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

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

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


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

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

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


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

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


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

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


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

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

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


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

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

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


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

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


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

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


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

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


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

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


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

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

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


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

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


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

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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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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.


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


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

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


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

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


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

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


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

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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


endogenous IRF2BP2 was immunoprecipitated by VGLL4 - FLAG in HEK293T

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


Correlation between expression of VGLL4 IRF2BP2 in 6 murine with indicated

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


Correlation between expression of VGLL4 IRF2BP2 i human with indicated

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


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

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


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

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


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

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


qRT - PCR of IRF2BP2 mRNA in control siVGLL4 A549

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


Suppression of IFNγ - inducible PD - L1 expression by YAP5SA is compromised in miR - 130a KO A549 . PD - L1 expression were analyzed by qRT - PCR in IFNγ - treated control or miR - 130a KO A549 pre - transduced with lenti - YAP5SA

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Suppression 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


Representative immunohistochemical for VGLL4 IRF2BP2 in human non - small lung cancer tissues . score is indicated . Scale bar : 50

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Representative immunohistochemical staining for VGLL4 and IRF2BP2 in human non-small cell lung cancer tissues. IHC score is indicated. Scale bar: 50 μm


of VGLL4 IRF2BP2 on human non - small lung cancer tissue arrays containing 71 samples was quantified as described in Materials Methods section . Correlation of VGLL4 IRF2BP2 expressions was significant ( Pearson correlation ; R = 0 . 6341 , P < 0 . 001 ) Correlation of VGLL4 PD - L1 expression on human non - small lung cancer tissue arrays containing 71

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Staining 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 samples


Kaplan - Meier survival of with high ( n = 74 ) or low ( n = 130 ) VGLL4 mRNA from GSE31210 , a lung cancer data set . Survival curves are calculated according to Kaplan - Meier

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Kaplan-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


B , C . Detection of CST at stalled active forks with SIRF in U2OS ( B ) HCT116 ( C ) . To detect CST at stalled forks , were - 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 of CTC1 , STN1 PCNA on normal or stalled forks are shown . Scale bars : 10 µm . Relative SIRF was calculated by normalizing to untreated samples then to their respective EdU - SIRF signals . Mean value in each sample is labeled in graph . N : number of analyzed in each sample .

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B, 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.


Western blot showing STN1 knockdown . shLUC ( siRNA targeting luciferase ) was used as control .

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Western blot showing STN1 knockdown. shLUC (siRNA targeting luciferase) was used as the control.


CST deficiency causes ssDNA accumulation upon fork stalling . HeLa with STN1 knockdown were incubated with 10 µM BrdU for 48 h , followed by 2 mM HU treatment for 3 h , subsequently stained with BrdU antibody under a non - denaturing condition to detect ssDNA . N , number of analyzed in each condition . Relative BrdU was quantitated by Image J .

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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.


fiber of U2OS with STN1 knockdown its RNAi - resistant Flag - STN1 WT re - expression with without mirin treatment .

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DNA fiber analysis of U2OS cells with STN1 knockdown and its RNAi-resistant Flag-STN1 WT re-expression with and without mirin treatment.


fiber of BJ with STN1 knockdown by two siRNA with without mirin treatment .

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DNA fiber analysis of BJ cells with STN1 knockdown by two siRNA sequence with and without mirin treatment.


fiber of HCT116 with STN1 knockdown with without mirin treatment .

DNA fiber analysis of HCT116 cells with STN1 knockdown with and without mirin treatment.


fiber of U2OS showing that SMARCAL1 - mediated fork reversal is needed for nascent - stand degradation observed in CST - deficient .

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DNA fiber analysis of U2OS cells showing that SMARCAL1-mediated fork reversal is needed for nascent-stand degradation observed in CST-deficient cells.


fiber of U2OS showing that ZRANB3 - mediated fork reversal is needed for nascent - strand degradation observed in CST - deficient .

DNA fiber analysis of U2OS cells showing that ZRANB3-mediated fork reversal is needed for nascent-strand degradation observed in CST-deficient cells.


SIRF detection of MRE11 at stalled forks in STN1 - deficient U2OS . Representative SIRF of MRE11 at normal or stalled replication forks are shown . Scale bars : 10 µm .

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SIRF 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 in STN1 - deficient U2OS with without mirin treatment . Representative of native BrdU are shown .

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ssDNA accumulation analysis in STN1-deficient U2OS cells with and without mirin treatment. Representative images of native BrdU staining are shown.


Coomassie blue stained SDS - PAGE gel ( 15 % ) of purified human MRE11 .

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Coomassie blue stained SDS-PAGE gel (15%) of purified human MRE11.


( i ) Scheme shows nuclease activity of MRE11 in degrading 5 ' Cy3 - labeled substrates ( 25 nt + 60 nt with phosphorothioate bonds on both ends ) . ( ii ) 5 ' Cy3 - labeled substrates ( 80 nM ) were incubated with indicated of MRE11 with or without Mn2 + cofactor at 37 ° C for 20 min . Reactions were stopped by SDS proteinase K resolved on 27 % denatured polyacrylamide gel . ( iii ) results are graphed , error bars represent standard deviation ( ± SD ) calculated from at least three independent experiments .

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(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 ( CTC1 wild - type ∆ 700N ) .

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Coomassie blue stained SDS-PAGE gel (15%) of purified human CST complex (CTC1 wild- type and ∆700N).


binding activity of wild - type CST Δ700N mutant . 5 ' Cy3 - labeled substrates ( 80 nM ) were incubated with indicated concentrations of CST . Samples were analyzed by 0 . 8 % agarose gel .

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 . 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 then stopped by SDS proteinase K . Samples were resolved in 27 % denatured polyacrylamide gel . results are graphed error bars represent standard deviation ( ± SD ) calculated from at least three independent experiments .

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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.


Deletion of N - terminal 700 aa of CTC1 abolishes CTC1 localization at telomeres . U2OS stably expressing vector ( V ) , WT Myc - CTC1 Myc - ∆ 700N were treated with or without HU co - stained with Myc ( red ) TRF2 ( green ) antibodies . Boxed areas are amplified in inserts to indicate CTC1 / TRF2 colocalization ( yellow ) . Scale bars : 10 μm . of of with > 3 CTC1 / TRF2 colocalization foci were from three independent experiments . In each experiment , > 150 from each sample were analyzed .

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Deletion 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 formation . HEK293T were co - transfected with Myc - CTC1 or Myc - ∆ 700N , His6 - STN1 HA - TEN1 . Co - IP was performed with Myc antibody to pulldown His6 - STN1 HA - TEN1 . Full - Myc - CTC1 was prone to degradation during immunoprecipitation .

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∆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 were co - transfected with Myc - CTC1 or Myc - ∆ 700N , His6 - STN1 , HA - TEN1 Flag - RAD51 or vector control ( V ) , treated with HU ( 2 mM , 16 h ) . Co - IP was performed with Flag antibody to pulldown Myc - CTC1 .

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∆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.


fiber of CTC1 depleted U2OS with RNAi - resistant WT or ∆ 700N re - expression . Mean values in each sample are listed at top of graph . N , number of analyzed in each condition , * * *

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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, ***


Micronuclei ( MN ) formation in BRCA2 - STN1 - deficient . Co - depletion was achieved by transfecting siBRCA2 in U2OS expressing shSTN1 . Representative DAPI 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 .

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Micronuclei (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 - STN1 - deficient U2OS . Scale bar : 10 µm . Average percentages of anaphase bridges from three independent experiments are presented .

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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 STN1 knockdown in U2OS . Nuclei containing ≥ 5 foci were considered as positive γ - H2AX . Results were from three independent knockdown experiments . In each experiment , > 80 were analyzed per sample .

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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 BRCA2 significantly impairs replication . Scale bar : 50 µm . Results were from three independent knockdown experiments . In each experiment , > 180 nuclei were analyzed per sample .

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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 BRCA2 increases chromosome instabilities . U2OS with siBRCA2 / or shSTN1 knockdown were treated with HU ( 2 mM , 3 h ) . Representative metaphase show aberrant chromosomes ( red arrows ) . Boxed areas are amplified shown at bottom of . Scale bars : 20 µm . Two independent knockdown chromosome spread experiments were performed . N , number of metaphase spreads analyzed in each sample .

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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.


SIRF of RAD51 binding to forks . Representative RAD51 SIRF at normal or stalled forks are shown . Scale bars : 10 µm . Two independent treatments were performed scatter plot of RAD51 - bound replication sites from one experiment is shown . Mean values in each sample are listed at top of graph . N , number of analyzed in each sample in one experiment .

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SIRF analysis of RAD51 binding to forks. Representative RAD51 SIRF images at normal or stalled forks are shown. Scale bars: 10 µm. Two independent treatments were performed and scatter plot of RAD51-bound replication sites from one experiment is shown. Mean values in each sample are listed at the top of the graph. N, number of cells analyzed in each sample in one experiment.


RAD51 foci formation in U2OS CTC1 knockdown was rescued by RNAi - resistant WT but not by ∆ 700N . were treated with HU ( 2 mM , 16 h ) stained with anti - RAD51 . Representative are shown . Scale bars : 10 µm . Scatter plot of RAD51 foci number with without HU treatment is shown . Mean values in each condition are listed at top of graph .

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RAD51 foci formation in U2OS CTC1 knockdown cells was rescued by RNAi-resistant WT but not by ∆700N. Cells were treated with HU (2 mM, 16 h) and stained with anti-RAD51. Representative images are shown. Scale bars: 10 µm. Scatter plot of RAD51 foci number with and without HU treatment is shown. Mean values in each condition are listed at the top of the graph.


fiber of nascent - strand degradation in U2OS with STN1 RAD51 co - depletion . Three independent treatments were performed results from one biological replicate are shown . N , number of analyzed in each sample in one experiment . Western blots showing knockdown in fiber assays are included .

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DNA fiber analysis of nascent-strand DNA degradation in U2OS cells with STN1 and RAD51 co-depletion. Three independent treatments were performed and results from one biological replicate are shown. N, the number of DNA fibers analyzed in each sample in one experiment. Western blots showing protein knockdown in DNA fiber assays are included.


F Whole - mount visualization of mitochondria in injured hypoglossal motor axon at 5 days after injury . G Immunostaining of injured hypoglossal nerves for GFP cytochrome c ( Cytc ) at 5 days after injury . H Number of mitochondria in injured axons of Atf3 : BAC2 Tg Rpt3 CKO mice at 5 days after injury ( n = 5 mice per group ) . I Total number of GFP - positive injured motor neurons in Atf3 : BAC2 Tg Rpt3 CKO mice at 5 days after injury ( n = 4 mice per group ) . J Number of GFP - positive mitochondria in soma at 5 days after injury . Data are shown as mean ± s . e . m . ( n = 3 mice ) . Data information : Data are shown as mean ± s . e . m . , * * P = 0 . 0005 in ( H ) , no significant difference ( P = 0 . 8915 ) in ( I ) * P = 0 . 0068 in ( J ) , determined by unpaired t - . Dashed lines reveal hypoglossal nucleus in axon in ( F G ) . Scale bars , 5 μm in ( F ) 2 . 5 μm in ( G ) .

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F Whole-mount visualization of mitochondria in injured hypoglossal motor axon at 5 days after injury. G Immunostaining of injured hypoglossal nerves for GFP and cytochrome c (Cytc) at 5 days after injury. H Number of mitochondria in injured axons of Atf3:BAC2 Tg and Rpt3 CKO mice at 5 days after injury (n= 5 mice per group). I Total number of GFP-positive injured motor neurons in Atf3:BAC2 Tg and Rpt3 CKO mice at 5 days after injury (n= 4 mice per group). J Number of GFP-positive mitochondria in soma at 5 days after injury. Data are shown as the mean ± s.e.m. (n= 3 mice). Data information: Data are shown as the mean ± s.e.m., ** P = 0.0005 in (H), no significant difference (P = 0.8915) in (I) and * P = 0.0068 in (J), determined by unpaired t-test. Dashed lines reveal hypoglossal nucleus in axon in (F and G). Scale bars, 5 μm in (F) and 2.5 μm in (G).


C Thionine - stained motor neurons of Atf3 : BAC2 Tg Rpt3 CKO mice at 7 , 14 17 days after hypoglossal nerve axotomy . Arrows indicate loss of motor neurons . D graph showing of surviving motor neurons on injured side compared with that on control side . Data are shown as mean ± s . e . m . ( * * P = 0 . 0002 , * * * P < 0 . 0001 , determined by unpaired t - , n = 5 mice per group at each time point ) . Data information : Dashed lines outline hypoglossal nucleus in XII , hypoglossal nucleus ; cc , central canal . Scale bars , 150 μm in C )

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C Thionine-stained motor neurons of Atf3:BAC2 Tg and Rpt3 CKO mice at 7, 14 and 17 days after hypoglossal nerve axotomy. Arrows indicate the loss of motor neurons. D The graph showing the percentage of surviving motor neurons on the injured side compared with that on the control side. Data are shown as the mean ± s.e.m. (** P = 0.0002, *** P < 0.0001, determined by unpaired t-test, n = 5 mice per group at each time point). Data information: Dashed lines outline hypoglossal nucleus in XII, hypoglossal nucleus; cc, central canal. Scale bars, 150 μm in C)


C Hypoglossal motor neurons of Atf3 : BAC2 Tg Rpt3 CKO mice before at 5 days ( 5d ) 28 days ( 28d ) after injury , immunostained by AnkG GFP . Arrows indicate AIS stained by AnkG . Scale bars , 20 μm . D of number of AIS in control ( Cont ) injured ( Inj ) hypoglossal motor neurons of Atf3 : BAC2 Rpt3 CKO mice . Data are expressed as mean ± s . e . m . * P = 0 . 043 , * * P = 0 . 0162 , * * * P = 0 . 008 , * * * * P < 0 . 0001 , determined by one - way ANOVA followed by Tukey post hoc , n . d . , not detected , n = 5 mice for each group . E of AIS in control ( Cont ) injured ( Inj ) hypoglossal motor neurons of Atf3 : BAC2 Rpt3 CKO mice . Data are expressed as mean ± s . e . m . * P = 0 . 0087 , * * P = 0 . 0024 , * * * P = 0 . 0007 , determined by one - way ANOVA followed by Tukey post hoc , n . d . , not detected , n = 5 mice for each group .

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C Hypoglossal motor neurons of Atf3:BAC2 Tg and Rpt3 CKO mice before and at 5 days (5d) and 28 days (28d) after injury, immunostained by AnkG and GFP. Arrows indicate the AIS stained by AnkG. Scale bars, 20 μm. D Quantification of the number of the AIS in control (Cont) and injured (Inj) hypoglossal motor neurons of Atf3:BAC2 and Rpt3 CKO mice. Data are expressed as mean ± s.e.m. * P = 0.043, ** P = 0.0162, *** P = 0.008, **** P < 0.0001, determined by one-way ANOVA followed by Tukey post hoc analysis, n.d., not detected, n = 5 mice for each group. E Quantification of the AIS length in control (Cont) and injured (Inj) hypoglossal motor neurons of Atf3:BAC2 and Rpt3 CKO mice. Data are expressed as mean ± s.e.m. * P = 0.0087, ** P = 0.0024, *** P = 0.0007, determined by one-way ANOVA followed by Tukey post hoc analysis, n.d., not detected, n = 5 mice for each group.


B Ubiquitination of AnkG . lysates of COS - 7 expressing indicated truncated AnkG tagged with GST HA - Ubiquitin were pulled down with glutathione Sepharose beads , followed by western blotting with anti - HA anti - GST antibodies .

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B Ubiquitination assay of AnkG protein. The lysates of COS-7 cells expressing the indicated truncated AnkG tagged with GST and HA-Ubiquitin were pulled down with glutathione Sepharose beads, followed by western blotting with anti-HA and anti-GST antibodies.


C Western blot of hypoglossal nuclei ( left ) nerves ( right ) at 5 days after injury with anti - AnkG , RPT3 GAPDH antibodies . AnkG products of 190 / 270 / 480 kDa are indicated by arrowheads . positions of degraded products by calpain are shown by asterisks at ~ 95 kDa 72 kDa .

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C Western blot of hypoglossal nuclei (left) and nerves (right) at 5 days after injury with anti-AnkG, RPT3 and GAPDH antibodies. AnkG products of 190/ 270/ 480 kDa are indicated by arrowheads. The positions of the degraded products by calpain are shown by asterisks at ~95 kDa and 72 kDa.


C localization of AnkG GFP - labeled mitochondria in AIS of uninjured MN of Thy1 - Mito Tg mice injured motor neurons of Atf3 : BAC2 Tg Rpt3 CKO mice . D Graphs show GFP AnkG scans corresponding to ( C ) over a 20 μm running from soma to axon . E Graph represents ( % ) of GFP in AIS , compared with that in AIS of Thy1 - Mito mouse ( n = 5 mice per group ) . Data information : For ( E , data are shown as mean ± s . e . m . , * * P = 0 . 0003 , * * * P < 0 . 0001 in ( E determined by one - way ANOVA followed by Tukey post - hoc , n . d . , not detected . In closed arrowheads denote presence of AIS , while open arrowheads show disappeared AIS . Scale bars , 5 μm in C ,

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C The localization of AnkG and GFP-labeled mitochondria in the AIS of uninjured MN of Thy1-Mito Tg mice and injured motor neurons of Atf3:BAC2 Tg and Rpt3 CKO mice. D Graphs show the GFP and AnkG fluorescent intensity scans corresponding to (C) over a 20 μm line running from the soma to the axon. E Graph represents the percentage (%) of GFP intensity in the AIS, compared with that in the AIS of Thy1-Mito mouse (n = 5 mice per group). Data information: For (E, data are shown as the mean ± s.e.m., ** P = 0.0003, *** P < 0.0001 in (E determined by one-way ANOVA followed by Tukey post-hoc test, n.d., not detected. In closed arrowheads denote the presence of the AIS, while open arrowheads show disappeared AIS. Scale bars, 5 μm in C,


F Whole - mount observation of GFP - or CFP - labeled mitochondria ( mtGFP or mtCFP ) in proximal region of peripheral hypoglossal ( XII ) nerve as shown in ( A ) . Dashed lines outline axon . G Graph shows mitochondrial number in hypoglossal ( XII ) axon ( n = 5 mice ) . Data information : For G , data are shown as mean ± s . e . m . , * * P = 0 . 0003 , * * * P < 0 . 0001 in G ) determined by one - way ANOVA followed by Tukey post - hoc , n . d . , not detected . Scale bars , 5 μm

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F Whole-mount observation of GFP- or CFP-labeled mitochondria (mtGFP or mtCFP) in the proximal region of peripheral hypoglossal (XII) nerve as shown in (A). Dashed lines outline the axon. G Graph shows mitochondrial number in hypoglossal (XII) axon (n = 5 mice). Data information: For G, data are shown as the mean ± s.e.m., ** P = 0.0003, *** P < 0.0001 in G) determined by one-way ANOVA followed by Tukey post-hoc test, n.d., not detected. Scale bars, 5 μm in


I Altered localization of GFP - labeled mitochondria in AIS of injured motor neurons of AnkG shRNA AAV - infected Rpt3 CKO mice at 5 days after injury . J scans of GFP AnkG corresponding to ( I ) . K Graph represents ( % ) of GFP in AIS , compared with that in AIS of Atf3 : BAC2 Tg mouse treated with AnkG shRNA ( n = 5 mice per group ) . Data information : For data are shown as mean ± s . e . m . , * * P = 0 . 0064 in ( K ) determined by one - way ANOVA followed by Tukey post - hoc , n . d . , not detected . In I ) , closed arrowheads denote presence of AIS , while open arrowheads show disappeared AIS . Scale bars , 5 μm in I )

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I Altered localization of the GFP-labeled mitochondria in the AIS of injured motor neurons of AnkG shRNA AAV-infected Rpt3 CKO mice at 5 days after injury. J Fluorescent intensity scans of GFP and AnkG corresponding to (I). K Graph represents the percentage (%) of GFP intensity in the AIS, compared with that in the AIS of Atf3:BAC2 Tg mouse treated with AnkG shRNA (n = 5 mice per group). Data information: For data are shown as the mean ± s.e.m. , ** P = 0.0064 in (K) determined by one-way ANOVA followed by Tukey post-hoc test, n.d., not detected. In I), closed arrowheads denote the presence of the AIS, while open arrowheads show disappeared AIS. Scale bars, 5 μm in I)


L Thionine - stained hypoglossal motor neurons of Rpt3 CKO mice at 10 days after injury . Rpt3 CKO mice are infected by AnkG shRNA or scramble shRNA AAVs . Dashed lines outline hypoglossal nucleus . XII , hypoglossal nucleus ; cc , central canal . M of surviving motor neurons on injured side compared with that on control side in Rpt3 CKO mice after infection of AnkG shRNA or scramble shRNA AAVs ( n = 4 mice per group ) . Data information : For M ) , data are shown as mean ± s . e . m . , * P = 0 . 0405 , * * P = 0 . 003 in ( M ) , determined by one - way ANOVA followed by Tukey post - hoc , n . d . , not detected . Scale bars , 100 μm in ( L ) .

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

L Thionine-stained hypoglossal motor neurons of Rpt3 CKO mice at 10 days after injury. Rpt3 CKO mice are infected by AnkG shRNA or scramble shRNA AAVs. Dashed lines outline hypoglossal nucleus. XII, hypoglossal nucleus; cc, central canal. M The percentage of surviving motor neurons on the injured side compared with that on the control side in Rpt3 CKO mice after infection of AnkG shRNA or scramble shRNA AAVs (n= 4 mice per group). Data information: For M), data are shown as the mean ± s.e.m. , * P = 0.0405, ** P = 0.003 in (M), determined by one-way ANOVA followed by Tukey post-hoc test, n.d., not detected. Scale bars, 100 μm in (L).


B Whole - mount visualization of GFP - or CFP - labeled mitochondria ( mtGFP or mtCFP ) in sciatic nerve . Dashed lines indicate boundary of axon . Scale bar , 5 μm . C Representative kymographs showing behavior of mtCFP in uninjured sciatic nerves of Thy1 - Mito Tg mtGFP in injured sciatic nerves of Atf3 ; BAC2 Rpt3 CKO mice .

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B Whole-mount visualization of GFP- or CFP-labeled mitochondria (mtGFP or mtCFP) in sciatic nerve. Dashed lines indicate the boundary of axon. Scale bar, 5 μm. C Representative kymographs showing the behavior of mtCFP in uninjured sciatic nerves of Thy1-Mito Tg and mtGFP in injured sciatic nerves of Atf3;BAC2 and Rpt3 CKO mice.


D - F number of mitochondria ( D ) , of motile mitochondria ( E ) stationary mitochondrial site ( F ) in axons . Data are shown as mean ± s . e . m . * * * P = 0 . 0001 , * * * * P < 0 . 0001 in ( D ) , * * P = 0 . 0006 , * * * P = 0 . 0001 , * * * * P < 0 . 0001 in ( E ) , determined by one - way ANOVA followed by Tukey post - hoc , n . d . , not detected , n = 5 mice for each group .

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D-F The number of mitochondria (D), percentage of motile mitochondria (E) and stationary mitochondrial site (F) in axons. Data are shown as the mean ± s.e.m. *** P = 0.0001, **** P < 0.0001 in (D), ** P = 0.0006, *** P = 0.0001, **** P < 0.0001 in (E), determined by one-way ANOVA followed by Tukey post-hoc test, n.d., not detected, n = 5 mice for each group.