Figure 6Prussian Perl blue staining in liver and skeletal muscle after iron injections . 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 test 24 h after iron injections . Glucose tolerance test ( GTT ) 24 h after iron injections . Insulin tolerance test 24 h after iron injections . Quantification of area under curve in ITT ( E ) . Glucose tolerance test ( GTT ) 24 h after iron injections . Quantification of area under curve for panel ( F ) . 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 .

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

Figure 6Prussian Perl blue staining in liver and skeletal muscle after iron injections.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 test 24 h after iron injections.Glucose tolerance test (GTT) 24 h after iron injections.Insulin tolerance test 24 h after iron injections. Quantification of area under curve in ITT (E).Glucose tolerance test (GTT) 24 h after iron injections. Quantification of area under curve for panel (F).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.


Figure 7Representative 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 TEM images of skeletal muscle 24 h after iron injections . Representative western blot images of phospho - UVRAG S550 and total UVRAG in skeletal muscle 24 h after iron injections . Representative western blot images and quantification of LC3 - B and p62 to tubulin in skeletal muscles 24 h after iron injections .

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

Figure 7Representative 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 TEM images of skeletal muscle 24 h after iron injections.Representative western blot images of phospho-UVRAG S550 and total UVRAG in skeletal muscle 24 h after iron injections.Representative western blot images and quantification of LC3-B and p62 to tubulin in skeletal muscles 24 h after iron injections.


Figure 1 ( 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 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 .

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

Figure 1(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 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.


Figure 2 ( 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 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 ) 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 ) 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 = * * *

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

Figure 2(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 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) 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) 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 = ***


Figure 3 ( 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 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 .

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

Figure 3(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 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


Figure 4 ( 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 .

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

Figure 4(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.


Figure 5 ( 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 .

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

Figure 5(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.


Figure 6 ( 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 ) 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 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 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 .

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

Figure 6(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) 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 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 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.


Figure 7 ( 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 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 and SMC ChIP occupancy in the vicinity of several typical EPODs .

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

Figure 7(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 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 and SMC ChIP occupancy in the vicinity of several typical EPODs.


Figure 1A . 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 individual patient samples .

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

Figure 1A. 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 individual patient samples.


Figure 2H . 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 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 ) .

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

Figure 2H. 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 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).


Figure 3A - 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 ) and Il6 ( L ) ; n = 3 ( LysM - Cre / Nrp1 + / + ) , n = 2 ( LysM - Cre / Nrp1fl / fl ) 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 .

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

Figure 3A-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) and Il6 (L); n = 3 (LysM-Cre/Nrp1+/+), n = 2 (LysM-Cre/Nrp1fl/fl)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.


Figure 4E . 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 . 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 ) .

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

Figure 4E. 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. 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).


Figure 1Representative 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 .

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

Figure 1Representative 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.


Figure 2A . 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 . 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 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 ) 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 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 .

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

Figure 2A. 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. 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 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) 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 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.


Figure 3Representative 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 .

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

Figure 3Representative 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.


Figure 4A , 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 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 . 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 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 and interscapular BAT immunostained for UCP1 of mice chronically treated with MP - 10 ( 10 mg / kg ) or vehicle control . Scale bars , 100µm .

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

Figure 4A, 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 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.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 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 and interscapular BAT immunostained for UCP1 of mice chronically treated with MP-10 (10 mg/kg) or vehicle control. Scale bars, 100µm.


Figure 5Representative 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 .

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

Figure 5Representative 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.


Figure 6A . 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 .

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

Figure 6A. 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.


Figure 1VGLL4 protein expression levels in 6 murine tumor cell lines . Total cell lysates were analyzed by western blotVGLL4 protein levels were reduced in Vgll4 depleted cells . Total cell lysates of Vgll4 - knockdown LLC and MB49 cells were analyzed by western blotControl 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 groupQuantification of tumor weights 21 days after transplantation of control or shVgll4 LLC cells into C57BL / 6 mice from ( C ) 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 weightControl 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 miceControl 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 groupImmunofluorescent 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 groupQuantification 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

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

Figure 1VGLL4 protein expression levels in 6 murine tumor cell lines. Total cell lysates were analyzed by western blotVGLL4 protein levels were reduced in Vgll4 depleted cells. Total cell lysates of Vgll4-knockdown LLC and MB49 cells were analyzed by western blotControl 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 groupQuantification of tumor weights 21 days after transplantation of control or shVgll4 LLC cells into C57BL/6 mice from (C)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 weightControl 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 miceControl 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 groupImmunofluorescent 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 groupQuantification 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


Figure 2Relative mRNA levels of immune related genes in Vgll4 - knockdown LLC or MB49 cells by qRT - PCR analysisKnockdown 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 antibodyRelative mRNA levels of immune related genes in Vgll4 - knockdown A549 cells by qRT - PCR analysisImmunoblot analysis revealed the reduced expression of PD - L1 protein in VGLL4 knockdown A549 , 16HBE and H358 cells compared with control cellsqRT - PCR analysis revealed a reduction of PD - L1 mRNA levels in VGLL4 - knockdown 16HBE and H358 cells by siRNAFlow cytometry analysis of surface PD - L1 levels in control and VGLL4 knockdown A549 cells treated with IFNγ for the indicated timesImmunoblot analysis revealed the reduced IFNγ - inducible PD - L1 expression in VGLL4 knockdown A549 and 16HBE cellsImmunoblot 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 / Cas9Loss 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 assayOverexpression 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 groupKnockdown 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

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

Figure 2Relative mRNA levels of immune related genes in Vgll4-knockdown LLC or MB49 cells by qRT-PCR analysisKnockdown 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 antibodyRelative mRNA levels of immune related genes in Vgll4-knockdown A549 cells by qRT-PCR analysisImmunoblot analysis revealed the reduced expression of PD-L1 protein in VGLL4 knockdown A549, 16HBE and H358 cells compared with control cellsqRT-PCR analysis revealed a reduction of PD-L1 mRNA levels in VGLL4-knockdown 16HBE and H358 cells by siRNAFlow cytometry analysis of surface PD-L1 levels in control and VGLL4 knockdown A549 cells treated with IFNγ for the indicated timesImmunoblot analysis revealed the reduced IFNγ-inducible PD-L1 expression in VGLL4 knockdown A549 and 16HBE cellsImmunoblot 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/Cas9Loss 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 assayOverexpression 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 groupKnockdown 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


Figure 3The endogenous complex of VGLL4 and IRF2BP2 was detected by immunoprecipitation using anti - VGLL4 antibody and visualized by anti - IRF2BP2 antibody in A549 cellsThe endogenous IRF2BP2 was immunoprecipitated by VGLL4 - FLAG proteins in HEK293T cellsInteraction between VGLL4 and IRF2BP1 or IRF2BP2 was revealed by co - immunoprecipitation of differentially tagged proteins in HEK293T cellsC - terminus of IRF2BP2 interacted with VGLL4 by co - immunoprecipitation assay in HEK293T cellsInteraction of IRF2BP2 with VGLL1 - 4 was revealed by co - immunoprecipitation of differentially tagged proteins in HEK293T cellsTDU domains in VGLL4 are required for the interaction with TEADs by co - immunoprecipitation assay in HEK293T cellsTDU - deleted or HF4A VGLL4 mutations interacted with IRF2BP by co - immunoprecipitation analysis in HEK293T cellsTDU - deleted or HF4A VGLL4 mutations interacted wit IRF2BP by co - immunoprecipitation analysis in HEK293T cellsExpression 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 antibodiesVGLL4 - 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 antibExpression 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 inoculationExpression 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

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

Figure 3The endogenous complex of VGLL4 and IRF2BP2 was detected by immunoprecipitation using anti-VGLL4 antibody and visualized by anti-IRF2BP2 antibody in A549 cellsThe endogenous IRF2BP2 was immunoprecipitated by VGLL4-FLAG proteins in HEK293T cellsInteraction between VGLL4 and IRF2BP1 or IRF2BP2 was revealed by co-immunoprecipitation of differentially tagged proteins in HEK293T cellsC-terminus of IRF2BP2 interacted with VGLL4 by co-immunoprecipitation assay in HEK293T cellsInteraction of IRF2BP2 with VGLL1-4 was revealed by co-immunoprecipitation of differentially tagged proteins in HEK293T cellsTDU domains in VGLL4 are required for the interaction with TEADs by co-immunoprecipitation assay in HEK293T cellsTDU-deleted or HF4A VGLL4 mutations interacted with IRF2BP by co-immunoprecipitation analysis in HEK293T cellsTDU-deleted or HF4A VGLL4 mutations interacted wit IRF2BP by co-immunoprecipitation analysis in HEK293T cellsExpression 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 antibodiesVGLL4-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 antibExpression 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 inoculationExpression 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


Figure 4Correlation between protein expression of VGLL4 and IRF2BP2 in 6 murine tumor cell line with the indicated antibodiesCorrelation between protein expression of VGLL4 and IRF2BP2 i human cell line with the indicated antibodiesImmunoblot analysis of A549 cells transfected with VGLL4 - FLAG or VGLL4 - HF4A plasmids revealed an increased IRF2BP2 protein levels compared with control cellsDeficiency of VGLL4 reduces IRF2BP2 protein levels . Immunoblot analysis of A549 and LLC cell lysates transfected with siRNA for control and VGLL4The indicated cell lines were transfected with control or VGLL4 siRNA and subjected to immunoblot analysis with the indicated antibodiesqRT - PCR analysis of IRF2BP2 mRNA levels in control and siVGLL4 A549 cellsA549 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 panelLys48 - 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 indicatedLys48 - 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

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

Figure 4Correlation between protein expression of VGLL4 and IRF2BP2 in 6 murine tumor cell line with the indicated antibodiesCorrelation between protein expression of VGLL4 and IRF2BP2 i human cell line with the indicated antibodiesImmunoblot analysis of A549 cells transfected with VGLL4-FLAG or VGLL4-HF4A plasmids revealed an increased IRF2BP2 protein levels compared with control cellsDeficiency of VGLL4 reduces IRF2BP2 protein levels. Immunoblot analysis of A549 and LLC cell lysates transfected with siRNA for control and VGLL4The indicated cell lines were transfected with control or VGLL4 siRNA and subjected to immunoblot analysis with the indicated antibodiesqRT-PCR analysis of IRF2BP2 mRNA levels in control and siVGLL4 A549 cellsA549 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 panelLys48-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 indicatedLys48-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


Figure 5IRF2 inhibits IRF1 transcriptional activity . A549 cells transfected with human PD - L1 promoter luciferase reporter and indicated plasmids were analyzed using a luciferase assayKnockdown 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 indicatedOverexpression 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 blotIRF2 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 controlIncreased 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 panelDynamic association of IRF2 with IRF2BP2 during IFNγ stimulation by immunoprecipitation analysis using anti - HA antibody in IRF2 - 3 × HA knockin A549 cellsLoss of IRF2BP2 attenuated IFNγ - inducible PD - L1 protein expression revealed by immunoblot in A549 control and IRF2BP2 knockout cells Quantification of PD - L1 protein levelLoss of IRF2BP2 attenuated IFNγ - inducible PD - L1 mRNA expression revealed by qRT - PCR analysis in A549 control and IRF2BP2 knockout

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

Figure 5IRF2 inhibits IRF1 transcriptional activity. A549 cells transfected with human PD-L1 promoter luciferase reporter and indicated plasmids were analyzed using a luciferase assayKnockdown 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 indicatedOverexpression 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 blotIRF2 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 controlIncreased 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 panelDynamic association of IRF2 with IRF2BP2 during IFNγ stimulation by immunoprecipitation analysis using anti-HA antibody in IRF2-3×HA knockin A549 cellsLoss of IRF2BP2 attenuated IFNγ-inducible PD-L1 protein expression revealed by immunoblot in A549 control and IRF2BP2 knockout cells Quantification of PD-L1 protein levelLoss of IRF2BP2 attenuated IFNγ-inducible PD-L1 mRNA expression revealed by qRT-PCR analysis in A549 control and IRF2BP2 knockout cells


Figure 6miR - 130a represses VGLL4 and PD - L1 protein levels . A549 cells were transfected with control and miR - 130a mimic and subjected to immunoblot analysisInhibition 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 immunoblotInhibition 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 analysisDecreased YAP target gene expression by inhibition of miR - 130a using its sponge in A549 cellsYAP5SA 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 analysisqRT - PCR analysis of cell lysates from control , YAP - 5SA or YAP - S94A expressing A549 cells treated with IFNγ for the indicated timesControl 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 panelmiR - 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 assayYAP5SA induces miR - 130a expression and represses IRF1 expression . A549 cells were transduced with control or YAP - 5SA lentivirus and subjected to qRT - PCR analysisInhibition 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 analysisSuppression of IFNγ - inducible PD - L1 expression by YAP5SA is compromised in miR - 130a KO A549 cells . PD - L1 expression levels were analyzed by qRT - PCR in IFNγ - treated control or miR - 130a KO A549 cells pre - transduced with lenti - YAP5SA

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

Figure 6miR-130a represses VGLL4 and PD-L1 protein levels. A549 cells were transfected with control and miR-130a mimic and subjected to immunoblot analysisInhibition 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 immunoblotInhibition 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 analysisDecreased YAP target gene expression by inhibition of miR-130a using its sponge in A549 cellsYAP5SA 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 analysisqRT-PCR analysis of cell lysates from control, YAP-5SA or YAP-S94A expressing A549 cells treated with IFNγ for the indicated timesControl 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 panelmiR-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 assayYAP5SA induces miR-130a expression and represses IRF1 expression. A549 cells were transduced with control or YAP-5SA lentivirus and subjected to qRT-PCR analysisInhibition 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 analysisSuppression of IFNγ-inducible PD-L1 expression by YAP5SA is compromised in miR-130a KO A549 cells. PD-L1 expression levels were analyzed by qRT-PCR in IFNγ-treated control or miR-130a KO A549 cells pre-transduced with lenti-YAP5SA virus


Figure 7Representative immunohistochemical staining for VGLL4 and IRF2BP2 in human non - small cell lung cancer tissues . IHC score is indicated . Scale bar : 50 μmStaining of VGLL4 and IRF2BP2 on human non - small cell lung cancer tissue arrays containing 71 samples was quantified as described in the Materials and Methods section . Correlation of VGLL4 and IRF2BP2 expressions was significant ( Pearson correlation test ; R = 0 . 6341 , P < 0 . 001 ) Correlation analysis of VGLL4 and PD - L1 expression on human non - small cell lung cancer tissue arrays containing 71 samplesKaplan - Meier survival analysis of patients with high ( n = 74 ) or low ( n = 130 ) VGLL4 mRNA levels from GSE31210 , a lung cancer data set . Survival curves are calculated according to the Kaplan - Meier

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

Figure 7Representative immunohistochemical staining for VGLL4 and IRF2BP2 in human non-small cell lung cancer tissues. IHC score is indicated. Scale bar: 50 μmStaining of VGLL4 and IRF2BP2 on human non-small cell lung cancer tissue arrays containing 71 samples was quantified as described in the Materials and Methods section. Correlation of VGLL4 and IRF2BP2 expressions was significant (Pearson correlation test; R = 0.6341, P < 0.001) Correlation analysis of VGLL4 and PD-L1 expression on human non-small cell lung cancer tissue arrays containing 71 samplesKaplan-Meier survival analysis of patients with high (n=74) or low (n=130) VGLL4 mRNA levels from GSE31210, a lung cancer data set. Survival curves are calculated according to the Kaplan-Meier method


Figure 1B , C . Detection of CST at stalled and active forks with SIRF assay in U2OS ( B ) and HCT116 ( C ) cells . To detect CST at stalled forks , cells were pulse - labeled with EdU for 8 min , followed by 4 mM HU treatment for 3 h . To detect CST at active forks , SIRF was performed without HU treatment . Representative SIRF images of CTC1 , STN1 and PCNA on normal or stalled forks are shown . Scale bars : 10 µm . Relative SIRF fluorescence intensity was calculated by normalizing to untreated samples and then to their respective EdU - SIRF signals . Mean value in each sample is labeled in the graph . N : the number of cells analyzed in each sample .

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

Figure 1B, C. Detection of CST at stalled and active forks with SIRF assay in U2OS (B) and HCT116 (C) cells. To detect CST at stalled forks, cells were pulse-labeled with EdU for 8 min, followed by 4 mM HU treatment for 3 h. To detect CST at active forks, SIRF was performed without HU treatment. Representative SIRF images of CTC1, STN1 and PCNA on normal or stalled forks are shown. Scale bars: 10 µm. Relative SIRF fluorescence intensity was calculated by normalizing to untreated samples and then to their respective EdU-SIRF signals. Mean value in each sample is labeled in the graph. N: the number of cells analyzed in each sample.


Figure 2Western blot showing STN1 knockdown . shLUC ( siRNA targeting luciferase ) was used as the control . CST deficiency causes ssDNA accumulation upon fork stalling . HeLa cells with STN1 knockdown were incubated with 10 µM BrdU for 48 h , followed by 2 mM HU treatment for 3 h , and subsequently stained with BrdU antibody under a non - denaturing condition to detect ssDNA . N , the number of cells analyzed in each condition . Relative BrdU fluorescence intensity was quantitated by Image J . Scheme of DNA fiber analysis and representative fiber images . DNA fiber analysis of U2OS cells with STN1 knockdown and its RNAi - resistant Flag - STN1 WT re - expression with and without mirin treatment . DNA fiber analysis of BJ cells with STN1 knockdown by two siRNA sequence with and without mirin treatment . DNA fiber analysis of HCT116 cells with STN1 knockdown with and without mirin treatment . DNA fiber analysis of U2OS cells showing that SMARCAL1 - mediated fork reversal is needed for nascent - stand degradation observed in CST - deficient cells . DNA fiber analysis of U2OS cells showing that ZRANB3 - mediated fork reversal is needed for nascent - strand degradation observed in CST - deficient cells .

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

Figure 2Western blot showing STN1 knockdown. shLUC (siRNA targeting luciferase) was used as the control.CST deficiency causes ssDNA accumulation upon fork stalling. HeLa cells with STN1 knockdown were incubated with 10 µM BrdU for 48 h, followed by 2 mM HU treatment for 3 h, and subsequently stained with BrdU antibody under a non-denaturing condition to detect ssDNA. N, the number of cells analyzed in each condition. Relative BrdU fluorescence intensity was quantitated by Image J.Scheme of DNA fiber analysis and representative fiber images.DNA fiber analysis of U2OS cells with STN1 knockdown and its RNAi-resistant Flag-STN1 WT re-expression with and without mirin treatment.DNA fiber analysis of BJ cells with STN1 knockdown by two siRNA sequence with and without mirin treatment.DNA fiber analysis of HCT116 cells with STN1 knockdown with and without mirin treatment.DNA fiber analysis of U2OS cells showing that SMARCAL1-mediated fork reversal is needed for nascent-stand degradation observed in CST-deficient cells.DNA fiber analysis of U2OS cells showing that ZRANB3-mediated fork reversal is needed for nascent-strand degradation observed in CST-deficient cells.


Figure 3SIRF detection of MRE11 at stalled forks in STN1 - deficient U2OS cells . Representative SIRF images of MRE11 at normal or stalled replication forks are shown . Scale bars : 10 µm . ssDNA accumulation analysis in STN1 - deficient U2OS cells with and without mirin treatment . Representative images of native BrdU staining are shown .

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

Figure 3SIRF detection of MRE11 at stalled forks in STN1-deficient U2OS cells. Representative SIRF images of MRE11 at normal or stalled replication forks are shown. Scale bars: 10 µm.ssDNA accumulation analysis in STN1-deficient U2OS cells with and without mirin treatment. Representative images of native BrdU staining are shown.


Figure 4Coomassie blue stained SDS - PAGE gel ( 15 % ) of purified human MRE11 . ( i ) Scheme shows the nuclease activity of MRE11 in degrading 5 ' Cy3 - labeled substrates ( 25 nt + 60 nt with phosphorothioate bonds on both ends ) . ( ii ) The 5 ' Cy3 - labeled substrates ( 80 nM ) were incubated with the indicated concentration of MRE11 with or without the Mn2 + cofactor at 37 ° C for 20 min . Reactions were stopped by SDS and proteinase K and resolved on 27 % denatured polyacrylamide gel . ( iii ) The results are graphed , and error bars represent the standard deviation ( ± SD ) calculated from at least three independent experiments . Coomassie blue stained SDS - PAGE gel ( 15 % ) of purified human CST complex ( CTC1 wild - type and ∆ 700N ) . DNA binding activity of wild - type CST complex and Δ700N mutant complex . The 5 ' Cy3 - labeled substrates ( 80 nM ) were incubated with the indicated concentrations of CST . Samples were analyzed by 0 . 8 % agarose gel . MRE11 degradation analysis . 5 ' Cy3 - labeled substrates ( 80 nM ) were pre - incubated with indicated concentrations of CST wild - type ( i ) or ∆ 700N mutant variant ( ii ) at 37 ° C for 5 min . Reactions were completed by adding MRE11 ( 200 nM ) for an additional 20 min incubation and then stopped by SDS and proteinase K . Samples were resolved in 27 % denatured polyacrylamide gel . The results are graphed and error bars represent the standard deviation ( ± SD ) calculated from at least three independent experiments .

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

Figure 4Coomassie blue stained SDS-PAGE gel (15%) of purified human MRE11.(i) Scheme shows the nuclease activity of MRE11 in degrading 5' Cy3-labeled substrates (25 nt + 60 nt with phosphorothioate bonds on both ends). (ii) The 5' Cy3-labeled substrates (80 nM) were incubated with the indicated concentration of MRE11 with or without the Mn2+ cofactor at 37°C for 20 min. Reactions were stopped by SDS and proteinase K and resolved on 27% denatured polyacrylamide gel. (iii) The results are graphed, and error bars represent the standard deviation (± SD) calculated from at least three independent experiments.Coomassie blue stained SDS-PAGE gel (15%) of purified human CST complex (CTC1 wild- type and ∆700N).DNA binding activity of wild-type CST complex and Δ700N mutant complex. The 5' Cy3- labeled substrates (80 nM) were incubated with the indicated concentrations of CST. Samples were analyzed by 0.8 % agarose gel.MRE11 degradation analysis. 5' Cy3-labeled substrates (80 nM) were pre-incubated with indicated concentrations of CST wild-type (i) or ∆700N mutant variant (ii) at 37°C for 5 min. Reactions were completed by adding MRE11 (200 nM) for an additional 20 min incubation and then stopped by SDS and proteinase K. Samples were resolved in 27% denatured polyacrylamide gel. The results are graphed and error bars represent the standard deviation (± SD) calculated from at least three independent experiments.


Figure 5Deletion of the N - terminal 700 aa of CTC1 abolishes CTC1 localization at telomeres . U2OS stably expressing vector ( V ) , WT Myc - CTC1 and Myc - ∆ 700N were treated with or without HU and co - stained with Myc ( red ) and TRF2 ( green ) antibodies . Boxed areas are amplified in inserts to indicate CTC1 / TRF2 colocalization ( yellow ) . Scale bars : 10 μm . Quantification of percent of cells with > 3 CTC1 / TRF2 colocalization foci were from three independent experiments . In each experiment , > 150 cells from each sample were analyzed . ∆ 700N does not affect CST complex formation . HEK293T cells were co - transfected with Myc - CTC1 or Myc - ∆ 700N , His6 - STN1 and HA - TEN1 . Co - IP was performed with Myc antibody to pulldown His6 - STN1 and HA - TEN1 . Full - length Myc - CTC1 was prone to degradation during immunoprecipitation . ∆ 700N retains RAD51 interaction . HEK293T cells were co - transfected with Myc - CTC1 or Myc - ∆ 700N , His6 - STN1 , HA - TEN1 and Flag - RAD51 or vector control ( V ) , and treated with HU ( 2 mM , 16 h ) . Co - IP was performed with Flag antibody to pulldown Myc - CTC1 . DNA fiber analysis of CTC1 depleted U2OS cells with RNAi - resistant WT or ∆ 700N re - expression . Mean values in each sample are listed at the top of the graph . N , the number of cells analyzed in each condition , * * *

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

Figure 5Deletion of the N-terminal 700 aa of CTC1 abolishes CTC1 localization at telomeres. U2OS stably expressing vector (V), WT Myc-CTC1 and Myc-∆700N were treated with or without HU and co-stained with Myc (red) and TRF2 (green) antibodies. Boxed areas are amplified in inserts to indicate CTC1/TRF2 colocalization (yellow).Scale bars: 10 μm. Quantification of percent of cells with >3 CTC1/TRF2 colocalization foci were from three independent experiments. In each experiment, >150 cells from each sample were analyzed.   ∆700N does not affect CST complex formation. HEK293T cells were co-transfected with Myc- CTC1 or Myc-∆700N, His6-STN1 and HA-TEN1. Co-IP was performed with Myc antibody to pulldown His6- STN1 and HA-TEN1. Full-length Myc-CTC1 was prone to degradation during immunoprecipitation.      ∆700N retains RAD51 interaction. HEK293T cells were co-transfected with Myc-CTC1 or Myc-∆700N, His6- STN1, HA-TEN1 and Flag-RAD51 or vector control (V), and treated with HU (2 mM, 16 h). Co-IP was performed with Flag antibody to pulldown Myc-CTC1.   DNA fiber analysis of CTC1 depleted U2OS cells with RNAi-resistant WT or ∆700N re-expression. Mean values in each sample are listed at the top of the graph. N, the number of cells analyzed in each condition, ***


Figure 6Micronuclei ( MN ) formation in BRCA2 - and STN1 - deficient cells . Co - depletion was achieved by transfecting siBRCA2 in U2OS cells expressing shSTN1 . Representative DAPI staining images are shown . Arrows point to MN . Scale bars : 20 µm . Average percentages of nuclei containing MN from three independent experiments are presented . In each experiment , > 300 nuclei were analyzed per sample . Anaphase bridges ( arrows ) in BRCA2 - and STN1 - deficient U2OS cells . Scale bar : 10 µm . Average percentages of anaphase bridges from three independent experiments are presented . γH2AX induced by BRCA2 knockdown and STN1 knockdown in U2OS cells . Nuclei containing ≥ 5 foci were considered as positive γ - H2AX staining . Results were from three independent knockdown experiments . In each experiment , > 80 cells were analyzed per sample . Co - depletion of STN1 and BRCA2 significantly impairs DNA replication . Scale bar : 50 µm . Results were from three independent knockdown experiments . In each experiment , > 180 nuclei were analyzed per sample . Co - depletion of STN1 and BRCA2 increases chromosome instabilities . U2OS cells with siBRCA2 and / or shSTN1 knockdown were treated with HU ( 2 mM , 3 h ) . Representative metaphase images show aberrant chromosomes ( red arrows ) . Boxed areas are amplified and shown at the bottom of images . Scale bars : 20 µm . Two independent knockdown and chromosome spread experiments were performed . N , the number of metaphase spreads analyzed in each sample .

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

Figure 6Micronuclei (MN) formation in BRCA2- and STN1-deficient cells. Co-depletion was achieved by transfecting siBRCA2 in U2OS cells expressing shSTN1. Representative DAPI staining images are shown. Arrows point to MN. Scale bars: 20 µm. Average percentages of nuclei containing MN from three independent experiments are presented. In each experiment, >300 nuclei were analyzed per sample.Anaphase bridges (arrows) in BRCA2- and STN1-deficient U2OS cells. Scale bar: 10 µm. Average percentages of anaphase bridges from three independent experiments are presented.   γH2AX induced by BRCA2 knockdown and STN1 knockdown in U2OS cells. Nuclei containing ≥ 5 foci were considered as positive γ-H2AX staining. Results were from three independent knockdown experiments. In each experiment, >80 cells were analyzed per sample.   Co-depletion of STN1 and BRCA2 significantly impairs DNA replication. Scale bar: 50 µm. Results were from three independent knockdown experiments. In each experiment, >180 nuclei were analyzed per sample.Co-depletion of STN1 and BRCA2 increases chromosome instabilities. U2OS cells with siBRCA2 and/or shSTN1 knockdown were treated with HU (2 mM, 3 h). Representative metaphase images show aberrant chromosomes (red arrows). Boxed areas are amplified and shown at the bottom of images. Scale bars: 20 µm. Two independent knockdown and chromosome spread experiments were performed. N, the number of metaphase spreads analyzed in each sample.