=2.5 µg/mL corresponding to the dashed lines in 3B.


E . coli BW25113 strains expressing a chromosomal copy of tet ( B ) WT or variant were grown in 2 % arabinose supplemented LB in absence of drug harvested at mid - exponential phase . samples were prepared from three colonies grown independently ( Figures S5 S6 ) . Compared to wild - type , strains expressing tet ( B ) L9F consistently have higher in vivo where as strains expressing tet ( B ) V339C tet ( B ) G366C have lower in vivo as detected by Western Blot . four variants located in interdomain loop exhibited in vivo similar to wild - type .

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

E. coli BW25113 strains expressing a chromosomal copy of tet(B)WT or variant were grown in 2% arabinose supplemented LB in the absence of drug and harvested at mid-exponential phase. Membrane samples were prepared from three individual colonies grown independently (Figures S5 and S6). Compared to wild-type, strains expressing tet(B)L9F consistently have higher in vivo protein levels where as strains expressing tet(B)V339C and tet(B)G366C have lower in vivo protein levels as detected by Western Blot analysis. All four variants located in the interdomain loop exhibited in vivo protein levels similar to wild-type.


A . spread of κ γ fits for 1000 randomly selected parameters A B from a set gamma distribution shows a linear correlation between κ γ .

O O O 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 spread of κ and γ fits for 1000 randomly selected parameters A and B from a set gamma distribution shows a linear correlation between κ and γ.


B . calculated mean error of γ / κ for sampled parameter fittings shown in Figure S2 . large error seen for TetBV339C TetBG366C in MCN reveals that we cannot make first order approximation within $ \ frac { \ gamma } { \ kappa } \ \ approx k _ { 1 } \ frac { { 2P } _ { \ text { tot } } } { D _ { 2 } } $ .

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 O O O O O O O O O O O O O O O O O O O O O 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. The calculated mean and error of γ/κ for the sampled parameter fittings shown in Figure S2. The large error seen for TetBV339C and TetBG366C in MCN reveals that we cannot make the first order approximation within $\frac{\gamma}{\kappa}\ \approx k_{1}\frac{{2P}_{\text{tot}}}{D_{2}}$.


A . Changes in drug binding affinity Δk1 pumping efficiency Δr for TetB variants relative to wild type in TET , DOX MCN . Error bars are comprised from a propagation of errors from A B global parameter modeling in vivo level determination .

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

A. Changes in drug binding affinity Δk1 and pumping efficiency Δr for TetB variants relative to wild type in TET, DOX and MCN. Error bars are comprised from a propagation of errors from A and B global parameter modeling and in vivo protein level determination.


A . Triplicate normalized fitness functions ( * ) for E . coli BW25113 strains harboring plasmid copies of tet ( B ) WT , tet ( B ) K183E , A290V , T326I ( variant 6 ) tet ( B ) F151I , F179L , T326A ( variant 7 ) in TET their corresponding least squares model fits ( solid ) where both variants display decreased fitness in TET compared to WT .

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

A. Triplicate normalized fitness functions (*) for E. coli BW25113 strains harboring plasmid copies of tet(B)WT, tet(B)K183E,A290V,T326I (variant 6) and tet(B)F151I,F179L,T326A (variant 7) in TET and their corresponding least squares model fits (solid line) where both variants display decreased fitness in TET compared to WT.


( B ) Representative of mCherry - optoYAP in HEK293T imaged with only 561 nm following protocol in ( A ) . Scale bars , 10 μm . ( B ' ) Fold - change in localisation of mCherry - optoYAP ( n = 50 from 3 independent experiments ) .

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(B) Representative images of mCherry-optoYAP in HEK293T cells imaged with only 561 nm laser following the protocol in (A). Scale bars, 10 μm. (B') Fold-change in nuclear localisation of mCherry-optoYAP (n=50 cells from 3 independent experiments).


( C ) Representative of same in ( B ) exposed to both 488 561 nm following protocol shown in ( A ) . Scale bars , 10 μm . ( C ' ) Fold - change in localisation of mCherry - optoYAP ( n = 50 from 3 independent experiments ) .

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(C) Representative images of the same cells in (B) exposed to both 488 and 561 nm following the protocol shown in (A). Scale bars, 10 μm. (C') Fold-change in nuclear localisation of the mCherry-optoYAP (n=50 cells from 3 independent experiments).


( D ) HEK293T transfected with optoYAP were subjected to three cycles of activation protocol recovery in . ( Top ) of mCherry - optoYAP signal in nucleus to mCherry - optoYAP signal ( n = 8 from 2 independent experiments ) . ( Bottom ) Representative of mCherry - optoYAP signal from same HEK293T at each cycle . Scale bars , 10 μm .

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(D) HEK293T cells transfected with optoYAP were subjected to three cycles of activation protocol and recovery in the dark. (Top) The ratio of mCherry-optoYAP signal in the nucleus to the cytoplasmic mCherry-optoYAP signal (n=8 cells from 2 independent experiments). (Bottom) Representative images of the mCherry-optoYAP signal from the same HEK293T cells at each cycle. Scale bars, 10 μm.


( B ) Example of expressing optofYap ( mCherry - tagged ) in EVL deep kept in or subjected to activation protocol at 6 hpf . Scale bars , 10 μm . ( B ' ) Fold - change in localisation of mCherry - optofYap ( n = 20 from 3 independent experiments ) .

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(B) Example images of embryos expressing optofYap (mCherry-tagged) in the EVL and deep cells kept in the dark or subjected to the activation protocol at 6 hpf. Scale bars, 10 μm. (B') Fold-change in nuclear localisation of mCherry-optofYap (n=20 cells from 3 independent experiments).


Soft agar colony formation for HFF ( A - B ) respectively grown on soft agar for one week under different conditions as described in Fig . 1A . Scale bars , 1 mm . Representative of colony growth in HFF respectively under different activation conditions . Diameter of colonies formed under different conditions genetic backgrounds . Colonies were counted measured from 3 biological replicates .

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Soft agar colony formation assay for HFF (A-B) cells respectively grown on soft agar for one week under different light conditions as described in Fig. 1A. Scale bars, 1 mm. Representative images of colony growth in HFF cells respectively under different activation conditions. Diameter of individual colonies formed under different light conditions and genetic backgrounds. Colonies were counted and measured from 3 biological replicates.


Soft agar colony formation for MKN28 ( C - D ) respectively grown on soft agar for one week under different conditions as described in Fig . 1A . Scale bars , 1 mm . Representative of colony growth in MKN28 respectively under different activation conditions . Diameter of colonies formed under different conditions genetic backgrounds . Colonies were counted measured from 3 biological replicates .

Soft agar colony formation assay for MKN28 (C-D) cells respectively grown on soft agar for one week under different light conditions as described in Fig. 1A. Scale bars, 1 mm. Representative images of colony growth in MKN28 cells respectively under different activation conditions. Diameter of individual colonies formed under different light conditions and genetic backgrounds. Colonies were counted and measured from 3 biological replicates.


Time - dependent acceptor changes due to FRET during co - translational insertion of acceptor - labeled LepB75 into donor - labeled translocon .

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Time-dependent acceptor fluorescence changes due to FRET during co-translational insertion of acceptor-labeled LepB75 into donor-labeled translocon.


of LepB94 translation products on SDS - PAGE . Translation products are visualized by of N - terminal Atto655 . Pausing intermediates are indicated P1 P2 .

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

Analysis of LepB94 translation products on SDS-PAGE. Translation products are visualized by the fluorescence of N-terminal Atto655. Pausing intermediates are indicated P1 and P2.


Fitting of time courses ( filled circles ) of translation of LepB mRNA constructs of increasing ( Appendix Fig S3 ) by delay - exponential functions ( red lines ) .

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Fitting of time courses (filled circles) of the translation of LepB mRNA constructs of increasing length (Appendix Fig S3) by delay-exponential functions (red lines).


Stopped - flow time courses of co - translational LepB insertion into SecYEG ( Atto488 donor - in at position 111 of SecY ) , as monitored by of Atto655 acceptor placed at N - terminus of nascent peptide . Fits ( red lines ) were obtained by global fitting

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Stopped-flow time courses of co-translational LepB insertion into SecYEG (Atto488 donor-in at position 111 of SecY), as monitored by the fluorescence of the Atto655 acceptor placed at the N-terminus of the nascent peptide. Fits (red lines) were obtained by global fitting


Stopped - flow time courses of co - translational LepB insertion into SecYEG ( Atto488 donor - in at position 111 of SecY ) , as monitored by of Atto655 acceptor placed at N - terminus of nascent peptide . Fits ( red lines ) were obtained by global

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Intrinsic intensities ( IFI ) derived from global fitting are shown as black lines filled circles along with amplitudes obtained from exponential fitting ( red squares ) . Confidence contours ( shaded areas ) were calculated using Fitspace

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Intrinsic fluorescence intensities (IFI) derived from global fitting are shown as black lines and filled circles along with amplitudes obtained from exponential fitting (red squares). Confidence contours (shaded areas) were calculated using the Fitspace algorithm


Time course of EmrD135 translation . Translation was monitored by SDS - PAGE as in Fig 2A . Time course of EmrD ( - ) 135 translation .

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Time course of EmrD135 translation. Translation was monitored by SDS-PAGE as in Fig 2A. Time course of EmrD(-)135 translation.


Stopped - flow time courses for translocon insertion of TM1 of EmrD ( black ) EmrD ( - ) ( blue ) with mRNA constructs of varying chain ( 50 to 135 aa ) . FRET acceptor ( Atto655 ) was placed at N - terminus of nascent peptide , donor ( Atto488 ) at position 111 ( donor - in ) of SecY . results of global fitting are indicated ( red lines ) .

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

Stopped-flow time courses for translocon insertion of TM1 of EmrD (black) and EmrD(-) (blue) with mRNA constructs of varying chain length (50 to 135 aa).The FRET acceptor (Atto655) was placed at the N-terminus of the nascent peptide, the donor (Atto488) at position 111 (donor-in) of SecY. The results of global fitting are indicated (red lines).


Intrinsic intensities ( IFIs ) calculated for EmrD ( black ) EmrD ( - ) ( blue ) . Error margins ( shaded areas ) are calculated using Fitspace

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

Intrinsic fluorescence intensities (IFIs) calculated for EmrD (black) and EmrD(-) (blue). Error margins (shaded areas) are calculated using the Fitspace algorithm


Stopped - flow traces for co - translational insertion of LepB94 ( red , from Fig 2 ) , EmrD135 ( black , from Fig 4E ) , EmrD ( - ) 135 ( blue , from Fig 4E ) , rescaled according to translation efficiency .

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

Stopped-flow traces for co-translational insertion of LepB94 (red, from Fig 2), EmrD135 (black, from Fig 4E), and EmrD(-)135 (blue, from Fig 4E), rescaled according to the translation efficiency.


A , B , C LepB ( A ) , EmrD ( B ) , EmrD ( - ) ( C ) are translated for times indicated subjected to PK treatment ( Materials Methods ) . Sensitivity to PK in absence of SecYEG ( black circles ) or in presence of SecYEG ( red squares ) , co - translational translocon insertion monitored by FRET ( blue trace ) are depicted . Error bars represent standard deviations ( n ≥ 3 ) corresponding lines represent fits to delay - exponential functions .

O O O O O B-MEASURED_VAR O O O O B-MEASURED_VAR O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O 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, C LepB (A), EmrD (B), and EmrD(-) (C) are translated for the times indicated and subjected to PK treatment (Materials and Methods). Sensitivity to PK in the absence of SecYEG (black circles) or in the presence of SecYEG (red squares), and co-translational translocon insertion monitored by FRET (blue trace) are depicted. Error bars represent standard deviations (n≥3) and the corresponding lines represent fits to delay-exponential functions.


DNase - Seq tag density plots showing peaks detected in replicate 1 of TB IL - 2 TB IL - 2nil ( 32694 ) ordered by increasing fold change of tag for TB IL - 2 compared to TB IL - 2nil . tag density is also shown in parallel at same sites for DNase - Seq analyses of CD4 naïve T ( TN ) , TB IL - 2 DMSO TB IL - 2 Ruxolitinib ATAC samples from in vivo derived Th2 ( Shih et al . , 2016 ) , Th1 Th17 . location of 1000 IL - 2 pDHSs is shown to right .

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

DNase-Seq tag density plots showing all peaks detected in replicate 1 of TB IL-2 and TB IL-2nil (32694) ordered by increasing fold change of tag count for TB IL-2 compared to TB IL-2nil. The sequence tag density is also shown in parallel at the same sites for the DNase-Seq analyses of CD4 naïve T cells (TN), TB IL-2 DMSO and TB IL-2 Ruxolitinib and ATAC samples from in vivo derived Th2 (Shih et al., 2016), Th1 and Th17 cells. The location of the 1000 IL-2 pDHSs is shown to the right.


Average DNase - Seq tag density profile for 1000 IL - 2 pDHSs in both replicates of TB IL - 2 TB IL - 2nil samples , for naïve T ( TN ) .

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

Average DNase-Seq tag density profile for the 1000 IL-2 pDHSs in both replicates of TB IL-2 and TB IL-2nil samples, and for naïve T cells (TN).


Number of IL - 2 pDHSs which are detected in in vivo purified CD4 T subsets ( Chen et al . , 2020 , Garg et al . , 2019 , Shih et al . , 2016 ) .

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Number of IL-2 pDHSs which are detected in in vivo purified CD4 T cell subsets (Chen et al., 2020, Garg et al., 2019, Shih et al., 2016).


UCSC genome browser tracks with examples of IL - 2 pDHSs within Locus Control Region ( LCR ) within Rad50 gene which controls Th2 cytokines Il13 , Il4 Il5 , plus IL - 2 pDHSs within Tnfrsf9 Cish loci . IL - 2 pDHSs are highlighted with blue boxes .

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

UCSC genome browser tracks with examples of IL-2 pDHSs within the Locus Control Region (LCR) within the Rad50 gene which controls the Th2 cytokines Il13, Il4 and Il5, plus IL-2 pDHSs within the Tnfrsf9 and Cish loci. IL-2 pDHSs are highlighted with blue boxes.


Jund mRNA in TB IL - 2 TB IL - 2nil . Standard deviation is shown from mean of 3 samples . P - values were calculated from - seq data using Limma with Benjamini - Hochberg correction for multiple testing .

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

Jund mRNA levels in TB IL-2 and TB IL-2nil. Standard deviation is shown from the mean of 3 samples. P-values were calculated from the RNA-seq data using Limma with Benjamini-Hochberg correction for multiple testing.


Western blot analyses showing JUND pSTAT5 in TB IL - 2 TB IL - 2nil ( left panel ) with signal calculated from 2 replicates relative to TB IL - 2 in right panel .

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

Western blot analyses showing JUND and pSTAT5 protein levels in TB IL-2 and TB IL-2nil (left panel) with the signal calculated from 2 replicates relative to TB IL-2 in the right panel.


motif density plots ( right ) showing location of binding motifs for RUNX , ETS , STAT AP - 1 transcription factors at 32694 DHSs detected in TB IL - 2 TB IL - 2nil , as depicted in Figure 2A , ordered according to fold enrichment of DNase - Seq tag in TB IL - 2 compared to TB IL - 2nil ( left ) .

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

DNA motif density plots (right) showing the location of binding motifs for RUNX, ETS, STAT and AP-1 transcription factors at the 32694 DHSs detected in TB IL-2 and TB IL-2nil, as depicted in Figure 2A, ordered according to fold enrichment of DNase-Seq tag counts in TB IL-2 compared to TB IL-2nil (left).


tag density plots from ChIP - Seq assays of RUNX1 , ETS1 , STAT5 JUND in TB IL - 2 , STAT5 JUND in TB IL - 2nil ordered as in ( D ) .

DNA sequence tag density plots from ChIP-Seq assays of RUNX1, ETS1, STAT5 and JUND in TB IL-2, and STAT5 and JUND in TB IL-2nil ordered as in (D).


UCSC genome browser tracks at Il13 / Il4 / Il5 Th2 cytokine locus LCR ( indicated by grey box within Rad50 gene ) , Gzmb Il24 showing DNase - Seq ChIP - Seq for STAT5 JUND in TB IL - 2 TB IL - 2nil . Replicate JUND STAT5 ChIP - Seq tracks are shown for TB IL - 2 .

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

UCSC genome browser tracks at the Il13/Il4/Il5 Th2 cytokine locus LCR (indicated by the grey box within the Rad50 gene), Gzmb and Il24 showing DNase-Seq and ChIP-Seq for STAT5 and JUND in TB IL-2 and TB IL-2nil. Replicate JUND and STAT5 ChIP-Seq tracks are shown for TB IL-2.


DNase - Seq tag density plots showing peaks detected in replicate 1 of TB IL - 2 TB + IL - 2 ( A , 38645 ) in TB + IL - 2 TB + IL - 2nil ( B , 36067 ) , ordered by increasing fold change in tag as indicated . position of 1015 IL - 2 - dependent inducible DHSs is shown alongside .

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

DNase-Seq tag density plots showing all peaks detected in replicate 1 of TB IL-2 and TB+ IL-2 (A, 38645) and in TB+ IL-2 and TB+ IL-2nil (B, 36067), ordered by increasing fold change in tag count as indicated. The position of the 1015 IL-2-dependent inducible DHSs is shown alongside.


Average DHS signal at 1015 IL - 2 - dependent inducible DHSs in TB IL - 2 , TB + IL - 2 , TB + IL - 2nil naïve T stimulated with PMA / I ( TN + ) .

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

Average DHS signal at the 1015 IL-2-dependent inducible DHSs in TB IL-2, TB+ IL-2, TB+ IL-2nil and naïve T cells stimulated with PMA/I (TN+).


Bar graph showing number of IL - 2 iDHSs found within 25 kb of an IL2 pDHS .

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

Bar graph showing the number of IL-2 iDHSs found within 25 kb of an IL2 pDHS.


Examples of IL - 2 iDHSs which are proximal to IL - 2 pDHSs at Csf2 / Il2 Il10 loci . IL - 2 iDHSs are highlighted in red IL - 2 pDHSs in blue .

Examples of IL-2 iDHSs which are proximal to IL-2 pDHSs at the Csf2/Il2 and Il10 loci. IL-2 iDHSs are highlighted in red and IL-2 pDHSs in blue.


Hierarchical clustering of - Seq data from 3 replicates as described in ( A ) .

Hierarchical clustering of RNA-Seq data from 3 replicates as described in (A).


MA plot showing genes differentially regulated between TB IL - 2nil TB IL - 2 . number of genes with a fold change > 3 adj p - value < 0 . 05 are highlighted . P - values were calculated from - seq data using Limma with Benjamini - Hochberg correction for multiple testing .

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

MA plot showing the genes differentially regulated between TB IL-2nil and TB IL-2. The number of genes with a fold change >3 and adj p-value <0.05 are highlighted. P-values were calculated from the RNA-seq data using Limma with Benjamini-Hochberg correction for multiple testing.


log2 fold change in gene expression in TB IL - 2nil compared to TB IL - 2 for nearest gene to DHSs ranked according to fold change in DNase - Seq tag for TB IL - 2 / TB IL - 2nil . DNase - Seq tag density plots are taken from Figure 2A for TB IL - 2 , TB IL - 2nil TN .

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

log2 fold change in gene expression in TB IL-2nil compared to TB IL-2 for the nearest gene to the DHSs ranked according to the fold change in DNase-Seq tag count for TB IL-2/TB IL-2nil. The DNase-Seq tag density plots are taken from Figure 2A for TB IL-2, TB IL-2nil and TN.


UCSC genome browser tracks showing DNase - seq - seq at Socs1 ( E ) Sell loci ( F ) .

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

UCSC genome browser tracks showing DNase-seq and RNA-seq at the Socs1 (E) and Sell loci (F).


Relative mRNA expression in TB + IL - 2 TB + IL - 2nil ( A ) TB + DMSO TB + Ruxolitinib ( B ) . expression level in IL - 2 / DMSO TB + sample is set to 1 . ( n = 3 , error bars represent standard deviation , P - values were calculated using a paired t - with * p < 0 . 05 , * * p < 0 . 005 , * * * p < 0 . 0005 . )

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

Relative mRNA expression levels in TB+ IL-2 and TB+ IL-2nil (A) and TB+ DMSO and TB+ Ruxolitinib (B). The expression level in the IL-2/DMSO TB+ sample is set to 1. (n=3, error bars represent standard deviation, P-values were calculated using a paired t-test with *p<0.05, **p<0.005, ***p<0.0005.)


UCSC genome browser tracks showing DNase - Seq - Seq at IL - 2 regulated genes Csf2 / Il3 at LCR within Rad50 gene ( grey box ) which controls Th2 cytokines Il13 , Il4 Il5 . IL - 2 pDHSs are highlighted in blue IL - 2 iDHSs in red .

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

UCSC genome browser tracks showing DNase-Seq and RNA-Seq at the IL-2 regulated genes Csf2/Il3 and at the LCR within the Rad50 gene (grey box) which controls the Th2 cytokines Il13, Il4 and Il5. IL-2 pDHSs are highlighted in blue and IL-2 iDHSs in red.


DNase - Seq tag density plots showing peaks detected in replicate 1 of TB IL - 2 TB IL ‑ 2nil ( data taken from Figure 2A ) , duplicate DNase - seq samples for TB IL - 7 TB IL - 7nil are shown in parallel for same sites .

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

DNase-Seq tag density plots showing all peaks detected in replicate 1 of TB IL-2 and TB IL‑2nil (data taken from Figure 2A), duplicate DNase-seq samples for TB IL-7 and TB IL-7nil are shown in parallel for the same sites.


Average DNase - Seq tag density profile for 1000 IL - 2 pDHSs in both replicates of TB IL - 7 TB IL - 7nil .

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

Average DNase-Seq tag density profile for the 1000 IL-2 pDHSs in both replicates of TB IL-7 and TB IL-7nil.


UCSC genome browser tracks with DNase - Seq data for TB IL - 7 TB IL - 7nil showing examples of IL - 2 pDHSs within Locus Control Region ( LCR ) within Rad50 gene which controls Th2 cytokines Il13 , Il4 Il5 , plus IL - 2 pDHSs within Tnfrsf9 Cish loci ( C ) IL - 2 pDHSs are highlighted with blue boxes .

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

UCSC genome browser tracks with DNase-Seq data for TB IL-7 and TB IL-7nil showing examples of IL-2 pDHSs within the Locus Control Region (LCR) within the Rad50 gene which controls the Th2 cytokines Il13, Il4 and Il5, plus IL-2 pDHSs within the Tnfrsf9 and Cish loci (C) IL-2 pDHSs are highlighted with blue boxes.


UCSC genome browser tracks with DNase - Seq data for TB IL - 7 TB IL - 7nil showing examples of IL - 2 pDHSs within Locus Control Region ( LCR ) within non - primed control regions from CD3 gene cluster Tbp locus ( D ) . IL - 2 pDHSs are highlighted with blue boxes .

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

UCSC genome browser tracks with DNase-Seq data for TB IL-7 and TB IL-7nil showing examples of IL-2 pDHSs within the Locus Control Region (LCR) within the non-primed control regions from the CD3 gene cluster and the Tbp locus (D). IL-2 pDHSs are highlighted with blue boxes.


Average DHS signal at 1015 IL - 2 regulated inducible DHSs in TB + IL - 7 TB + IL - 7nil .

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

Average DHS signal at the 1015 IL-2 regulated inducible DHSs in TB+ IL-7 and TB+ IL-7nil.


Relative mRNA expression in TB + IL - 7 TB + IL - 7nil . expression level in TB + IL - 7 is set to 1 . ( n = 3 , error bars represent standard deviation , P - values were calculated using a paired t - with * p < 0 . 05 , * * p < 0 . 005 , * * * p < 0 . 0005 . )

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

Relative mRNA expression levels in TB+ IL-7 and TB+ IL-7nil. The expression level in the TB+ IL-7 is set to 1. (n=3, error bars represent standard deviation, P-values were calculated using a paired t-test with *p<0.05, **p<0.005, ***p<0.0005.)


Flow cytometric of IL - 7Rα expression by 2W1S - specific CD44hi CD4 T in presence or absence of cre recombinase .

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

Flow cytometric analysis of IL-7Rα expression by 2W1S-specific CD44hi CD4 T cells in the presence or absence of cre recombinase.


Average number of 2W1S - specific recovered / mouse after sorting 3 replicate samples of Il7rf / f compared to CD4creERT2 x Il7rf / f . Errors bars indicate standard deviation .

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

Average number of 2W1S-specific cells recovered/mouse after sorting 3 replicate samples of Il7rf/f compared to CD4creERT2 x Il7rf/f . Errors bars indicate the standard deviation.


Average ATAC - Seq signal at 411 IL - 2 pDHSs which contain a STAT binding motif .

Average ATAC-Seq signal at the 411 IL-2 pDHSs which contain a STAT binding motif.


UCSC genome browser tracks at loci with IL - 2 pDHSs showing replicate ATAC - Seq samples for CD4creERT2 x Il7rf / f memory T , Il7rf / f control memory T , replicate DNase - Seq samples for TB IL - 2 TB IL - 2nil TB IL - 2 TB IL ‑ 2nil STAT5 ChIP - Seq samples . grey boxes signify IL - 2 pDHSs which diminish after disruption of Il7r gene .

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

UCSC genome browser tracks at loci with IL-2 pDHSs showing replicate ATAC-Seq samples for CD4creERT2 x Il7rf/f memory T cells, Il7rf/f control memory T cells, replicate DNase-Seq samples for TB IL-2 and TB IL-2nil and TB IL-2 and TB IL‑2nil STAT5 ChIP-Seq samples. The grey boxes signify IL-2 pDHSs which diminish after disruption of the Il7r gene.