In vivo Ahp1 conjugation analyses with extracts obtained from indicated yeast strains expressing HA - URM1 . Unconjugated Urm1 Urm1 - Ahp1 conjugates were detected by anti - HA ( top panels ) Western blots . Anti - Ahp1 blots ( middle ) detect unmodified Ahp1 . Anti - Cdc19 ( bottom panels ) served as loading control .

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In vivo Ahp1 conjugation analyses with protein extracts obtained from the indicated yeast strains expressing HA-URM1. Unconjugated Urm1 and Urm1-Ahp1 conjugates were detected by anti-HA (top panels) Western blots. Anti-Ahp1 blots (middle) detect unmodified Ahp1. Anti-Cdc19 (bottom panels) served as loading control.


Northern - blot for thiolation of tRNA < sub > UUC < / sub > < sup > Glu < / sup > in genetic background of indicated yeast strains . Total tRNA was resolved on denaturing PAGE supplemented with APM to retard migration of thiolated tRNAUUCGlu . APM : ( [ N - Acryloyl - ] phenyl ) mercuric chloride . ( bottom ) Quantified thiolation from 3 independent biological replicates .

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Northern-blot analysis for thiolation levels of tRNAUUCGlu in the genetic background of the indicated yeast strains. Total tRNA was resolved on denaturing PAGE supplemented with APM to retard the migration of thiolated tRNAUUCGlu. APM: ([N-Acryloyl-amino] phenyl) mercuric chloride. (bottom) Quantified thiolation levels from 3 independent biological replicates.


Ferrozine - intracellular iron measurement in L6 after temporal of iron treatment at 100 or 250 µM for multiple time points . * P < 0 . 05 ( unpaired Student ' s t - versus each time points with FeSO4 100 µM ) .

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Ferrozine-based intracellular iron concentration measurement in L6 cells after temporal analysis of iron treatment at 100 or 250 µM for multiple time points. *P < 0.05 (unpaired Student's t-test versus each time points with FeSO4 100 µM).


Representative confocal microscope of L6 stained with IP - 1 ( Iron Probe 1 ) or transfected with IRE - CFP ( Iron Regulatory Element ) reporter after iron treatment ( FeSO4 , 250 µM ) for 24 h .

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Representative confocal microscope images of L6 cells stained with IP-1 (Iron Probe 1) or transfected with IRE-CFP (Iron Regulatory Element) reporter after iron treatment (FeSO4, 250 µM) for 24 h.


Representative confocal of L6 using iron sensitive PGSK dye after iron treatment ( 250 µM , 24 h ) with iron chelator DPD ( 500 µM ) .

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Representative confocal images of L6 cells using iron sensitive fluorescent PGSK dye after iron treatment (250 µM, 24 h) with iron chelator DPD (500 µM).


Relative gene expressions - ferritin heavy chain ( FTH ) , ferritin chain ( FTL ) , ferroportin ( SLC40A1 , transferrin receptor 1 ( tfr1 , TFRC ) - normalized to 18sr expression after iron treatment ( FeSO4 , 250 µM ) for 24 h . * P < 0 . 05 ( unpaired Student ' s t - versus Basal ) .

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Relative gene expressions - ferritin heavy chain (FTH), ferritin light chain (FTL), ferroportin (SLC40A1, transferrin receptor 1 (tfr1, TFRC) - normalized to 18sr RNA expression after iron treatment (FeSO4, 250 µM) for 24 h. *P < 0.05 (unpaired Student's t-test versus Basal).


Representative western blot of ferritin ( heavy chain ) tfr1 ( indicated with arrowhead ) over GAPDH after iron treatment ( 250 µM , 24 h ) .

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Representative western blot images and quantification of ferritin (heavy chain) and tfr1 (indicated with arrowhead) over GAPDH after iron treatment (250 µM, 24 h).


Glucose uptake of L6 with insulin stimulation ( 10 nM or 100 nM , 20 min ) after iron treatment ( 250 µM , 1 h or 24 h ) .

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Glucose uptake of L6 cells with insulin stimulation (10 nM or 100 nM, 20 min) after iron treatment (250 µM, 1 h or 24 h).


Representative western blot of phospho - IRS1 ( Y612 ) phospho - AKT ( T308 ) over GAPDH with insulin stimulation ( 10 nM or 100 nM , 5 min ) after iron treatment ( 250 µM , 24 h ) .

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Representative western blot images and quantification of phospho-IRS1 (Y612) and phospho-AKT (T308) over GAPDH with insulin stimulation (10 nM or 100 nM, 5 min) after iron treatment (250 µM, 24 h).


Representative confocal microscope image of L6 stably expressing tandem - eGFP - mCherry LC3B treated with iron ( FeSO4 , 250 µM ) for indicated time points ( 4 h , 8 h , 24 h ) . of mean LC3B per from ( A ) . Experiments were repeated three times , one representative experiment is presented here .

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Representative confocal microscope image of L6 cells stably expressing tandem fluorescent-eGFP-mCherry LC3B treated with iron (FeSO4, 250 µM) for the indicated time points (4 h, 8 h, 24 h). Quantification of mean LC3B puncta per cell from (A). Experiments were repeated three times, and one representative experiment is presented here.


Representative western blot of p62 to actin in L6 after iron treatment ( FeSO4 , 250 µM ) at multiple time points .

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Representative western blot images and quantification of p62 to actin in L6 cells after iron treatment (FeSO4, 250 µM) at multiple time points.


Representative western blot of LC3B - II to GAPDH in L6 after 4h iron treatment stimulated with chloroquine ( CQ , 30 µM ) . * Representative western blot of LC3B - II to GAPDH in L6 after 24 h iron treatment stimulated with CQ 30 µM .

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Representative western blot images and quantification of LC3B-II to GAPDH in L6 cells after 4h iron treatment stimulated with chloroquine (CQ, 30 µM). * Representative western blot images and quantification of LC3B-II to GAPDH in L6 cells after 24 h iron treatment stimulated with CQ 30 µM.


Representative confocal microscope z - stack image of mCherry - LC3B L6 after iron treatment ( 250 µM , 24 h ) .

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Representative confocal microscope z-stack image of mCherry-LC3B L6 cells after iron treatment (250 µM, 24 h).


of LC3B from ( F ) . Experiments were performed three times , one representative experiment is presented here .

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Quantification of LC3B puncta size from (F). Experiments were performed three times, and one representative experiment is presented here.


of autophagosomes motility from live microscopy ( Supplementary Movie 1 ) from mCherry - LC3B L6 under basal , iron ( 250 µM , 24 h ) starvation ( acid free ) condition . Experiments were performed three times , one representative experiment is presented here . * P < 0 . 05 ( unpaired Student ' s t - compared to Iron 24h ) .

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Quantification of autophagosomes motility from live cell microscopy (Supplementary Movie 1) from mCherry-LC3B L6 cells under basal, iron (250 µM, 24 h) and starvation (amino acid free) condition. Experiments were performed three times, and one representative experiment is presented here. *P < 0.05 (unpaired Student's t-test compared to Iron 24h).


Representative of autophagosome autolysosomes in L6 after iron treatment ( 250 µM , 24 h ) .

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Representative TEM images of autophagosome and autolysosomes in L6 cells after iron treatment (250 µM, 24 h).


Representative confocal microscope of eGFP - mCherry - LC3B L6 with Lysotracker DeepRed after iron treatment ( 250 µM , 24 h ) .

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Representative confocal microscope images of eGFP-mCherry-LC3B L6 cells with Lysotracker DeepRed after iron treatment (250 µM, 24 h).


Representative western blot of phospho - ULK1 ( S757 ) , phospho - S6Kp70 ( T389 ) , total S6K , GAPDH after iron treatment ( FeSO4 , 250 µM ) for multiple time points . of mTORC1 activity analyzed through phosphorylation of S6K T389 to total S6k ULK1 S757 to GAPDH after iron treatment ( FeSO4 , 250 µM ) for multiple time points .

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Representative western blot images of phospho-ULK1 (S757), phospho-S6Kp70 (T389), total S6K, GAPDH after iron treatment (FeSO4, 250 µM) for multiple time points. Quantification of mTORC1 activity analyzed through phosphorylation of S6K T389 to total S6k and ULK1 S757 to GAPDH after iron treatment (FeSO4, 250 µM) for multiple time points.


Representative epi - immunofluorescent of L6 transfected with myc - RHEB Q46L immuno - stained against LC3B myc after iron treatment ( FeSO4 , 250 µM ) for 24 h .

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Representative epi-immunofluorescent images of L6 cells transfected with myc-RHEB Q46L and immuno-stained against LC3B and myc after iron treatment (FeSO4, 250 µM) for 24 h.


Ferrozine - colorimetric measurement of intracellular iron in wild - type ( wt ) L6 RHEB - Q64L L6 after iron treatment ( 50 µM or 250 µM ) for 24 h .

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Ferrozine-based colorimetric measurement of intracellular iron in wild-type (wt) L6 and RHEB-Q64L L6 cells after iron treatment (50 µM or 250 µM) for 24 h.


Representative confocal of wt L6 RHEB - Q64L L6 pulsed with Lysotracker Deep Red immuno - stained against LC3B after iron treatment ( 250 µM , 24 h ) .

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Representative confocal images of wt L6 and RHEB-Q64L L6 cells pulsed with Lysotracker Deep Red and immuno-stained against LC3B after iron treatment (250 µM, 24 h).


of LC3B - free lysosome numbers in ( E ) . Experiments were repeated three times , one representative experiment is presented here . * P < 0 . 05 ( multiple unpaired Student ' s t - versus Basal in wt RHEB Q64L )

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Quantification of LC3B-free lysosome numbers in (E). Experiments were repeated three times, and one representative experiment is presented here. *P < 0.05 (multiple unpaired Student's t-test versus Basal in wt and RHEB Q64L cells)


Representative of wt L6 RHEB - Q64L after iron treatment ( 250 µM , 24 h ) .

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Representative TEM images of wt L6 and RHEB-Q64L cells after iron treatment (250 µM, 24 h).


Representative western blot quantifications of phospho - IRS1 ( Y612 , indicated by arrowhead ) AKT ( T308 , indicated by arrowhead ) to GAPDH in wt RHEB - Q64L L6 stimulated with insulin ( 100 nM , 5 min ) after iron treatment ( 250 µM , 24 h ) . * P < 0 . 05 ( multiple unpaired Student ' s t - versus Basal in wt RHEB Q64L )

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Representative western blot images and quantifications of phospho-IRS1 (Y612, indicated by arrowhead) and AKT (T308, indicated by arrowhead) to GAPDH in wt and RHEB-Q64L L6 cells stimulated with insulin (100 nM, 5 min) after iron treatment (250 µM, 24 h). *P < 0.05 (multiple unpaired Student's t-test versus Basal in wt and RHEB Q64L cells)


Representative confocal microscope of L6 pulsed with PGSK dye after iron treatment ( 250 µM , 24 h ) followed by 24 h iron withdrawal . Ferrozine colorimetric measurement of intracellular iron in L6 after iron treatment ( 250 µM , 24 h ) followed by 24 h withdrawal .

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Representative confocal microscope images of L6 cells pulsed with PGSK dye after iron treatment (250 µM, 24 h) followed by 24 h iron withdrawal. Ferrozine based colorimetric measurement of intracellular iron in L6 cells after iron treatment (250 µM, 24 h) followed by 24 h withdrawal.


Representative western blot of phospho - IRS1 ( Y612 ) phospho - AKT ( T308 ) to GAPDH in L6 iron treatment ( 250 µM , 24 h ) followed by 24 h withdrawal .

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Representative western blot images and quantification of phospho-IRS1 (Y612) and phospho-AKT (T308) to GAPDH in L6 cells iron treatment (250 µM, 24 h) followed by 24 h withdrawal.


Representative epi - immunofluorescent microscope of L6 immuno - stained against LC3B LAMP1 after iron treatment ( 250 µM , 24 h ) followed by 3 h withdrawal .

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Representative epi-immunofluorescent microscope images of L6 cells immuno-stained against LC3B and LAMP1 after iron treatment (250 µM, 24 h) followed by 3 h withdrawal.


of autophagosome - free lysosomes in ( E ) . Experiments were performed three times , one representative experiment is presented here .

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Quantification of autophagosome-free lysosomes in (E). Experiments were performed three times, and one representative experiment is presented here.


Representative western blot of ferritin to vinculin in L6 after iron treatment ( 250 µM , 24 h ) followed by withdrawal for 4 h or 24 h .

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Representative western blot images and quantification of ferritin to vinculin in L6 cells after iron treatment (250 µM, 24 h) followed by withdrawal for 4 h or 24 h.


Representative western blot of phospho - mTOR S2448 , total mTOR actin in L6 after iron treatment ( 250 µM , 24 h ) followed by withdrawal for 4 h .

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Representative western blot images and quantification of phospho-mTOR S2448, total mTOR and actin in L6 cells after iron treatment (250 µM, 24 h) followed by withdrawal for 4 h.


Representative western blot of phospho - mTOR S2448 , total mTOR phsophpo - TSC2 T1462 , total TSC2 , phospho - AKT S473 , total AKT , actin in L6 after iron treatment ( 250 µM , 24 h ) followed by withdrawal for 4 h with or without MK2206 ( AKT inhibitor . )

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Representative western blot images of phospho-mTOR S2448, total mTOR phsophpo-TSC2 T1462, total TSC2, phospho-AKT S473, total AKT, actin in L6 cells after iron treatment (250 µM, 24 h) followed by withdrawal for 4 h with or without MK2206 (AKT inhibitor.)


Representative western blot of phospho - UVRAG S550 total UVRAG in L6 transfected with FLAG - UVRAG FLAG pulldown after iron treatment ( 250 µM , 4 h 24 h ) followed by withdrawal for 1 h 3 h .

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Representative western blot images of phospho-UVRAG S550 and total UVRAG in L6 cells transfected with FLAG-UVRAG and FLAG pulldown after iron treatment (250 µM, 4 h and 24 h) followed by withdrawal for 1 h and 3 h.


Representative western blot of endogenous phospho - UVRAG S550 , total UVRAG expression in L6 after iron treatment ( 250 µM , 24 h ) followed by withdrawal for 4 h with or without torin1 ( 200 nM ) .

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Representative western blot images of endogenous phospho-UVRAG S550, total UVRAG expression in L6 cells after iron treatment (250 µM, 24 h) followed by withdrawal for 4 h with or without torin1 (200 nM).


Live imaging of L6 transfected with LAMP1 - RFP from Movie EV2 : lysosomal number in L6 after iron treatment ( 250 µM , 24 h ) followed by withdrawal for 4 h with or without Torin1 ( 200 nM ) .

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Live cell imaging analysis of L6 cells transfected with LAMP1-RFP from Movie EV2: lysosomal number in L6 cells after iron treatment (250 µM, 24 h) followed by withdrawal for 4 h with or without Torin1 (200 nM).


Live imaging of L6 transfected with LAMP1 - RFP from Movie EV2 : speed ( F ) in L6 after iron treatment ( 250 µM , 24 h ) followed by withdrawal for 4 h with or without Torin1 ( 200 nM ) .

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Live cell imaging analysis of L6 cells transfected with LAMP1-RFP from Movie EV2: speed (F) in L6 cells after iron treatment (250 µM, 24 h) followed by withdrawal for 4 h with or without Torin1 (200 nM).


Representative confocal of time - lapse captures of lysosomes at 6 sec intervals for 30 sec . yellow arrows mark positions of two representative LAMP1 over 30 seconds , traces of were shown as lines in final panel .

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Representative confocal images of time-lapse captures of lysosomes at 6 sec intervals for 30 sec. White and yellow arrows mark positions of two representative LAMP1 puncta over 30 seconds, and the traces of puncta were shown as lines in final panel.


Prussian Perl blue in liver skeletal muscle after iron injections .

Prussian Perl blue staining in liver and skeletal muscle after iron injections.


Representative western blot of ferritin tfr1 ( indicated by arrowhead ) to tubulin in skeletal 24 h after iron injections .

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Representative western blot images and quantification of ferritin and tfr1 (indicated by arrowhead) to tubulin in skeletal muscles 24 h after iron injections.


Insulin tolerance 24 h after iron injections .

Insulin tolerance test 24 h after iron injections.


Glucose tolerance ( GTT ) 24 h after iron injections .

Glucose tolerance test (GTT) 24 h after iron injections.


Insulin tolerance 24 h after iron injections . of under curve in ITT ( E ) .

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Insulin tolerance test 24 h after iron injections. Quantification of area under curve in ITT (E).


Glucose tolerance ( GTT ) 24 h after iron injections . of under curve for panel ( F ) .

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Glucose tolerance test (GTT) 24 h after iron injections. Quantification of area under curve for panel (F).


Representative western blot of phospho - IRS1 ( Y612 ) phospho - AKT ( T308 ) to GAPDH in skeletal 24 h after iron injection followed by i . p . insulin injection .

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Representative western blot images and quantification of phospho-IRS1 (Y612) and phospho-AKT (T308) to GAPDH in skeletal muscles 24 h after iron injection followed by i.p. insulin injection.


Representative confocal microscope of skeletal muscle tissue sections immuno - stained against LC3B ( Alexa 647 ) LAMP1 ( Alexa 555 ) of lysosomes ( LAMP1 ) in skeletal 24 h after iron injections .

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Representative confocal microscope images of skeletal muscle tissue sections immuno-stained against LC3B (Alexa 647) and LAMP1 (Alexa 555) Quantification of lysosomes (LAMP1 puncta) in skeletal muscles 24 h after iron injections.


Representative of skeletal muscle 24 h after iron injections .

Representative TEM images of skeletal muscle 24 h after iron injections.


Representative western blot of phospho - UVRAG S550 total UVRAG in skeletal muscle 24 h after iron injections .

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Representative western blot images of phospho-UVRAG S550 and total UVRAG in skeletal muscle 24 h after iron injections.


Representative western blot of LC3 - B p62 to tubulin in skeletal 24 h after iron injections .

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Representative western blot images and quantification of LC3-B and p62 to tubulin in skeletal muscles 24 h after iron injections.


( A ) Example regions exhibiting IPOD - HR occupancy where EPOD calls are made . Loose EPODs ( pink ) Strict EPODs ( teal ) represent spans of high occupancy at lower higher thresholds , respectively . Neighboring areas showing lower robust z scores are not EPODs . Note that following definitions in [ 12 ] , presence of an EPOD requires a sustained region of high average occupancy , without any regions in which occupancy drops to background . Thus , breaks in EPOD calls represent cases where a rolling average occupancy drops below calling threshold ( e . g . , breakpoint in middle of pinQ in top panel ) , regions of apparent occupancy may nevertheless not be called due to being below minimum threshold ( as is case for nohA ) .

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(A) Example regions exhibiting the IPOD-HR occupancy where EPOD calls are made. Loose EPODs (pink) and Strict EPODs (teal) represent spans of high protein occupancy at lower and higher thresholds, respectively. Neighboring areas showing lower robust z scores are not EPODs. Note that following the definitions in [12], the presence of an EPOD requires a sustained region of high average occupancy, without any regions in which occupancy drops to background levels. Thus, breaks in EPOD calls represent cases where a rolling average occupancy drops below the calling threshold (e.g., the breakpoint in the middle of pinQ in the top panel), and regions of apparent occupancy may nevertheless not be called due to being below the minimum length threshold (as is the case for nohA).


( E ) Specific locations across conditions remain occupied by where EPODs across a representative region , waa operon , were called ( left ) unoccupied by polymerase ( right ) . quantile normalized robust z scores of occupancy at each 5 bp are represented by IPOD - HR occupancy .

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(E) Specific locations across all conditions remain occupied by protein where EPODs across a representative region, the waa operon, were called (left) and unoccupied by RNA polymerase (right). The quantile normalized robust z scores of the protein occupancy at each 5 bp are represented by the IPOD-HR occupancy.


( A ) Number of EPODs called in different NAP deletions . D . S . denotes genotypes where were collected in deep stationary phase of growth .

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(A) Number of EPODs called in different NAP deletions. D.S. denotes genotypes where cells were collected in the deep stationary phase of growth.


( D ) Distributions of mean occupancy at WT EPOD boundaries background . blue dots denote median black displays interquartile ranges in each condition . dashed pink represents WT median . ( * ) indicate Wilcoxon Rank Sum p value comparing change in pseudomedian vs . WT for each condition that has been adjusted using Benjamini Hochberg method ( against a null hypothesis of no difference in pseudomedians ) . grey denotes same comparison between D . S . conditions . Underlined ( * ) ' s indicate a gain in pseudomedian compared to baseline conditions , while no underline indicates a loss in pseudomedian compared to baseline conditions . p value < 0 . 05 = * , < 0 . 005 = * * , < 0 . 0005 = * * * . Three biological replicates were used for " WT , Rich " case " WT ( deep stat . ) " case , two biological replicates for " WT , Min " " WT ( 2 ) , Rich " conditions .

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(D) Distributions of mean protein occupancy at WT EPOD boundaries and background. The blue dots denote the median and the black line displays the interquartile ranges in each condition. The dashed pink line represents the WT median. (*) indicate the Wilcoxon Rank Sum test p value comparing the change in pseudomedian vs. WT for each condition that has been adjusted using the Benjamini and Hochberg method (against a null hypothesis of no difference in pseudomedians). The grey line denotes the same comparison between the D.S. conditions. Underlined (*)'s indicate a gain in the pseudomedian compared to baseline conditions, while no underline indicates a loss in the pseudomedian compared to baseline conditions. p value < 0.05 = *, <0.005=**, <0.0005=***. Three biological replicates were used for the "WT, Rich" case and "WT (deep stat.)" case, and two biological replicates for the "WT, Min" and "WT (2), Rich" conditions.


( E ) occupancy over waa operon . quantile normalized robust z scores of occupancy at each 5 bp are represented by IPOD - HR occupancy . EPOD over waa operon is lost in ∆ stpA ∆ hns condition which results in increased accessibility polymerase

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(E) Protein occupancy over the waa operon. The quantile normalized robust z scores of the protein occupancy at each 5 bp are represented by the IPOD-HR occupancy. The EPOD over the waa operon is lost in the ∆stpA∆hns condition which results in increased accessibility and RNA polymerase occupancy


( G ) - seq shows log fold change compared to WT of waa operon expression upon deletion of hns stpA . ( * ) indicate q value < 0 . 05 = * , < 0 . 005 = * * , < 0 . 0005 = * * *

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(G) RNA-seq analysis shows log fold change compared to WT of waa operon expression upon deletion of hns and stpA. (*) indicate q value < 0.05 = *, < 0.005 = **, < 0.0005 = ***


( B ) occupancy over idn operon for each condition ( colors are denoted in ( A ) ) . quantile normalized robust z scores of occupancy at each 5 bp are represented by IPOD - HR occupancy . There is a large loss in occupancy when are shifted to 5KDG , leading to loss of call ed EPOD . occupancy is restored once are grown in second glucose condition . 500bp normalized average of previously published H - NS ChIP - seq [ 20 ] exhibits high H - NS binding on idnD promoter region .

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(B) Protein occupancy over the idn operon for each condition (colors are denoted in (A)). The quantile normalized robust z scores of the protein occupancy at each 5 bp are represented by the IPOD-HR occupancy. There is a large loss in protein occupancy when cells are shifted to 5KDG, leading to the loss of the call ed EPOD. Protein occupancy is restored once cells are grown in the second glucose condition. The 500bp normalized average of previously published H-NS ChIP-seq [20] exhibits high H-NS binding on the idnD promoter region.


( C ) To examine expression of idn operon at each shift , - seq was performed . - seq expression estimates log2 scaled for idn operon genes for WT grown in each condition ( as colored in ( A ) ) . Comparisons are denoted with colored dots with significance stars representing adjusted p - value where ( * * * ) signify q - values < 0 .

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(C) To examine the expression of the idn operon at each shift, RNA-seq was performed. RNA-seq expression estimates log2 scaled for idn operon genes for WT cells grown in each condition (as colored in (A)). Comparisons are denoted with colored dots with significance stars representing the adjusted p-value where (***) signify q-values <0.0005


( B ) Difference in Log2 of fitnesses of KDG - exposed with unexposed lacZ : : cat relative to that observed prior to KDG exposure , given as a function of time since exposure to KDG . Day - 1 corresponds to day before KDG exposure , Day 0 is taken immediately after growth in KDG , subsequent timepoints reflect different durations of growth in M9 Min Glu prior to competition ( See ( A ) ) . Points show medians across replicates ( 3 biological replicates for 0 . 5 days , 6 biological replicates for other timepoints ) ; error bars show 95 % confidence intervals for difference relative to Day - 1 timepoint , calculated using R wilcox . function . Significance was assessed using a Wilcoxon rank sum comparing distribution at each timepoint to - 1 day ( naive ) timepoint : * , p < 0 . 05 ; * * , p < 0 . 005 .

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(B) Difference in Log2 ratio of fitnesses of the KDG-exposed cells with unexposed lacZ::cat relative to that observed prior to KDG exposure, given as a function of time since exposure to KDG. Day -1 corresponds to the day before KDG exposure, Day 0 is cells taken immediately after growth in KDG, and subsequent timepoints reflect different durations of growth in M9 Min Glu prior to competition (See (A)). Points show medians across replicates (3 biological replicates for 0.5 days, 6 biological replicates for all other timepoints); error bars show 95% confidence intervals for the difference relative to the Day -1 timepoint, calculated using the R wilcox.test function. Significance was assessed using a Wilcoxon rank sum test comparing the distribution at each timepoint to the -1 day (naive) timepoint: *, p<0.05; **, p<0.005.


( C ) Mean occupancy was calculated across WT EPOD locations that contain annotated prophages . change in mean occupancy compared to WT was calculated for each condition , where anything negative is a loss in occupancy compared to WT . Hierarchical clustering was performed to examine which genotypes clustered together were more similar . Permutation comparing change in occupancy over EPODs containing prophages were performed against of genome . P value < 0 . 05 = * , < 0 . 005 = * * indicate a negative mean change in occupancy compared to WT .

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(C) Mean protein occupancy was calculated across all WT EPOD locations that contain annotated prophages. The change in mean protein occupancy compared to WT was calculated for each condition, where anything negative is a loss in occupancy compared to WT. Hierarchical clustering was performed to examine which genotypes clustered together and were more similar. Permutation tests comparing the change in occupancy over EPODs containing prophages were performed against the rest of the genome. P value <0.05 = *, <0.005=** indicate a negative mean change in occupancy compared to WT.


( A ) Example prophage region that is annotated with Fis - Hfq - associated HMM class 5 in our genome - wide HMM classification quantile normalized robust z scores of occupancy at each 5 bp are represented by IPOD - HR occupancy . Prophage genes are highlighted with a red box . major peak associated with WT IPOD - HR occupancy is represented in a grey dashed as a reference to compare IPOD - HR occupancy in other genotypes . Modest loss of occupancy was observed at same prophage - containing EPOD for ∆ fis ( purple ; see color key in ( B ) ) ∆ hfq ( purple ; see color key in ( B ) ) conditions compared to WT ( gold ) .

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

(A) Example prophage region that is annotated with the Fis- and Hfq-associated HMM class 5 in our genome-wide HMM classification The quantile normalized robust z scores of the protein occupancy at each 5 bp are represented by the IPOD-HR occupancy. Prophage genes are highlighted with a red box. The major peak associated with WT IPOD-HR occupancy is represented in a grey dashed line as a reference to compare IPOD-HR occupancy in the other genotypes. Modest loss of protein occupancy was observed at the same prophage-containing EPOD for ∆fis (light purple; see color key in (B)) and ∆hfq (dark purple; see color key in (B)) conditions compared to WT (gold).


( B ) - seq of WT , ∆ fis , ∆ hfq were performed . log fold change compared to WT was calculated at prophage genes contained in dashed box in ( A ) . Induction of prophages across region where loss in occupancy is observed . ( * ) indicate adjusted p - value : < 0 . 10 = + , < 0 . 05 = * , < 0 . 005 = * * , < 0 . 0005 = * * *

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(B) RNA-seq of WT, ∆fis, and ∆hfq were performed. The log fold change compared to WT was calculated at prophage genes contained in the dashed box in (A). Induction of prophages across the region where loss in occupancy is observed. (*) indicate the adjusted p-value: <0.10 = +, < 0.05 = *, <0.005=**, <0.0005=***


( F ) Rate ratios of genes ( grey ) prophage genes ( red ) in each quadrant outline in ( E ) , showing a higher rate of genes that resided in + / + category , indicating that many prophages are de - repressed in both ∆ fis ∆ hfq . ( * ) indicate p - value calculated from testing null that rate ratios are same . P - value < 0 . 05 = * , < 0 . 005 = * * , < 0 . 0005 = * * *

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(F) Rate ratios of all genes (grey) and prophage genes (red) in each quadrant outline in (E), showing a higher rate of genes that resided in the +/+ category, indicating that many prophages are de-repressed in both ∆fis and ∆hfq. (*) indicate the p-value calculated from testing the null that the rate ratios are the same. P-value < 0.05 = *, <0.005=**, <0.0005=***


( G ) P1 vir transduction experiment to viability of ∆ fis ∆ hfq . - Hfq indicates deleting Hfq - CspE indicates deleting - CspE as a control . identities are indicated in box . Number of transductions were counted on LB + Kan plates ; efficiencies are relative to WT , thus log - scaled relative transduction rate for WT itself is 0 by definition . - R1 indicates that prophage region in ( A ) was deleted to whether loss of prophages silenced by Fis Hfq restored viability of a ∆ fis ∆ hfq genotype . R2 R3 were other regions in genome that contained prophages that appeared to have Fis / Hfq dependent EPODs . Plotted values are mean efficiencies across replicates , with error bars showing a 95 % credible interval obtained via Bayesian inference , assuming replicate - level colony are Poisson distributed with a ( conjugate ) Gamma ( 0 , 0 ) prior ; log ratios ( including error bounds ) are plotted relative to mean WT value . Data obtained from 5 biological replicates for hfq : : kan transductions 4 biological replicates for cspE : : kan transductions .

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

(G) P1 vir transduction experiment to test the viability of ∆fis and ∆hfq. -Hfq indicates deleting Hfq and -CspE indicates deleting -CspE as a control. Strain identities are indicated in the box. Number of transductions were counted on LB + Kan plates; all efficiencies are relative to WT, and thus the log-scaled relative transduction rate for the WT itself is 0 by definition. -R1 indicates that the prophage region in (A) was deleted to test whether the loss of prophages silenced by Fis and Hfq restored viability of a ∆fis ∆hfq genotype. R2 and R3 were other regions in the genome that contained prophages that appeared to have Fis/Hfq dependent EPODs. Plotted values are mean efficiencies across replicates, with error bars showing a 95% credible interval obtained via Bayesian inference, assuming the replicate-level colony counts are Poisson distributed with a (conjugate) Gamma(0,0) prior; all log ratios (including error bounds) are plotted relative to the mean WT value. Data obtained from 5 biological replicates for hfq::kan transductions and 4 biological replicates for cspE::kan transductions.


( A ) IPOD - HR ChIP - subtracted z - scores polymerase ChIP in vicinity of an extended occupancy domain ( EPOD ) . ChIP tracks are shown as log2 extracted / input ratios ; z - score is ChIP - subtracted robust z - score smoothed with a 512 bp rolling median . ( B ) IPOD - HR polymerase ChIP tracks in vicinity of a negative EPOD ( nEPOD ) .

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(A) IPOD-HR ChIP-subtracted z-scores and RNA polymerase ChIP in the vicinity of an extended protein occupancy domain (EPOD). ChIP tracks are shown as log2 extracted/input ratios; the z-score is the ChIP-subtracted robust z-score smoothed with a 512 bp rolling median. (B) IPOD-HR and RNA polymerase ChIP tracks in the vicinity of a negative EPOD (nEPOD).


( C ) Effects of deletion of rok in vicinity of an nEPOD ; Rok ChIP data from [ 70 ] shows a strong overlap with nEPOD boundary , whereas that occupancy region is lost in Δrok .

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(C) Effects of deletion of rok in the vicinity of an nEPOD; Rok ChIP data from[70] shows a strong overlap with the nEPOD boundary, whereas that occupancy region is lost in Δrok cells.


( E ) Comparison of IPOD occupancy SMC ChIP occupancy in vicinity of several typical EPODs .

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(E) Comparison of IPOD occupancy and SMC ChIP occupancy in the vicinity of several typical EPODs.


A . Optical coherence tomography ( OCT ) horizontal B - scan thickness map of neovascular related macular degeneration ( NV AMD ) with active lesions ( 1 ) , cystoid macular edema ( 2 ) , subretinal fibrin deposits ( 3 ) serous retinal detachment ( 4 ) . B . Optical coherence tomography ( OCT ) horizontal B - scan thickness map of control with a medium sized , stage 3 , full thickness macular hole ( 5 ) .

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A. Optical coherence tomography (OCT) horizontal B-scan and thickness map of neovascular age related macular degeneration (NV AMD) patient with active lesions (1), cystoid macular edema (2), subretinal fibrin deposits (3) and serous retinal detachment (4). B. Optical coherence tomography (OCT) horizontal B-scan and thickness map of control patient with a medium sized, stage 3, full thickness macular hole (5).


C - G . Vitreous humor analyzed by ELISA for VEGF - A ( C ) ; n = 7 ( Ctrl ) , 9 ( NV AMD ) , SEMA3A ( D ) ; n = 10 ( Ctrl ) , 10 ( NV AMD ) , TGFβ ( E ) ; n = 8 ( Ctrl ) , 7 ( NV AMD ) , PDGF - BB ( F ) ; n = 5 ( Ctrl ) , 6 ( NV AMD ) , PGF ( G ) : n = 5 ( Ctrl ) , 5 ( NV AMD ) . Dots represent concentrations of samples .

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C-G. Vitreous humor analyzed by ELISA for VEGF-A (C); n = 7 (Ctrl), 9 (NV AMD), SEMA3A (D); n = 10 (Ctrl), 10 (NV AMD), TGFβ (E); n = 8 (Ctrl), 7 (NV AMD), PDGF-BB (F); n = 5 (Ctrl),6 (NV AMD), PGF (G): n = 5 (Ctrl), 5 (NV AMD). Dots represent concentrations of individual patient samples.


H . Representative confocal of IBA - 1 - stained mononuclear phagocytes on choroidal flat mounts from LysM - Cre / Nrp1 + / + LysM - Cre / Nrp1fl / fl mice at D7 D14 . Examples of macrophage ( yellow stars ) are presented in side panels . Scale bar : 20μm . I , J . Total number of IBA - 1 - positive mononuclear phagocytes counted around impact on confocal of choroidal flat mounts at D7 ( I ) D14 ( J ) ; n = 19 burns ( D7 LysM - Cre / Nrp1 + / + ) , n = 25 burns ( D7 LysM - Cre / Nrp1fl / fl ) n = 37 burns ( D7 LysM - Cre / Nrp1 + / + ) , n = 23 burns ( D7 LysM - Cre / Nrp1fl / fl ) , 3 - 5 mice with ~ 4 burns per eye .

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H. Representative confocal images of IBA-1-stained mononuclear phagocytes on choroidal flat mounts from LysM-Cre/Nrp1+/+ and LysM-Cre/Nrp1fl/fl mice at D7 and D14. Examples of macrophage quantification (yellow stars) are presented in side panels. Scale bar: 20μm. I, J. Total number of IBA-1-positive mononuclear phagocytes counted around laser impact area on confocal images of choroidal flat mounts at D7 (I) and D14 (J); n = 19 burns (D7 LysM-Cre/Nrp1+/+), n = 25 burns (D7 LysM-Cre/Nrp1fl/fl) n = 37 burns (D7 LysM-Cre/Nrp1+/+), n = 23 burns (D7 LysM-Cre/Nrp1fl/fl), 3-5 mice with ~4 burns per eye.


K . Compilation of representative compressed Z - stack confocal of FITC - dextran - labeled CNV IB4 - stained impact from LysM - Cre / Nrp1 + / + LysM - Cre / Nrp1fl / fl mice at D14 . Scale bar : 20μm . L - N . of of FITC - dextran - labeled CNV ( L ) , isolectin B4 ( IB4 ) - stained impact ( M ) of FITC / IB4 per - burn ( N ) relative to LysM - Cre / Nrp1 + / + at D14 ; n = 23 burns ( LysM - Cre / Nrp1 + / + ) , n = 27 burns ( LysM - Cre / Nrp1fl / fl ) .

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K. Compilation of representative compressed Z-stack confocal images of FITC-dextran-labeled CNV and IB4-stained laser impact area from LysM-Cre/Nrp1+/+ and LysM-Cre/Nrp1fl/fl mice at D14. Scale bar: 20μm. L-N. Quantification of area of FITC-dextran-labeled CNV (L), isolectin B4 (IB4 )-stained laser impact area (M) and the ratio of FITC/IB4 per laser-burn (N) relative to LysM-Cre/Nrp1+/+ at D14; n = 23 burns (LysM-Cre/Nrp1+/+), n = 27 burns ( LysM-Cre/Nrp1fl/fl).


A - D . mRNA expression of inflammation markers relative to LysM - Cre / Nrp1 + / + in mouse RPE - choroid - sclera complexes at D3 for Il1b ( A ) ; n = 10 ( LysM - Cre / Nrp1 + / + LysM - Cre / Nrp1fl / fl ) , Il6 ( B ) ; n = 7 ( LysM - Cre / Nrp1 + / + LysM - Cre / Nrp1fl / fl ) , Vegfa ( C ) ; n = 10 ( LysM - Cre / Nrp1 + / + LysM - Cre / Nrp1fl / fl ) , Tnf ( D ) ; n = 5 ( LysM - Cre / Nrp1 + / + ) , n = 10 ( LysM - Cre / Nrp1fl / fl ) .

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A-D. mRNA expression of inflammation markers relative to LysM-Cre/Nrp1+/+ in mouse RPE-choroid-sclera complexes at D3 for Il1b (A); n = 10 (LysM-Cre/Nrp1+/+ and LysM-Cre/Nrp1fl/fl), Il6 (B); n = 7 (LysM-Cre/Nrp1+/+ and LysM-Cre/Nrp1fl/fl), Vegfa (C); n = 10 (LysM-Cre/Nrp1+/+ and LysM-Cre/Nrp1fl/fl), Tnf (D); n = 5 (LysM-Cre/Nrp1+/+), n = 10 (LysM-Cre/Nrp1fl/fl).


K , L . mRNA expression relative to LysM - Cre / Nrp1 + / + of inflammation markers in mouse BMDMs for Il1b ( K ) ; n = 4 ( LysM - Cre / Nrp1 + / + ) , n = 5 ( LysM - Cre / Nrp1fl / fl ) Il6 ( L ) ; n = 3 ( LysM - Cre / Nrp1 + / + ) , n = 2 ( LysM - Cre / Nrp1fl / fl )

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K, L. mRNA expression relative to LysM-Cre/Nrp1+/+ of inflammation markers in mouse BMDMs for Il1b (K); n = 4 (LysM-Cre/Nrp1+/+), n = 5 (LysM-Cre/Nrp1fl/fl) and Il6 (L); n = 3 (LysM-Cre/Nrp1+/+), n = 2 (LysM-Cre/Nrp1fl/fl)


P . ( left ) enrichment plot ( right ) of GO Angiogenesis gene set enrichment ( GSEA ) of wildtype LysM - Cre / Nrp1fl / fl peritoneal macrophages ; n = 2 . NES , normalized enrichment score ; FDR , false discovery rate .

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P. Heatmap (left) and enrichment plot (right) of GO Angiogenesis gene set enrichment analysis (GSEA) of wildtype and LysM-Cre/Nrp1fl/fl peritoneal macrophages; n = 2. NES, normalized enrichment score; FDR, false discovery rate.


E . Compilation of representative compressed Z - stack confocal of FITC - dextran - labeled CNV isolectin B4 ( IB4 ) - stained impact from Vehicle NRP1 - derived trap treated wildtype mice . Scale bar : 20μm . F - H . of of FITC - dextran - labeled CNV ( F ) , IB4 - stained impact ( G ) of FITC / IB4 per - burn ( H ) relative to Vehicle at D14 ; n = 24 burns ( Vehicle ) , n = 26 burns ( NRP1 - derived trap ) .

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

E. Compilation of representative compressed Z-stack confocal images of FITC-dextran-labeled CNV and isolectin B4 (IB4)-stained laser impact area from Vehicle and NRP1-derived trap treated wildtype mice. Scale bar: 20μm. F-H. Quantification of area of FITC -dextran-labeled CNV (F), IB4-stained laser impact area (G) and the ratio of FITC/IB4 per laser-burn (H) relative to Vehicle at D14; n = 24 burns (Vehicle), n = 26 burns (NRP1-derived trap).


J - L . of of FITC - dextran - labeled CNV ( J ) , isolectin B4 ( IB4 ) - stained impact ( K ) of FITC / IB4 per - burn ( L ) relative to LysM - Cre / Nrp1 + / + + Vehicle in Vehicle NRP1 - derived trap treated LysM - Cre / Nrp1 + / + LysM - Cre / Nrp1fl / fl mice at D14 ; n = 16 burns ( LysM - Cre / Nrp1 + / + + Vehicle ) , n = 16 burns ( LysM - Cre / Nrp1 + / + + Trap ) , n = 13 burns ( LysM - null / Nrp1fl / fl + Vehicle ) , n = 19 burns ( LysM - Cre / Nrp1fl / fl + Trap ) .

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

J-L. Quantification of area of FITC-dextran-labeled CNV (J), isolectin B4 (IB4)-stained laser impact area (K) and the ratio of FITC/IB4 per laser-burn (L) relative to LysM-Cre/Nrp1+/+ + Vehicle in Vehicle and NRP1-derived trap treated LysM-Cre/Nrp1+/+ and LysM-Cre/Nrp1fl/fl mice at D14; n = 16 burns (LysM-Cre/Nrp1+/+ + Vehicle), n = 16 burns (LysM-Cre/Nrp1+/+ + Trap), n = 13 burns (LysM-null/Nrp1fl/fl + Vehicle) , n = 19 burns (LysM-Cre/Nrp1fl/fl + Trap).


Representative PET , MRI fused PET / MRI of thoracic region of a mouse that received PDE10A radioligand [ 18F ] - AQ28A . Interscapular brown adipose tissue ( BAT ) in each image is highlighted by crosshairs . mean standardized uptake ( SUV ) value of [ 18F ] - AQ28A in BAT skeletal muscle throughout dynamic scan was calculated compared for each ( n = 5 ) . * P = 0 . 0488 ( 20 mins ) , * P = 0 . 0236 ( 25 mins ) , * P = 0 . 0167 ( 30 mins ) , * * P = 0 . 00301 ( 35 mins ) , * * P = 0 . 00269 ( 40 mins ) , * * P = 0 . 00215 ( 45 mins ) , * * P = 0 . 00129 ( 50mins ) * * P = 0 . 00141 ( 45mins ) using unpaired 2 - Student ' s t .

O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 PET, MRI and fused PET/MRI images of the thoracic region of a mouse that received the PDE10A radioligand [18F]-AQ28A. Interscapular brown adipose tissue (BAT) in each image is highlighted by the crosshairs. The mean standardized uptake (SUV) value of [18F]-AQ28A in BAT and skeletal muscle throughout the dynamic scan was calculated and compared for each animal (n = 5). * P = 0.0488 (20 mins), * P = 0.0236 (25 mins), * P = 0.0167 (30 mins), ** P = 0.00301 (35 mins), ** P = 0.00269 (40 mins), ** P = 0.00215 (45 mins), ** P = 0.00129 (50mins) and ** P = 0.00141 (45mins) using unpaired 2-tailed Student's t tests.


A . Representative PET of a sagittal view of thoracic region of fasted lean mice treated with either MP - 10 ( 30mg / kg ) ( n = 3 ) or vehicle ( n = 4 ) prior to receiving [ 18F ] - FDG . Interscapular brown adipose tissue ( BAT ) in each image is highlighted by crosshairs . mean standardized uptake value ( SUV ) of [ 18F ] - FDG in BAT after both treatments was calculated for each group . * * P = 0 . 0013 using unpaired 2 - Student ' s t .

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

A. Representative PET images of a sagittal view of the thoracic region of fasted lean mice treated with either MP-10 (30mg/kg) (n = 3) or vehicle (n = 4) prior to receiving [18F]-FDG. Interscapular brown adipose tissue (BAT) in each image is highlighted by the crosshairs. The mean standardized uptake value (SUV) of [18F]-FDG in BAT after both treatments was calculated for each group. ** P = 0.0013 using unpaired 2-tailed Student's t test.


B . ( % ) change in associated under curve ( AUC ) of mice treated with MP - 10 ( 30mg / kg ) ( n = 4 ) or vehicle ( n = 4 ) prior to a cold challenge study for 4 hours at 8oC . * P = 0 . 0108 ( 1st hour ) , * P = 0 . 0218 ( 2nd hour ) , * P = 0 . 0121 ( 3rd hour ) , * P = 0 . 0168 ( 4th hour ) * P = 0 . 0264 ( AUC ) using unpaired 2 - Student ' s t .

O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 percentage (%) change in core body temperature and associated area under the curve (AUC) of mice treated with MP-10 (30mg/kg) (n = 4) or vehicle (n=4) prior to a cold challenge study for 4 hours at 8oC. * P = 0.0108 (1st hour), * P = 0.0218 (2nd hour), * P = 0.0121 (3rd hour), * P = 0.0168 (4th hour) and * P = 0.0264 (AUC) using unpaired 2-tailed Student's t tests.


C . RT - qPCR of relative mRNA expression of Pde10a in BAT , peri - ovarian visceral adipose tissue ( VAT ) inguinal subcutaneous adipose tissue ( SAT ) of lean mice ( n = 5 ) . * * P = 0 . 0063 ( SAT vs VAT ) , * * P = 0 . 0011 ( SAT vs BAT ) * * P = 0 . 0037 ( VAT vs BAT ) using unpaired 2 - Student ' s t .

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

C. RT-qPCR analysis of relative mRNA expression of Pde10a in BAT, peri-ovarian visceral white adipose tissue (VAT) and inguinal subcutaneous white adipose tissue (SAT) of lean mice (n = 5). ** P = 0.0063 (SAT vs VAT), ** P = 0.0011 (SAT vs BAT) and ** P = 0.0037 (VAT vs BAT) using unpaired 2-tailed Student's t test.


D Cyclic GMP ( cGMP ) cyclic AMP ( cGMP ) concentrations normalized to in SAT , VAT BAT 30 minutes after treatment of lean mice with MP - 10 ( 30 mg / kg ) ( n = 6 ) or vehicle control ( n = 6 ) . * * P = 0 . 0085 ( cGMP in VAT ) , * * P = 0 . 0011 ( cGMP in BAT ) * P = 0 . 04 ( cAMP in VAT ) using unpaired 2 - Student ' s t .

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

D Cyclic GMP (cGMP) and cyclic AMP (cGMP) concentrations normalized to protein concentration in SAT, VAT and BAT 30 minutes after treatment of lean mice with MP-10 (30 mg/kg) (n=6) or vehicle control (n = 6). ** P = 0.0085 (cGMP in VAT), ** P = 0.0011 (cGMP in BAT) and * P = 0.04 (cAMP in VAT) using unpaired 2-tailed Student's t tests.


E RT - qPCR of relative mRNA expression of thermogenic genes in SAT , VAT BAT after treatment of lean mice treated with MP - 10 ( 30 mg / kg ) ( n = 7 ) or vehicle control ( n = 6 ) . * * * * P < 0 . 0001 ( Pgc1alpha in VAT ) , * * P = 0 . 0012 ( Ucp1 in VAT ) , * * P = 0 . 0071 ( Pgc1alpha in BAT ) * P = 0 . 0486 ( Ucp1 in BAT ) using unpaired 2 - Student ' s t .

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

E RT-qPCR analysis of relative mRNA expression of thermogenic genes in SAT, VAT and BAT after treatment of lean mice treated with MP-10 (30 mg/kg) (n=7) or vehicle control (n = 6). **** P < 0.0001 (Pgc1alpha in VAT), ** P = 0.0012 (Ucp1 in VAT), ** P = 0.0071 (Pgc1alpha in BAT) and * P = 0.0486 (Ucp1 in BAT) using unpaired 2-tailed Student's t tests.


Representative fused PET / MRI of transverse view of thoracic region of lean ( n = 5 ) , diet induced obese ( DIO ) ( n = 4 ) leptin deficient ( ob / ob ) ( n = 5 ) mice that received PDE10A radioligand [ 18F ] - AQ28A . Interscapular brown adipose tissue ( BAT ) in each image is highlighted by crosshairs . mean standardized uptake ( SUV ) value of [ 18F ] - AQ28A in BAT throughout dynamic scan was calculated with associated under curve ( AUC ) . * P = 0 . 0128 ( lean vs DIO ) * * P = 0 . 0031 ( lean vs ob / ob ) using unpaired 2 - Student ' s t .

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

Representative fused PET/MRI images of the transverse view of the thoracic region of lean (n = 5), diet induced obese (DIO) (n = 4) and leptin deficient (ob/ob) (n = 5) mice that received the PDE10A radioligand [18F]-AQ28A. Interscapular brown adipose tissue (BAT) in each image is highlighted by the crosshairs. The mean standardized uptake (SUV) value of [18F]-AQ28A in BAT throughout the dynamic scan was calculated with associated area under the curve (AUC). * P = 0.0128 (lean vs DIO) and ** P = 0.0031 (lean vs ob/ob) using unpaired 2-tailed Student's t test.


A , B ( % ) change in cumulative food intake of diet induced obese ( DIO ) mice in response to daily treatment with MP - 10 ( 10mg / kg ) or vehicle for a week ( n = 4 per group ) . * P = 0 . 0264 using ( day 4 ) , * * * P = 0 . 0002 ( day 5 ) , * * * P < 0 . 0001 ( day 6 ) * * * P < 0 . 0001 ( day 7 ) using Two - Way of variance ( ANOVA ) with Sidak ' s post - hoc .

O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 The percentage (%) change in body weight and cumulative food intake of diet induced obese (DIO) mice in response to daily treatment with MP-10 (10mg/kg) or vehicle for a week (n= 4 per group). * P = 0.0264 using (day 4), *** P = 0.0002 (day 5), *** P < 0.0001 (day 6) and *** P < 0.0001 (day 7) using Two-Way analysis of variance (ANOVA) with Sidak's post-hoc test.


C . Upon completion of chronic feeding study , an insulin tolerance was performed on mice with single treatment of 0 . 75 U / kg insulin ( n = 4 per group ) . * P = 0 . 0246 * P = 0 . 0305 using unpaired 2 - Student ' s t .

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

C. Upon completion of the chronic feeding study, an insulin tolerance test was performed on mice with single treatment of 0.75 U/kg insulin (n = 4 per group). * P = 0.0246 and * P = 0.0305 using unpaired 2-tailed Student's t tests.


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

D, E, F Upon completion of the chronic feeding study, resting energy expenditure (REE), respiratory quotient (RQ) and locomotor activity of DIO mice treated with MP-10 (30mg/kg) compared to vehicle control were measured in metabolic cage experiments (n = 4 per group). * P = 0.0239 (02:00, RQ), * P = 0.0424 (02:30, RQ), * P = 0.0420 (03:00, RQ), * P = 0.0230 (03:30, RQ), * P = 0.0213 (04:00, RQ), ** P = 0.0116 (00:00, REE), ** P = 0.00823 (00:30, REE), ** P = 0.00969 (01:00, REE), ** P = 0.00274 (01:30, REE), ** P = 0.00323 (02:00, REE), ** P = 0.000441 (02:30, RE), ** P = 0.00390 (03:00, REE), ** P = 0.000211 (03:30, REE), ** P = 0.00216 (04:00, REE) and ** P = 0.0185 (04:30, REE) using unpaired 2-tailed Student's t tests.


G . RT - qPCR of relative mRNA expression of candidate genes in inguinal subcutaneous adipose tissue ( SAT ) , periovarian visceral adipose tissue ( VAT ) interscapularbrown adipose tissue ( BAT ) ( n = 8 per group ) * * * P = 0 . 0004 ( Pgc1alpha VAT ) , * * P = 0 . 0036 ( Ucp1 VAT ) , * * P = 0 . 0068 ( Cidea VAT ) , * P = 0 . 0411 ( Tmem26 VAT ) * * P = 0 . 0070 ( Pgc1alpha BAT ) using unpaired 2 - Student ' s t .

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

G. RT-qPCR analysis of relative mRNA expression of candidate genes in inguinal subcutaneous white adipose tissue (SAT), periovarian visceral white adipose tissue (VAT) and interscapularbrown adipose tissue (BAT) (n = 8 per group) *** P = 0.0004 (Pgc1alpha VAT), ** P = 0.0036 (Ucp1 VAT), ** P = 0.0068 (Cidea VAT), * P = 0.0411 (Tmem26 VAT) and ** P = 0.0070 (Pgc1alpha BAT) using unpaired 2-tailed Student's t tests.


H . Representative sections of inguinal SAT , peri - ovarian VAT interscapular BAT immunostained for UCP1 of mice chronically treated with MP - 10 ( 10 mg / kg ) or vehicle control . Scale bars , 100µm .

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

H. Representative sections of inguinal SAT, peri-ovarian VAT and interscapular BAT immunostained for UCP1 of mice chronically treated with MP-10 (10 mg/kg) or vehicle control. Scale bars, 100µm.


Representative PET image of upper of a human subject who received PDE10A radioligand [ 18F ] - MNI - 1869 . Supraclavicular brown adipose tissue ( BAT ) is highlighted by crosshairs . mean standardized uptake value ( SUV ) of [ 18F ] - MNI - 1869 in supraclavicular BAT skeletal muscle was calculated in human subjects ( n = 5 ) . * * P = 0 . 00210 ( 5 mins ) , * * * * P < 0 . 0001 ( 15 mins ) , * * * * P < 0 . 0001 ( 30 mins ) , * * * * P < 0 . 0001 ( 50 mins ) * * P = 0 . 00462 ( 75 mins ) using unpaired 2 - Student ' s t .

O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 PET image of the upper body of a human subject who received the PDE10A radioligand [18F]-MNI-1869. Supraclavicular brown adipose tissue (BAT) is highlighted by the crosshairs. The mean standardized uptake value (SUV) of [18F]-MNI-1869 in supraclavicular BAT and skeletal muscle was calculated in human subjects (n = 5). ** P = 0.00210 (5 mins), **** P < 0.0001 (15 mins), **** P < 0.0001 (30 mins), **** P < 0.0001 (50 mins) and ** P = 0.00462 (75 mins) using unpaired 2-tailed Student's t tests.


A . RT - qPCR of relative mRNA expression of thermogenic genes in cultured human brown adipocytes in response to chronic treatments ( 8 hours ) with DMSO ( control ) , MP - 10 ( 1nM 100nM ) , cyclic AMP ( cAMP - 10µM ) , cyclic GMP ( cGMP - 10µM ) cAMP + cGMP ( cA + cG - 10µM ( n = 4 separate cultures ) . * P = 0 . 0278 vs ( 100nM MP - 10 vs control ; Pgc1alpha ) , * * P = 0 . 0097 ( 10µM cA + cG vs control ; Pgc1alpha ) , * * P = 0 . 0063 ( 1nM MP - 10 vs control ; Ucp1 ) , * * * * P < 0 . 0001 ( 100nM MP - 10 vs control ; Ucp1 ) , # # P = 0 . 0017 ( 1nM MP - 10 vs 100nM MP - 10 ; Ucp1 ) , § § § § P < 0 . 0001 ( 10µM cAMP vs 100nM MP - 10 ; Ucp1 ) , § § § § P < 0 . 0001 ( 10µM cGMP vs 100nM MP - 10 ; Ucp1 ) , § § § § P < 0 . 0001 ( 10µM cA + cG vs 100nM MP - 10 ; Ucp1 ) , * P = 0 . 0112 vs ( 100nM MP - 10 vs control ; Cidea ) , * * * P = 0 . 0007 ( 1nM MP - 10 vs control ; Dio2 ) , * * * * P < 0 . 0001 ( 100nM MP - 10 vs control ; Dio2 ) , # P = 0 . 0267 ( 1nM MP - 10 vs 10µM cAMP ; Dio2 ) , § § § P = 0 . 0001 ( 10µM cAMP vs 100nM MP - 10 ; Dio2 ) , § § § P = 0 . 0007 ( 10µM cGMP vs 100nM MP - 10 ; Dio2 ) , * P = 0 . 0232 ( 10µM cA + cG vs control ; Dio2 ) § § P = 0 . 0058 ( 10µM cA + cG vs 100nM MP - 10 ; Dio2 ) , * P = 0 . 0416 ( 10µM cGMP vs control ; Prdm16 ) * P = 0 . 0105 ( 10µM cA + cG vs control ; Prdm16 ) using one - way of variance ( ANOVA ) with Tukey ' s post - hoc . B . RT - qPCR of relative mRNA expression of thermogenic genes in cultured human adipocytes in response to chronic treatments ( 8 hours ) with DMSO ( control ) , MP - 10 ( 1nM 100nM ) , cyclic AMP ( cAMP - 10µM ) , cyclic GMP ( cGMP - 10µM ) cAMP + cGMP ( cA + cG - 10µM ( n = 4 separate cultures ) . * P = 0 . 0186 vs ( 1nM MP - 10 vs control ; Ucp1 ) , * * * * P < 0 . 0001 ( 100nM MP - 10 vs control ; Ucp1 ) , # # P = 0 . 0016 ( 1nM MP - 10 vs 100nM MP - 10 ; Ucp1 ) , * * * P = 0 . 0003 ( 10µM cAMP vs control ; Ucp1 ) , § § § P = 0 . 0002 ( 10µM cGMP vs 100nM MP - 10 ; Ucp1 ) * * * P = 0 . 0003 ( cA + cG vs control ; Ucp1 ) using one - way of variance ( ANOVA ) with Tukey ' s post - hoc .

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

A. RT-qPCR analysis of relative mRNA expression of thermogenic genes in cultured primary human brown adipocytes in response to chronic treatments (8 hours) with DMSO (control), MP-10 (1nM and 100nM), cyclic AMP (cAMP - 10µM), cyclic GMP (cGMP - 10µM) and cAMP + cGMP (cA + cG - 10µM (n = 4 separate cultures). * P = 0.0278 vs (100nM MP-10 vs control; Pgc1alpha), **P = 0.0097 (10µM cA + cG vs control; Pgc1alpha), ** P = 0.0063 (1nM MP-10 vs control; Ucp1), **** P < 0.0001 (100nM MP-10 vs control; Ucp1), ## P = 0.0017 (1nM MP-10 vs 100nM MP-10; Ucp1), §§§§ P < 0.0001 (10µM cAMP vs 100nM MP-10; Ucp1), §§§§ P < 0.0001 (10µM cGMP vs 100nM MP-10; Ucp1), §§§§ P < 0.0001 (10µM cA + cG vs 100nM MP-10; Ucp1), * P = 0.0112 vs (100nM MP-10 vs control; Cidea), *** P = 0.0007 (1nM MP-10 vs control; Dio2), **** P < 0.0001 (100nM MP-10 vs control; Dio2), # P = 0.0267 (1nM MP-10 vs 10µM cAMP; Dio2), §§§ P = 0.0001 (10µM cAMP vs 100nM MP-10; Dio2), §§§ P = 0.0007 (10µM cGMP vs 100nM MP-10; Dio2), * P = 0.0232 (10µM cA + cG vs control; Dio2) §§ P = 0.0058 (10µM cA + cG vs 100nM MP-10; Dio2), * P = 0.0416 (10µM cGMP vs control; Prdm16) and * P = 0.0105 (10µM cA + cG vs control; Prdm16) using one-way analysis of variance (ANOVA) with Tukey's post-hoc test.B. RT-qPCR analysis of relative mRNA expression of thermogenic genes in cultured primary human white adipocytes in response to chronic treatments (8 hours) with DMSO (control), MP-10 (1nM and 100nM), cyclic AMP (cAMP - 10µM), cyclic GMP (cGMP - 10µM) and cAMP + cGMP (cA + cG - 10µM (n = 4 separate cultures). * P = 0.0186 vs (1nM MP-10 vs control; Ucp1), **** P < 0.0001 (100nM MP-10 vs control; Ucp1), ## P = 0.0016 (1nM MP-10 vs 100nM MP-10; Ucp1), *** P = 0.0003 (10µM cAMP vs control; Ucp1), §§§ P = 0.0002 (10µM cGMP vs 100nM MP-10; Ucp1) and *** P = 0.0003 (cA + cG vs control; Ucp1) using one-way analysis of variance (ANOVA) with Tukey's post-hoc test.


VGLL4 expression in 6 murine lines . Total lysates were analyzed by western

VGLL4 protein expression levels in 6 murine tumor cell lines. Total cell lysates were analyzed by western blot


VGLL4 were reduced in Vgll4 depleted . Total lysates of Vgll4 - knockdown LLC MB49 were analyzed by western

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VGLL4 protein levels were reduced in Vgll4 depleted cells. Total cell lysates of Vgll4-knockdown LLC and MB49 cells were analyzed by western blot


Control or shVgll4 LLC were transplanted into C57BL / 6 mice , - growth kinetics was measured at indicated times . n = 12 tumors for each

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Control or shVgll4 LLC cells were transplanted into C57BL/6 mice, and tumor-growth kinetics was measured at the indicated times. n=12 tumors for each group


of weights 21 days after transplantation of control or shVgll4 LLC into C57BL / 6 mice from ( C )

Quantification of tumor weights 21 days after transplantation of control or shVgll4 LLC cells into C57BL/6 mice from (C)


Control or shVgll4 LLC were transplanted into nude mice , - growth kinetics was measured at indicated times . n = 8 tumors for each group . ( F ) of weights 15 days after transplantation of control or shVgll4 LLC into nude mice from ( E ) . n = 8 tumors for each group . n . s . p > 0 . 05 , two - Student " s t - . solid represents average

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Control or shVgll4 LLC cells were transplanted into nude mice, and tumor-growth kinetics was measured at the indicated times. n=8 tumors for each group. (F) Quantification of tumor weights 15 days after transplantation of control or shVgll4 LLC cells into nude mice from (E). n=8 tumors for each group. n.s. p>0.05, two-tailed Student"s t-test. The solid line represents the average weight


Control or shVgll4 MB49 were transplanted into C57BL / 6 mice , - growth kinetics was measured at indicated times . n = 9 tumors for each group of weights 30 days after transplantation of control or shVgll4 MB49 into C57BL / 6

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Control or shVgll4 MB49 cells were transplanted into C57BL/6 mice, and tumor-growth kinetics was measured at the indicated times. n=9 tumors for each group Quantification of tumor weights 30 days after transplantation of control or shVgll4 MB49 cells into C57BL/6 mice


Control or shVgll4 MB49 were transplanted into nude mice , - growth kinetics was measured at indicated times . n = 8 tumors for each group of weights 15 days after transplantation of control or shVgll4 MB49 into nude mice from ( I ) . n = 8 tumors for each

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Control or shVgll4 MB49 cells were transplanted into nude mice, and tumor-growth kinetics was measured at the indicated times. n=8 tumors for each group Quantification of tumor weights 15 days after transplantation of control or shVgll4 MB49 cells into nude mice from (I). n=8 tumors for each group


Immunofluorescent of CD3 , CD8 CD45 ( red ) counterstained with DAPI for ( blue ) using control or shVgll4 LLC tumors from C57BL / 6 mice . Statistical of of CD3 , CD8 CD45 positive in tumors is shown in right panel respectively . n = 3 tumors for each

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Immunofluorescent staining of CD3, CD8 and CD45 (red) counterstained with DAPI for DNA (blue) using control or shVgll4 LLC tumors from C57BL/6 mice. Statistical analysis of the percentage of CD3, CD8 and CD45 positive cells in the tumors is shown in the right panel respectively. n=3 tumors for each group


of weights 30 days after transplantation of shVgll4 MB49 into C57BL / 6 mice receiving anti - CD4 alone , anti - CD8 alone , or both anti - CD4 anti - CD8 blocking antibodies . n = 20 tumors for each

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Quantification of tumor weights 30 days after transplantation of shVgll4 MB49 cells into C57BL/6 mice receiving anti-CD4 alone, anti-CD8 alone, or both anti-CD4 and anti-CD8 blocking antibodies. n=20 tumors for each group


Relative mRNA of immune related genes in Vgll4 - knockdown LLC or MB49 by qRT - PCR

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Relative mRNA levels of immune related genes in Vgll4-knockdown LLC or MB49 cells by qRT-PCR analysis


Knockdown of Vgll4 in MB49 decreased surface expression of PD - L1 on control or shVgll4 MB49 measured by flow cytometry with anti - mPD - L1

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Knockdown of Vgll4 in MB49 cells decreased the surface expression of PD-L1 protein on control or shVgll4 MB49 cells measured by flow cytometry analysis with anti-mPD-L1 antibody


Relative mRNA of immune related genes in Vgll4 - knockdown A549 by qRT - PCR

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Relative mRNA levels of immune related genes in Vgll4-knockdown A549 cells by qRT-PCR analysis


Immunoblot revealed reduced expression of PD - L1 in VGLL4 knockdown A549 , 16HBE H358 compared with control

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Immunoblot analysis revealed the reduced expression of PD-L1 protein in VGLL4 knockdown A549, 16HBE and H358 cells compared with control cells


qRT - PCR revealed a reduction of PD - L1 mRNA in VGLL4 - knockdown 16HBE H358 by

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qRT-PCR analysis revealed a reduction of PD-L1 mRNA levels in VGLL4-knockdown 16HBE and H358 cells by siRNA


Flow cytometry of surface PD - L1 in control VGLL4 knockdown A549 treated with IFNγ for indicated

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Flow cytometry analysis of surface PD-L1 levels in control and VGLL4 knockdown A549 cells treated with IFNγ for the indicated times


Immunoblot revealed reduced IFNγ - inducible PD - L1 expression in VGLL4 knockdown A549 16HBE

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Immunoblot analysis revealed the reduced IFNγ-inducible PD-L1 expression in VGLL4 knockdown A549 and 16HBE cells


Immunoblot of total lysates with indicated antibodies revealed attenuated IFNγ - inducible PD - L1 expression in VGLL4 knockout ( KO ) A549 generated by CRISPR /

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Immunoblot analysis of total cell lysates with the indicated antibodies revealed the attenuated IFNγ-inducible PD-L1 expression in VGLL4 knockout (KO) A549 cells generated by CRISPR/Cas9


Loss of VGLL4 dampened activity of PD - L1 promoter reporter in a luciferase . A549 were transfected with human PD - L1 - promoter luciferase plasmid indicated siRNA , treated with IFNγ for 12 h , subjected to a luciferase

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Loss of VGLL4 dampened the activity of PD-L1 promoter reporter in a luciferase assay. A549 cells were transfected with human PD-L1-promoter luciferase plasmid and indicated siRNA, treated with IFNγ for 12 h, and subjected to a luciferase assay


Overexpression of mouse PD - L1 in Vgll4 knockdown LLC restored growth in C57BL / 6 mice . weights of shVgll4 or shVgll4 - lenti - mPD - L1 LLC were determined 21 days after transplantation into C57BL / 6 mice ( left panel ) . Expression of mPD - L1 was shown in right panel by immunoblot with anti - mPD - L1 antibody . n = 10 tumors for each

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Overexpression of mouse PD-L1 in Vgll4 knockdown LLC cells restored the tumor growth in C57BL/6 mice. Tumor weights of shVgll4 or shVgll4-lenti-mPD-L1 LLC cells were determined 21 days after tumor cell transplantation into C57BL/6 mice (left panel). Expression of mPD-L1 was shown in the right panel by immunoblot analysis with anti-mPD-L1 antibody. n=10 tumors for each group


Knockdown of VGLL4 in A549 enhances T - meditated killing . Activated T A549 were co - cultured in 24 - well plates for 4 days then surviving were visualized by crystal violet . Relative fold ratios of surviving intensities are shown in right

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Knockdown of VGLL4 in A549 cells enhances T cell-meditated tumor cell killing. Activated T cells and A549 cells were co-cultured in 24-well plates for 4 days and then surviving tumor cells were visualized by crystal violet staining. Relative fold ratios of surviving cell intensities are shown in right panel


endogenous of VGLL4 IRF2BP2 was detected by immunoprecipitation using anti - VGLL4 antibody visualized by anti - IRF2BP2 antibody in A549

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The endogenous complex of VGLL4 and IRF2BP2 was detected by immunoprecipitation using anti-VGLL4 antibody and visualized by anti-IRF2BP2 antibody in A549 cells


endogenous IRF2BP2 was immunoprecipitated by VGLL4 - FLAG in HEK293T

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The endogenous IRF2BP2 was immunoprecipitated by VGLL4-FLAG proteins in HEK293T cells


Interaction between VGLL4 IRF2BP1 or IRF2BP2 was revealed by co - immunoprecipitation of differentially tagged in HEK293T

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Interaction between VGLL4 and IRF2BP1 or IRF2BP2 was revealed by co-immunoprecipitation of differentially tagged proteins in HEK293T cells


C - terminus of IRF2BP2 interacted with VGLL4 by co - immunoprecipitation in HEK293T

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C-terminus of IRF2BP2 interacted with VGLL4 by co-immunoprecipitation assay in HEK293T cells


Interaction of IRF2BP2 with VGLL1 - 4 was revealed by co - immunoprecipitation of differentially tagged in HEK293T

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Interaction of IRF2BP2 with VGLL1-4 was revealed by co-immunoprecipitation of differentially tagged proteins in HEK293T cells


TDU domains in VGLL4 are required for interaction with TEADs by co - immunoprecipitation in HEK293T

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TDU domains in VGLL4 are required for the interaction with TEADs by co-immunoprecipitation assay in HEK293T cells


TDU - deleted or HF4A VGLL4 mutations interacted with IRF2BP by co - immunoprecipitation in HEK293T

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TDU-deleted or HF4A VGLL4 mutations interacted with IRF2BP by co-immunoprecipitation analysis in HEK293T cells


TDU - deleted or HF4A VGLL4 mutations interacted wit IRF2BP by co - immunoprecipitation in HEK293T

TDU-deleted or HF4A VGLL4 mutations interacted wit IRF2BP by co-immunoprecipitation analysis in HEK293T cells


Expression of VGLL4 - WT or VGLL4 - HF4A enhances PD - L1 expression in A549 by immunoblot . A549 were transfected with indicated plasmids subjected to immunoblot with indicated

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Expression of VGLL4-WT or VGLL4-HF4A enhances PD-L1 expression in A549 cells by immunoblot analysis. A549 cells were transfected with indicated plasmids and subjected to immunoblot with indicated antibodies


VGLL4 - HF4A rescues PD - L1 expression in VGLL4 - knockdown A549 . A549 were transfected with siRNA targeting to 3 " UTR of VGLL4 mRNA followed by transduction with lenti - VGLL4 - HF4A . lysates were analyzed by western blot with indicated

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VGLL4-HF4A rescues PD-L1 expression in VGLL4-knockdown A549 cells. A549 cells were transfected with siRNA targeting to the 3"UTR of VGLL4 mRNA and followed by transduction with lenti-VGLL4-HF4A virus. Cell lysates were analyzed by western blot with indicated antib


Expression of VGLL4 - HF4A rescues growth of VGLL4 - knockdown LLC in C57BL / 6 mice . Control , Vgll4 knockdown or Vgll4 knockdown together with VGLL4 - HF4A overexpressing LLC were transplanted into nude mice or C57BL6 mice . volumes were measured 15 days for nude mice 21 days for C57BL / 6 mice after

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Expression of VGLL4-HF4A rescues tumor growth of VGLL4-knockdown LLC cells in C57BL/6 mice. Control, Vgll4 knockdown or Vgll4 knockdown together with VGLL4-HF4A overexpressing LLC cells were transplanted into nude mice or C57BL6 mice. Tumor volumes were measured 15 days for nude mice and 21 days for C57BL/6 mice after tumor cell inoculation


Expression of VGLL4 - HF4A attenuates T - meditated killing . Activated T A549 were co - cultured in 24 - well plates for 4 days Relative fold ratios of surviving are shown by measuring intensities of surviving stained with crystal

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Expression of VGLL4-HF4A attenuates the T cell-meditated tumor cell killing. Activated T cells and A549 cells were co-cultured in 24-well plates for 4 days and Relative fold ratios of surviving cells are shown by measuring the intensities of surviving cells stained with crystal violet


Correlation between expression of VGLL4 IRF2BP2 in 6 murine with indicated

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Correlation between protein expression of VGLL4 and IRF2BP2 in 6 murine tumor cell line with the indicated antibodies


Correlation between expression of VGLL4 IRF2BP2 i human with indicated

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Correlation between protein expression of VGLL4 and IRF2BP2 i human cell line with the indicated antibodies


Immunoblot of A549 transfected with VGLL4 - FLAG or VGLL4 - HF4A plasmids revealed an increased IRF2BP2 compared with control

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Immunoblot analysis of A549 cells transfected with VGLL4-FLAG or VGLL4-HF4A plasmids revealed an increased IRF2BP2 protein levels compared with control cells


Deficiency of VGLL4 reduces IRF2BP2 . Immunoblot of A549 LLC lysates transfected with siRNA for control

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Deficiency of VGLL4 reduces IRF2BP2 protein levels. Immunoblot analysis of A549 and LLC cell lysates transfected with siRNA for control and VGLL4


indicated lines were transfected with control or VGLL4 siRNA subjected to immunoblot with indicated

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The indicated cell lines were transfected with control or VGLL4 siRNA and subjected to immunoblot analysis with the indicated antibodies


qRT - PCR of IRF2BP2 mRNA in control siVGLL4 A549

qRT-PCR analysis of IRF2BP2 mRNA levels in control and siVGLL4 A549 cells


A549 or 16HBE were transfected with control or VGLL4 siRNA treated with MG132 for indicated times subjected to immunoblot with indicated antibodies . Relative IRF2BP2 expression were quantified in right

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A549 or 16HBE cells were transfected with control or VGLL4 siRNA and treated with MG132 for the indicated times and subjected to immunoblot analysis with the indicated antibodies. Relative IRF2BP2 expression levels were quantified in the right panel


Lys48 - linked ubiquitylation of IRF2BP2 is strongly inhibited when VGLL4 was overexpressed in HEK293T . HEK293T were transfected with indicated plasmids , treated with MG132 for 3 h before harvest , subjected to immunoprecipitation immunoblot as

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Lys48-linked ubiquitylation of IRF2BP2 is strongly inhibited when VGLL4 was overexpressed in HEK293T cells. HEK293T cells were transfected with indicated plasmids, treated with MG132 for 3 h before harvest, and subjected to immunoprecipitation and immunoblot analysis as indicated


Lys48 - linked ubiquitylation of IRF2BP2 is increased in absence of VGLL4 in HEK293T . HEK293T were transfected with indicated siRNA plasmids , subjected to immunoprecipitation immunoblot as

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Lys48-linked ubiquitylation of IRF2BP2 is increased in the absence of VGLL4 in HEK293T cells. HEK293T cells were transfected with indicated siRNA and plasmids, and subjected to immunoprecipitation and immunoblot analysis as indicated


IRF2 inhibits IRF1 transcriptional activity . A549 transfected with human PD - L1 promoter luciferase reporter indicated plasmids were analyzed using a luciferase

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IRF2 inhibits IRF1 transcriptional activity. A549 cells transfected with human PD-L1 promoter luciferase reporter and indicated plasmids were analyzed using a luciferase assay


Knockdown IRF2 restores PD - L1 expression in VGLL4 - knockdown A549 . Gene expression were analyzed by qRT - PCR in A549 transfected or transduced as

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Knockdown IRF2 restores PD-L1 expression in VGLL4-knockdown A549 cells. Gene expression levels were analyzed by qRT-PCR in A549 cells transfected or transduced as indicated


Overexpression of IRF1 increases PD - L1 expression in VGLL4 - knockdown A549 . A549 were transfected with VGLL4 siRNA or together with HA - IRF1 plasmid total lysates were analyzed by western

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Overexpression of IRF1 increases PD-L1 expression in VGLL4-knockdown A549 cells. A549 cells were transfected with VGLL4 siRNA or together with HA-IRF1 plasmid and total cell lysates were analyzed by western blot


IRF2 is released from PD - L1 promoter upon IFNγ stimulation . IRF2 - 3 × HA knockin A549 were stimulated with IFNγ subjected to ChIP - qPCR in PD - L1 promoter using control HA antibodies . Normal mouse IgG was used as

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IRF2 is released from PD-L1 promoter upon IFNγ stimulation. IRF2-3×HA knockin A549 cells were stimulated with IFNγ and subjected to ChIP-qPCR analysis in the PD-L1 promoter using control and HA antibodies. Normal mouse IgG was used as control


Increased association of IRF2 with PD - L1 promoter in IRF2BP2 knockout ( KO ) A549 revealed by ChIP - qPCR . Immunoblot showed loss of IRF2BP2 in IRF2BP2 KO A549 in lower

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Increased association of IRF2 with PD-L1 promoter in IRF2BP2 knockout (KO) A549 cells revealed by ChIP-qPCR analysis. Immunoblot showed the loss of IRF2BP2 in IRF2BP2 KO A549 cells in the lower panel


Dynamic association of IRF2 with IRF2BP2 during IFNγ stimulation by immunoprecipitation using anti - HA antibody in IRF2 - 3 × HA knockin A549

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Dynamic association of IRF2 with IRF2BP2 during IFNγ stimulation by immunoprecipitation analysis using anti-HA antibody in IRF2-3×HA knockin A549 cells


Loss of IRF2BP2 attenuated IFNγ - inducible PD - L1 expression revealed by immunoblot in A549 control IRF2BP2 knockout of PD - L1

Loss of IRF2BP2 attenuated IFNγ-inducible PD-L1 protein expression revealed by immunoblot in A549 control and IRF2BP2 knockout cells Quantification of PD-L1 protein level


Loss of IRF2BP2 attenuated IFNγ - inducible PD - L1 mRNA expression revealed by qRT - PCR in A549 control IRF2BP2 knockout

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Loss of IRF2BP2 attenuated IFNγ-inducible PD-L1 mRNA expression revealed by qRT-PCR analysis in A549 control and IRF2BP2 knockout cells


miR - 130a represses VGLL4 PD - L1 . A549 were transfected with control miR - 130a mimic subjected to immunoblot

miR-130a represses VGLL4 and PD-L1 protein levels. A549 cells were transfected with control and miR-130a mimic and subjected to immunoblot analysis


Inhibition of miR - 130a by its sponge enhances IFNγ - inducible PD - L1 expression . A549 were transduced with control or miR - 130a sponge lentivirus subjected to

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Inhibition of miR-130a by its sponge enhances IFNγ-inducible PD-L1 expression. A549 cells were transduced with control or miR-130a sponge lentivirus and subjected to immunoblot


Inhibition of miR - 130a by its sponge enhances IFNγ - inducible PD - L1 expression . A549 were transduced with control or miR - 130a sponge lentivirus subjected t qRT - PCR

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Inhibition of miR-130a by its sponge enhances IFNγ-inducible PD-L1 expression. A549 cells were transduced with control or miR-130a sponge lentivirus and subjected t qRT-PCR analysis


Decreased YAP target gene expression by inhibition of miR - 130a using its sponge in A549

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Decreased YAP target gene expression by inhibition of miR-130a using its sponge in A549 cells


YAP5SA expression attenuates IFNγ - inducible PD - L1 expression . A549 were transduced with control , YAP - 5SA or YAP - S94A lentivirus treated with IFNγ for indicated times subjected to immunoblot

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YAP5SA expression attenuates IFNγ-inducible PD-L1 expression. A549 cells were transduced with control, YAP-5SA or YAP-S94A lentivirus and treated with IFNγ for the indicated times and subjected to immunoblot analysis


qRT - PCR of lysates from control , YAP - 5SA or YAP - S94A expressing A549 treated with IFNγ for indicated

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qRT-PCR analysis of cell lysates from control, YAP-5SA or YAP-S94A expressing A549 cells treated with IFNγ for the indicated times


Control or YAP - overexpressing LLC were transplanted into nude mice ( upper panel ) or C57BL / 6 ( lower panel ) mice , - growth kinetics was measured at indicated times . volumes at end time points were analyzed shown at right

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Control or YAP-overexpressing LLC cells were transplanted into nude mice (upper panel) or C57BL/6 (lower panel) mice, and tumor-growth kinetics was measured at the indicated times. Tumor volumes at the end time points were analyzed and shown at the right panel


miR - 130a regulates IRF1 3 ′ UTR sensor activity . A549 were transfected with WT or mutant IRF1 3 ′ UTR sensor plasmids together with miR - 130a mimic . Sensor activities were determined by dual luciferase

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miR-130a regulates IRF1 3′UTR sensor activity. A549 cells were transfected with WT or mutant IRF1 3′UTR sensor plasmids together with miR-130a mimic. Sensor activities were determined by dual luciferase assay


YAP5SA induces miR - 130a expression represses IRF1 expression . A549 were transduced with control or YAP - 5SA lentivirus subjected to qRT - PCR

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YAP5SA induces miR-130a expression and represses IRF1 expression. A549 cells were transduced with control or YAP-5SA lentivirus and subjected to qRT-PCR analysis


Inhibition of miR - 130a by its sponge enhances IFNγ - inducible IRF1 expression . A549 were transduced with control or miR - 130a sponge lentivirus subjected to qRT - PCR

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Inhibition of miR-130a by its sponge enhances IFNγ-inducible IRF1 expression. A549 cells were transduced with control or miR-130a sponge lentivirus and subjected to qRT-PCR analysis