Twelve - week - old wild type mice were infected with an adenovirus harboring GFP or FERM for 3 days , fasted for 4 hours intraperitoneally injected with glucagon . ( G ) Blood glucose ( mg / dL ) were measured 0 , 15 , 30 , 60 120 minutes after glucagon stimulation ( 0 . 2 mg / kg , n = 10 mice / group ) .

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Twelve-week-old male wild type mice were infected with an adenovirus harboring GFP or FERM for 3 days, fasted for 4 hours and intraperitoneally injected with glucagon. (G) Blood glucose levels (mg/dL) were measured 0, 15, 30, 60 and 120 minutes after glucagon stimulation (0.2 mg/kg body weight, n = 10 mice/ group).


Twelve - week - old wild type mice were infected with an adenovirus harboring GFP or FERM for 3 days , fasted for 4 hours intraperitoneally injected with glucagon . ( H ) of p - CREB , CREB , p - PKA substrates , FERM in liver 10 minutes after glucagon stimulation ( 2 mg / kg ) were determined by immunoblotting ( n = 3 mice / group ) .

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Twelve-week-old male wild type mice were infected with an adenovirus harboring GFP or FERM for 3 days, fasted for 4 hours and intraperitoneally injected with glucagon. (H) Levels of p-CREB, CREB, p-PKA substrates, and FERM in the liver 10 minutes after glucagon stimulation (2 mg/kg body weight) were determined by immunoblotting (n = 3 mice/ group).


Twelve - week - old wild type mice were infected with an adenovirus harboring GFP or FERM for 3 days , fasted for 4 hours intraperitoneally injected with glucagon . ( I ) Glucose disposal rates in GFP - or FERM - overexpressing mice were measured using glucose , pyruvate insulin tolerance ( n = 10 mice / group ) .

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Twelve-week-old male wild type mice were infected with an adenovirus harboring GFP or FERM for 3 days, fasted for 4 hours and intraperitoneally injected with glucagon. (I) Glucose disposal rates in GFP- or FERM-overexpressing mice were measured using glucose, pyruvate and insulin tolerance tests (n = 10 mice/group).


( A ) Wild type hepatocytes were infected with an adenovirus harboring sh - control ( sh - Con ) , sh - radixin ( sh - Rdx ) , GFP ( Ad - GFP ) , / or sh - resistant wildtype radixin ( Ad - RdxR ) or AKAP - dead LPTD mutant of radixin ( Ad - LPTDR ) for 24 hours .

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(A) Wild type primary hepatocytes were infected with an adenovirus harboring sh-control (sh-Con), sh-radixin (sh-Rdx), GFP (Ad-GFP), and/or sh-resistant wildtype radixin (Ad-RdxR) or AKAP-dead LPTD mutant of radixin (Ad-LPTDR) for 24 hours.


( B ) Increasing amount of Ad - RdxR or Ad - LPTDR were infected to WT hepatocytes for 24 hours . Hepatocytes were further serum - starved for 18 hours stimulated with glucagon ( 10 nM ) for 10 minutes . of p - PKA substrates , p - CREB , CREB , radixin , Myc - radixin GAPDH were determined by immunoblotting .

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(B) Increasing amount of Ad-RdxR or Ad-LPTDR were infected to WT primary hepatocytes for 24 hours. Hepatocytes were further serum-starved for 18 hours and stimulated with glucagon (10 nM) for 10 minutes. Levels of p-PKA substrates, p-CREB, CREB, radixin, Myc-radixin and GAPDH were determined by immunoblotting.


Four - week - old WT ( Prom1 + / + ) KO ( Prom1 - / - ) mice were fed a high - fat diet for 8 ( Diet - Induced Obesity ; DIO ) , fasted for 4 hours before experiments . ( C ) DIO WT ( Prom1 + / + ) KO ( Prom1 - / - ) mice were intraperitoneally injected with glucagon . Blood glucose ( mg / dL ) were measured 0 , 15 , 30 , 60 120 minutes after glucagon ( 100 µg / kg ) stimulation ( n = 10 mice / group ) .

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Four-week-old male WT (Prom1+/+) and KO (Prom1-/-) mice were fed a high-fat diet for 8 weeks (Diet-Induced Obesity; DIO), and fasted for 4 hours before experiments. (C) DIO WT (Prom1+/+) and KO (Prom1-/-) mice were intraperitoneally injected with glucagon. Blood glucose levels (mg/dL) were measured 0, 15, 30, 60 and 120 minutes after glucagon (100 µg/kg body weight) stimulation (n = 10 mice/ group).


Four - week - old WT ( Prom1 + / + ) KO ( Prom1 - / - ) mice were fed a high - fat diet for 8 ( Diet - Induced Obesity ; DIO ) , fasted for 4 hours before experiments . ( D ) of p - PKA substrates , p - CREB , CREB , Prom1 GAPDH were determined by immunoblotting 10 minutes after glucagon ( 2 mg / kg ) stimulation ( n = 3 mice / group ) .

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Four-week-old male WT (Prom1+/+) and KO (Prom1-/-) mice were fed a high-fat diet for 8 weeks (Diet-Induced Obesity; DIO), and fasted for 4 hours before experiments. (D) Levels of p-PKA substrates, p-CREB, CREB, Prom1 and GAPDH were determined by immunoblotting 10 minutes after glucagon (2 mg/kg body weight) stimulation (n = 3 mice/group).


Four - week - old WT ( Prom1 + / + ) KO ( Prom1 - / - ) mice were fed a high - fat diet for 8 ( Diet - Induced Obesity ; DIO ) , fasted for 4 hours before experiments . ( E ) Glucose disposal rates in DIO WT ( Prom1 + / + ) KO ( Prom1 - / - ) mice were measured using glucose insulin tolerance ( n = 10 mice / group ) .

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

Four-week-old male WT (Prom1+/+) and KO (Prom1-/-) mice were fed a high-fat diet for 8 weeks (Diet-Induced Obesity; DIO), and fasted for 4 hours before experiments. (E) Glucose disposal rates in DIO WT (Prom1+/+) and KO (Prom1-/-) mice were measured using glucose and insulin tolerance tests (n = 10 mice/group).


Four - week - old WT ( Prom1 + / + ) KO ( Prom1 - / - ) mice were fed a high - fat diet for 8 ( Diet - Induced Obesity ; DIO ) , fasted for 4 hours before experiments . ( F ) DIO KO ( Prom1 - / - ) mice were infected with an adenovirus harboring LacZ or Prom1 for 24 hours , fasted for 4 hours intraperitoneally injected with glucagon ( 100 µg / kg ) . Blood glucose ( mg / dL ) were measured 0 , 15 , 30 , 60 120 minutes after glucagon stimulation ( GST ) . Glucose disposal rates were measured using pyruvate tolerance ( PTT ) ( n = 10 mice / group ) .

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

Four-week-old male WT (Prom1+/+) and KO (Prom1-/-) mice were fed a high-fat diet for 8 weeks (Diet-Induced Obesity; DIO), and fasted for 4 hours before experiments. (F) DIO KO (Prom1-/-) mice were infected with an adenovirus harboring LacZ or Prom1 for 24 hours, fasted for 4 hours and intraperitoneally injected with glucagon (100 µg/kg body weight). Blood glucose levels (mg/dL) were measured 0, 15, 30, 60 and 120 minutes after glucagon stimulation (GST). Glucose disposal rates were measured using the pyruvate tolerance test (PTT) (n = 10 mice/group).


( a ) ( top ) Experimental design . Cultured cortical neurons are stimulated with BDNF , KCl or no treatment ( Control ) activated neurons are prepared in three specific time points for - seq ATAC - seq at early ( 1h ) late time points ( 6 10h ) ; ( bottom ) Blot for pMAPK in neurons treated with KCl 55 mM several concentrations of BDNF ( Con = no stimulation ) . 44 42 KDa bands indicated with lines . Nucleolin shown as internal control .

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(a) (top) Experimental design. Cultured cortical neurons are stimulated with BDNF, KCl or no treatment (Control) and activated neurons are prepared in three specific time points for RNA-seq and ATAC-seq at early (1h) and late time points (6 and 10h); (bottom) Blot for pMAPK in neurons treated with KCl 55 mM and several concentrations of BDNF (Con = no stimulation). 44 and 42 KDa bands indicated with lines. Nucleolin shown as internal control.


( b ) Hierarchical clustering for differentially - expressed genes ( DE - genes , adjusted P - value < 0 . 1 , using Benjamini Hochberg ' s correction ) using correlation of log2 - fold changes when compared to unstimulated control samples at each time point .

O O O O O O O O O O O O O O O O O O O O O O O O O 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) Hierarchical clustering for all differentially-expressed genes (DE-genes, adjusted P-value < 0.1, using Benjamini Hochberg's correction) using correlation of log2-fold changes when compared to unstimulated control samples at each time point.


( d ) Partitioning around medoids clustering of expression dynamics using genewise - scaled Z - scores of top 5 , 000 DE - genes from ( c ) with lowest adjusted p - value ( k = 10 clusters ) . Numbers indicate clusters . Left plots indicate expression for signature genes selected for each cluster . 4 , 907 DE - genes are also selected with FDR = 1 % more than 100 across samples . ( e ) Gene Ontology enrichment for clusters shown in ( d ) . Each ribbon shows clusters with their respective significant gene groups using topGO ( Alexa et al , 2006 ) . Up to five significant terms per cluster are shown ( P - values adjusted with BH ) . Circle sizes indicate enrichment of ontology genes in each cluster versus other clusters . Names on right y - axis indicate ontology common names .

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

(d) Partitioning around medoids clustering of expression dynamics using genewise-scaled Z-scores of top 5,000 DE-genes from (c) with lowest adjusted p-value (k = 10 clusters). Numbers indicate clusters. Left line plots indicate expression levels for signature genes selected for each cluster. 4,907 DE-genes are also selected with FDR=1% and more than 100 counts across samples. (e) Gene Ontology enrichment analysis for clusters shown in (d). Each ribbon shows clusters with their respective significant gene groups using topGO(Alexa et al, 2006). Up to five significant terms per cluster are shown (P-values adjusted with BH). Circle sizes indicate enrichment of ontology genes in each cluster versus all other clusters. Names on the right y-axis indicate ontology common names.


( a ) Hierarchical clustering for differentially accessible peaks ( DA - peaks , adjusted P < 0 . 1 , using Benjamini Hochberg ' s correction ) using correlation of log2 - fold changes when compared versus matched control samples .

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(a) Hierarchical clustering for all differentially accessible peaks (DA-peaks, adjusted P < 0.1, using Benjamini Hochberg's correction) using correlation of log2-fold changes when compared versus matched control samples.


( d ) ( top ) Association between distal regulatory elements ( DREs ) gene expression at BDNF 1h time - point . Each point indicates log2 - fold change of an ATAC - seq peak ( x - axis ) gene expression of a closest gene ( y - axis ) with between 2 - 20 Kbp . Colors indicate whether none ( gray ) , only peak ( orange ) , or both peak gene ( red ) show significant changes versus control neurons . ( bottom ) Enrichment for paired DA - peak DE - gene in four quadrants are summarized for BDNF KCl . Asterisks indicate P values as corrected by a Benjamini Hochberg procedure ( * = P < 0 . 05 ; * * = P < 0 . 01 ) .

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(d) (top) Association between distal regulatory elements (DREs) and gene expression at the BDNF 1h time-point. Each point indicates the log2-fold change of an ATAC-seq peak (x-axis) and the gene expression of a closest gene (y-axis) with distance between 2-20 Kbp. Colors indicate whether none (gray), only the peak (orange), or both peak and gene (red) show significant changes versus control neurons. (bottom) Enrichment for paired DA-peak and DE-gene in the four quadrants are summarized for BDNF and KCl. Asterisks indicate P values as corrected by a Benjamini Hochberg procedure (* = P < 0.05; ** = P < 0.01).


( c ) expression changes for TFs related to 8 - mer groups bZIP , CTCF , HIC1 , EGR . Significant changes relative to control samples are shown with asterisks ( Wilcoxon rank sums , two - sided ) .

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(c) RNA expression changes for TFs related to 8-mer groups bZIP, CTCF, HIC1, and EGR. Significant changes relative to control samples are shown with asterisks (Wilcoxon rank sums test, two-sided).


( c ) Chromosome 15 genome tracks neighboring Arc , displaying ATAC - seq read per million ( CPM ) ; - seq CPM ; H3K27ac normalized signal upon KCl stimulation , H3K4me1 ( Malik et al , 2014 ) ; CTCF ( Sams et al , 2016 ) ; Cortical neurons Hi - C data ( Bonev et al , 2017 ) . Red bars in ATAC - seq tracks indicate gained DA - peaks in BDNF , red bars in - seq tracks indicate Arc differential expression in BDNF KCl 1h . Green blocks in Hi - C tracks indicate anchor points for calculation of contact scores , using shaman ( Cohen et al , 2017 ) . Spearman ' s rho value indicate rank - correlation between highlighted peaks across samples ( Appendix Figure S4c ) . TF module names indicate presence of 8 - mers in those peaks .

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(c) Chromosome 15 genome tracks neighboring Arc, displaying ATAC-seq read counts per million (CPM); RNA-seq CPM; H3K27ac normalized signal upon KCl stimulation, H3K4me1(Malik et al, 2014); CTCF(Sams et al, 2016); and Cortical neurons Hi-C data(Bonev et al, 2017). Red bars in ATAC-seq tracks indicate gained DA-peaks in BDNF, and red bars in RNA-seq tracks indicate Arc differential expression in BDNF and KCl 1h. Green blocks in Hi-C tracks indicate anchor points for the calculation of contact scores, using shaman(Cohen et al, 2017). Line and Spearman's rho value indicate counts rank-based correlation between highlighted peaks across all samples (Appendix Figure S4c). TF module names indicate the presence of 8-mers in those peaks.


( a ) Associations between accessibility DA - peaks GWAS summary statistics . DA - peaks ( DA ) , gained DA - peaks ( gained ) , closing DA - peaks , consensus peaks ( ) are fitted to summary statistics data of multiple GWAS studies ( y - axis ) . Circle color indicates LDSC regression coefficient ( effect ) , circle indicates association significance . Shadings indicate treatments . Time points are shown ( orange = 1h , brown = 6h ) . Red lines indicate LD score regression coefficient adjusted P < 0 . 1 after BH correction . Orange lines indicate P < 0 . 1 . ( BD = Bipolar Disorder ; SZ = Schizophrenia ; BD + SZ : BP SZ samples combined ; MDD = Major Depressive Disorder ; ADHD = Attention deficit hyperactivity disorder ; AD = Alzheimer ' s Disease ) .

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(a) Associations between chromatin accessibility DA-peaks and GWAS summary statistics. All DA-peaks (DA), gained DA-peaks (gained), closing DA-peaks, and consensus peaks (all) are fitted to summary statistics data of multiple GWAS studies (y-axis). Circle color indicates LDSC regression coefficient (effect size), and circle size indicates association significance. Shadings indicate treatments. Time points are shown (orange = 1h, brown = 6h). Red lines indicate LD score regression coefficient adjusted P < 0.1 after BH correction. Orange lines indicate P < 0.1. (BD = Bipolar Disorder; SZ = Schizophrenia; BD+SZ: BP and SZ samples combined; MDD = Major Depressive Disorder; ADHD = Attention deficit hyperactivity disorder; AD = Alzheimer's Disease).


( b ) Correlations between differentially accessible elements conserved mouse human regions across conditions ( N = 16 , 580 ) . Asterisks indicate significance of Pearson ' s R correlation after correction with BH - procedure .

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(b) Correlations between differentially accessible elements conserved mouse and human regions across conditions (N=16,580). Asterisks indicate significance of Pearson's R correlation after correction with BH-procedure.


( D ) Validation by western blot of changes in specific phosphorylation events on kinases . Left , Immunoblots of cortical lysates of CTL micTNFα - KO at ZT6 for : ( top ) phosphorylated GSK3α / β ( pGSK3α at Ser21 pGSK3β at Ser9 ) total GSK3α ; ( bottom ) phosphorylated MAPK ( pMAPK , MAPK3 at Thr203 / Tyr205 MAPK1 at Thr183 / Tyr185 ) total MAPK . Right , between phosphorylated total kinase normalized to CTL at ZT18 ZT6 . n = 5 mice per group . * * * p = 0 . 000009 ( GSK3α ) , multiple t - with Benjamini - Hochberg correction .

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(D) Validation by western blot of changes in specific phosphorylation events on kinases. Left, Immunoblots of cortical lysates of CTL and micTNFα-KO at ZT6 for: (top) phosphorylated GSK3α/β (pGSK3α at Ser21 and pGSK3β at Ser9) and total GSK3α; and (bottom) phosphorylated MAPK (pMAPK, MAPK3 at Thr203/Tyr205 and MAPK1 at Thr183/Tyr185) and total MAPK. Right, Ratio between phosphorylated and total kinase normalized to CTL at ZT18 and ZT6. n=5 mice per group. ***p=0.000009 (GSK3α), multiple t-test with Benjamini-Hochberg correction.


( E ) Kinases with altered activity between CTL micTNF - KOs predicted by robust kinase activity inference ( RoKAI ) at ZT18 ZT6 . Graph shows z - score value of each kinase assigned by RoKAI . Kinases above below dotted represent prediction of up - downregulated activity in micTNFα - KO , respectively . Kinases are color - coded according to kinase group as described in ( C ) .

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(E) Kinases with altered activity between CTL and micTNF-KOs predicted by robust kinase activity inference (RoKAI) at ZT18 and ZT6. Graph shows the z-score value of each kinase assigned by RoKAI. Kinases above and below the dotted line represent prediction of up- and downregulated activity in micTNFα-KO, respectively. Kinases are color-coded according to kinase group as described in (C).


( F Validation by western blot of altered activity of predicted kinases between CTL micTNF - KOs . ( F ) Left , Immunoblots of cortical lysates of CTL micTNFα - KO at ZT6 using antibodies specific to target phosphorylation motifs of MAPK / CDK ( PXS * P , S * PX ( K / R ) ) PKC ( ( K / R ) XS * X ( K / R ) ) . Dashed boxes indicate quantified signal . Right , between phosphorylated substrates loading control normalized to CTL at ZT18 ZT6 . ( F , n = 5 mice per group . * p = 0 . 022 ( MAPK / CDK phospho sub ) , * p = 0 . 014 ( PKC phospho sub ) multiple t - with Benjamini - Hochberg correction .

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(F Validation by western blot of altered activity of predicted kinases between CTL and micTNF-KOs. (F) Left, Immunoblots of cortical lysates of CTL and micTNFα-KO at ZT6 using antibodies specific to target phosphorylation motifs of MAPK/CDK (PXS*P, S*PX(K/R)) and PKC ((K/R)XS*X(K/R)). Dashed boxes indicate quantified signal. Right, Ratio between phosphorylated substrates and loading control normalized to CTL at ZT18 and ZT6. (F, n=5 mice per group. *p=0.022 (MAPK/CDK phospho sub), *p=0.014 (PKC phospho sub) multiple t-test with Benjamini-Hochberg correction.


G ) Validation by western blot of altered activity of predicted kinases between CTL micTNF - KOs . ( G ) Left , Immunoblots show signal of phosphorylated threonine in MARKs activation loop . Right , between phosphorylated MARK activation loop loading control normalized to CTL at ZT18 ZT6 . G ) n = 5 mice per group . * * p = 0 . 0091 ( MARK ) , multiple t - with Benjamini - Hochberg correction .

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G) Validation by western blot of altered activity of predicted kinases between CTL and micTNF-KOs. (G) Left, Immunoblots show signal of phosphorylated threonine in MARKs activation loop. Right, Ratio between phosphorylated MARK activation loop and loading control normalized to CTL at ZT18 and ZT6. G) n=5 mice per group. **p=0.0091 (MARK), multiple t-test with Benjamini-Hochberg correction.


A MOAP - 1 co - localizes with p62 in inclusion bodies upon proteasome inhibition . HCT116 were transfected with plasmid encoding GFP - MOAP - 1 or GFP only control . 14 hours later , were treated with proteasome inhibitor MG132 ( 5 µM ) for 8 hours analyzed by immunofluorescence ( IF ) with anti - GFP ( in green ) anti - p62 ( in red ) antibodies . Data information : , nuclei were counterstained with DAPI ( blue )

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A MOAP-1 co-localizes with p62 in the cytoplasmic inclusion bodies upon proteasome inhibition. HCT116 cells were transfected with plasmid encoding GFP-MOAP-1 or GFP only control. 14 hours later, cells were treated with the proteasome inhibitor MG132 (5 µM) for 8 hours and analyzed by immunofluorescence (IF) with anti-GFP (in green) and anti-p62 (in red) antibodies. Data information: , nuclei were counterstained with DAPI (blue)


B Endogenous MOAP - 1 resides at p62 bodies upon proteasome inhibition . HCT116 were treated with MG132 ( 5 µM ) for 8 hours analyzed by IF with anti - MOAP - 1 ( in green ) anti - p62 ( in red ) antibodies . Data information : nuclei were counterstained with DAPI ( blue )

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

B Endogenous MOAP-1 resides at the p62 bodies upon proteasome inhibition. HCT116 cells were treated with MG132 (5 µM) for 8 hours and analyzed by IF with anti-MOAP-1 (in green) and anti-p62 (in red) antibodies. Data information: nuclei were counterstained with DAPI (blue)


C MOAP - 1 is recruited to p62 bodies upon exposure to stresses . LO2 hepatocytes were transfected with plasmid encoding Myc - MOAP - 1 . 14 hours later , were then treated with MG132 ( 5 µM ) , arsenic trioxide ( As2O3 , 10 µM ) or diethylnitrosamine ( DEN , 200 µM ) for 8 hours each . were then processed for IF with anti - Myc ( in green ) anti - p62 ( in red ) antibodies . Data information : nuclei were counterstained with DAPI ( blue )

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

C MOAP-1 is recruited to the p62 bodies upon exposure to cellular stresses. LO2 hepatocytes were transfected with plasmid encoding Myc-MOAP-1. 14 hours later, cells were then treated with MG132 (5 µM), arsenic trioxide (As2O3, 10 µM) or diethylnitrosamine (DEN, 200 µM) for 8 hours each. Cells were then processed for IF analysis with anti-Myc (in green) and anti-p62 (in red) antibodies. Data information: nuclei were counterstained with DAPI (blue)


D MOAP - 1 spontaneously localizes to p62 bodies at resting state in liver cancer lines , HepG2 , Huh - 1 , JHH5 JHH7 . liver cancer lines were transfected with plasmid encoding Myc - MOAP - 1 . 14 hours later , transfected were subjected to IF with anti - Myc ( in green ) anti - p62 ( in red ) antibodies . Data information : nuclei were counterstained with DAPI ( blue )

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

D MOAP-1 spontaneously localizes to the p62 bodies at resting state in the liver cancer cell lines, HepG2, Huh-1, JHH5 and JHH7. The liver cancer cell lines were transfected with the plasmid encoding Myc-MOAP-1. 14 hours later, transfected cells were subjected to IF analysis with anti-Myc (in green) and anti-p62 (in red) antibodies. Data information: nuclei were counterstained with DAPI (blue)


E Absence of aggregated patterns of MOAP - 1 in p62 deficient . WT p62 KO HepG2 were transfected with plasmid encoding Myc - MOAP - 1 for 14 hours were subjected to IF as in ( D ) . Data information : , nuclei were counterstained with DAPI ( blue )

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

E Absence of the aggregated patterns of MOAP-1 in the p62 deficient cells. WT and p62 KO HepG2 cells were transfected with plasmid encoding Myc-MOAP-1 for 14 hours and the cells were subjected to IF analysis as in (D). Data information: , nuclei were counterstained with DAPI (blue)


F Western blotting of p62 Myc - MOAP - 1 in WT p62 KO HepG2 as described in ( E ) . Actin as loading control .

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

F Western blotting analysis of p62 and Myc-MOAP-1 protein levels in the WT and p62 KO HepG2 cells as described in (E). Actin as loading control.


A MOAP - 1 downregulates of diethylnitrosamine ( DEN ) - induced p62 bodies in a time - dependent manner . LO2 were transfected with plasmid encoding Myc - MOAP - 1 . 14 hours later , were treated with DEN ( 200 µM ) for indicated period of times , before being subjected to IF with anti - Myc ( in green ) anti - p62 ( in red ) antibodies . expressing Myc - MOAP - 1 were marked by dashed lines . Data information : nuclei were counterstained with DAPI ( blue )

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

A MOAP-1 downregulates the levels of the diethylnitrosamine (DEN)-induced p62 bodies in a time-dependent manner. LO2 cells were transfected with plasmid encoding Myc-MOAP-1. 14 hours later, cells were treated with DEN (200 µM) for the indicated period of times, before being subjected to IF with anti-Myc (in green) and anti-p62 (in red) antibodies. Cells expressing Myc-MOAP-1 were marked by dashed lines. Data information: nuclei were counterstained with DAPI (blue)


B MOAP - 1 downregulates p62 bodies in resting HepG2 liver cancer . HepG2 were transfected with plasmid encoding Myc - MOAP - 1 . At indicated time post - transfection , transfected were subjected to IF as in ( A ) . Data information : nuclei were counterstained with DAPI ( blue )

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B MOAP-1 downregulates the p62 bodies in the resting HepG2 liver cancer cells. HepG2 were transfected with plasmid encoding Myc-MOAP-1. At the indicated time post-transfection, transfected cells were subjected to IF as in (A). Data information: nuclei were counterstained with DAPI (blue)


C Loss of MOAP - 1 leads to spontaneous elevation of basal of p62 bodies in LO2 . WT , MOAP - 1 KO p62 KO LO2 were subjected to IF with anti - p62 antibody ( in red ) . p62 KO were included as a negative control . Insets in upper panels are enlarged in lower panels . D of p62 bodies in WT MOAP - 1 KO LO2 . ( Left panel ) number of p62 bodies per . n = 7 independently plated samples . ( Right panel ) of p62 bodies . n = 442 1014 bodies detected in WT MOAP - 1 KO LO2 , respectively , from three independently plated samples . Data information : nuclei were counterstained with DAPI ( blue )

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

C Loss of MOAP-1 leads to spontaneous elevation of the basal levels of p62 bodies in the LO2 cells. WT, MOAP-1 KO and p62 KO LO2 cells were subjected to IF with anti-p62 antibody (in red). p62 KO cells were included as a negative control. Insets in the upper panels are enlarged in the lower panels. D Quantification of the p62 bodies in the WT and MOAP-1 KO LO2 cells. (Left panel) number of p62 bodies per cell. n = 7 independently plated samples. (Right panel) size of p62 bodies. n = 442 and 1014 bodies detected in the WT and MOAP-1 KO LO2 cells, respectively, from three independently plated samples. Data information: nuclei were counterstained with DAPI (blue)


C Loss of MOAP - 1 leads to spontaneous elevation of basal of p62 bodies in LO2 . WT , MOAP - 1 KO p62 KO LO2 were subjected to IF with anti - p62 antibody ( in red ) . p62 KO were included as a negative control . Insets in upper panels are enlarged in lower panels .

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

C Loss of MOAP-1 leads to spontaneous elevation of the basal levels of p62 bodies in the LO2 cells. WT, MOAP-1 KO and p62 KO LO2 cells were subjected to IF with anti-p62 antibody (in red). p62 KO cells were included as a negative control. Insets in the upper panels are enlarged in the lower panels.


E Re - expression of MOAP - 1 reduces of p62 bodies in MOAP - 1 deficient LO2 . MOAP - 1 KO LO2 were transfected with plasmid encoding Myc - MOAP - 1 for 24 hours subjected to IF with anti - p62 ( in red ) anti - Myc ( green ) antibodies . expressing Myc - MOAP - 1 were marked by dashed lines . F of p62 bodies in MOAP - 1 KO LO2 re - introduced with Myc - MOAP - 1 . ( Left panel ) number of p62 bodies per . n = 7 independently plated samples of MOAP - 1 KO transfected with empty vector control ( Ctrl ) or plasmid encoding Myc - MOAP - 1 . ( Right panel ) of p62 bodies . n = 178 69 bodies detected in Ctrl Myc - MOAP - 1 expressing , respectively , from three independently plated samples . Data information : , nuclei were counterstained with DAPI ( blue )

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

E Re-expression of MOAP-1 reduces levels of p62 bodies in the MOAP-1 deficient LO2 cells. MOAP-1 KO LO2 cells were transfected with plasmid encoding Myc-MOAP-1 for 24 hours and subjected to IF with anti-p62 (in red) and anti-Myc (green) antibodies. Cells expressing Myc-MOAP-1 were marked by dashed lines. F Quantification of the p62 bodies in the MOAP-1 KO LO2 cells re-introduced with Myc-MOAP-1. (Left panel) number of p62 bodies per cell. n = 7 independently plated samples of MOAP-1 KO cells transfected with empty vector control (Ctrl) or plasmid encoding Myc-MOAP-1. (Right panel) size of p62 bodies. n = 178 and 69 bodies detected in the Ctrl and Myc-MOAP-1 expressing cells, respectively, from three independently plated samples. Data information: , nuclei were counterstained with DAPI (blue)


E Re - expression of MOAP - 1 reduces of p62 bodies in MOAP - 1 deficient LO2 . MOAP - 1 KO LO2 were transfected with plasmid encoding Myc - MOAP - 1 for 24 hours subjected to IF with anti - p62 ( in red ) anti - Myc ( green ) antibodies . expressing Myc - MOAP - 1 were marked by dashed lines .

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

E Re-expression of MOAP-1 reduces levels of p62 bodies in the MOAP-1 deficient LO2 cells. MOAP-1 KO LO2 cells were transfected with plasmid encoding Myc-MOAP-1 for 24 hours and subjected to IF with anti-p62 (in red) and anti-Myc (green) antibodies. Cells expressing Myc-MOAP-1 were marked by dashed lines.


G Increased level of p62 bodies in MOAP - 1 KO MEFs upon treatment with arsenic trioxide . WT MOAP - 1 KO MEFs were treated with arsenic trioxide ( As2O3 , 10 µM ) for indicated durations subjected to IF with anti - p62 antibody ( in red ) . H of p62 bodies in WT MOAP - 1 KO MEFs treated with arsenic trioxide . ( Left panel ) number of p62 bodies per . n ≥ 6 independently plated samples . ( Right panel ) of p62 bodies . n = 10 ( WT , basal ) , 17 ( MOAP - 1 KO , basal ) , 104 ( WT , As2O3 2h ) , 137 ( MOAP - 1 KO , As2O3 2h ) , 605 ( WT , As2O3 4h ) , 977 ( MOAP - 1 KO , As2O3 4h ) , 721 ( WT , As2O3 6h ) 487 bodies ( MOAP - 1 KO , As2O3 6h ) from three independently plated samples . Data information : nuclei were counterstained with DAPI ( blue )

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

G Increased level of p62 bodies in MOAP-1 KO MEFs upon treatment with arsenic trioxide. WT and MOAP-1 KO MEFs were treated with arsenic trioxide (As2O3, 10 µM) for the indicated durations and subjected to IF with anti-p62 antibody (in red). H Quantification of the p62 bodies in the WT and MOAP-1 KO MEFs treated with arsenic trioxide. (Left panel) number of p62 bodies per cell. n ≥ 6 independently plated samples. (Right panel) size of p62 bodies. n = 10 (WT, basal), 17 (MOAP-1 KO, basal), 104 (WT, As2O3 2h), 137 (MOAP-1 KO, As2O3 2h), 605 (WT, As2O3 4h), 977 (MOAP-1 KO, As2O3 4h), 721 (WT, As2O3 6h) and 487 bodies (MOAP-1 KO, As2O3 6h) from three independently plated samples. Data information: nuclei were counterstained with DAPI (blue)


G Increased level of p62 bodies in MOAP - 1 KO MEFs upon treatment with arsenic trioxide . WT MOAP - 1 KO MEFs were treated with arsenic trioxide ( As2O3 , 10 µM ) for indicated durations subjected to IF with anti - p62 antibody ( in red ) .

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

G Increased level of p62 bodies in MOAP-1 KO MEFs upon treatment with arsenic trioxide. WT and MOAP-1 KO MEFs were treated with arsenic trioxide (As2O3, 10 µM) for the indicated durations and subjected to IF with anti-p62 antibody (in red).


I MOAP - 1 deficiency does not alter total of p62 in LO2 . Western blotting of p62 in WT , MOAP - 1 KO p62 KO LO2 lysed in 1x Laemmli sample buffer containing 2 % SDS . Actin as loading control .

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I MOAP-1 deficiency does not alter total protein levels of p62 in the LO2 cells. Western blotting analysis of p62 protein levels in the WT, MOAP-1 KO and p62 KO LO2 cells lysed in 1x Laemmli sample buffer containing 2% SDS. Actin as loading control.


J MOAP - 1 deficiency does not affect total of p62 in MEFs under basal As2O3 - treated conditions . Western blotting of p62 MOAP - 1 in WT MOAP - 1 KO MEFs treated with As2O3 as indicated . MOAP - 1 was immunoprecipitated from lysates . Actin as loading control .

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

J MOAP-1 deficiency does not affect total protein levels of p62 in MEFs under basal and As2O3-treated conditions. Western blotting analysis of p62 and MOAP-1 in the WT and MOAP-1 KO MEFs treated with As2O3 as indicated. MOAP-1 was immunoprecipitated from the lysates. Actin as loading control.


K Loss of MOAP - 1 does not alter turnover of p62 in LO2 . WT MOAP - 1 KO LO2 stably expressing Halo - tagged p62 were subjected to TMR labelling harvested at time points post - labelling as indicated . TMR signals of Halo - p62 were visualized by a Bio - rad Imager at 565 nM wavelength , whereas total Halo - p62 was detected by Western blotting using anti - Halo or anti - p62 antibodies . L of TMR signals in WT MOAP - 1 KO LO2 by densitometric . Average integrated density values of TMR signals from three independent experiments were presented , relative to at 0 hour .

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K Loss of MOAP-1 does not alter protein turnover of p62 in the LO2 cells. WT and MOAP-1 KO LO2 cells stably expressing Halo-tagged p62 were subjected to TMR labelling and harvested at the time points post-labelling as indicated. TMR signals of Halo-p62 were visualized by a Bio-rad Imager at 565 nM wavelength, whereas the total Halo-p62 protein was detected by Western blotting using anti-Halo or anti-p62 antibodies. L Quantification of the TMR signals in the WT and MOAP-1 KO LO2 cells by densitometric analysis. Average integrated density values of the TMR signals from three independent experiments were presented, relative to the levels at 0 hour.


K Loss of MOAP - 1 does not alter turnover of p62 in LO2 . WT MOAP - 1 KO LO2 stably expressing Halo - tagged p62 were subjected to TMR labelling harvested at time points post - labelling as indicated . TMR signals of Halo - p62 were visualized by a Bio - rad Imager at 565 nM wavelength , whereas total Halo - p62 was detected by Western blotting using anti - Halo or anti - p62 antibodies .

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

K Loss of MOAP-1 does not alter protein turnover of p62 in the LO2 cells. WT and MOAP-1 KO LO2 cells stably expressing Halo-tagged p62 were subjected to TMR labelling and harvested at the time points post-labelling as indicated. TMR signals of Halo-p62 were visualized by a Bio-rad Imager at 565 nM wavelength, whereas the total Halo-p62 protein was detected by Western blotting using anti-Halo or anti-p62 antibodies.


A Overexpression of MOAP - 1 downregulates of p62 bodies in ATG5 - depleted . HepG2 were infected with lentivirus encoding short hairpin for ATG5 ( shATG5 ) or scramble control ( shCtrl ) for 48 hours before being transfected with Myc - MOAP - 1 or empty vector ( EV ) control for 24 hours subjected to IF using anti - p62 ( in red ) anti - Myc ( in green ) antibodies . expressing Myc - MOAP - 1 were marked by dashed lines . B of p62 bodies in ATG5 - depleted transfected with Myc - MOAP - 1 or empty vector . ( Upper panel ) number of p62 bodies per . n ≥ 3 independently plated samples . ( Lower panel ) of p62 bodies . n = 135 , 133 65 bodies in shCtrl + EV , shATG5 + EV shATG5 + Myc - MOAP - 1 , respectively , from three independently plated samples . Data information : nuclei were counterstained with DAPI ( blue )

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

A Overexpression of MOAP-1 downregulates levels of p62 bodies in the ATG5-depleted cells. HepG2 cells were infected with lentivirus encoding short hairpin RNA for ATG5 (shATG5) or scramble control (shCtrl) for 48 hours before being transfected with Myc-MOAP-1 or empty vector (EV) control for 24 hours and subjected to IF analysis using anti-p62 (in red) and anti-Myc (in green) antibodies. Cells expressing Myc-MOAP-1 were marked by dashed lines. B Quantification of the p62 bodies in the ATG5-depleted cells transfected with Myc-MOAP-1 or empty vector. (Upper panel) number of p62 bodies per cell. n ≥ 3 independently plated samples. (Lower panel) size of p62 bodies. n = 135, 133 and 65 bodies in the shCtrl+EV, shATG5+EV and shATG5+Myc-MOAP-1 cells, respectively, from three independently plated samples. Data information: nuclei were counterstained with DAPI (blue)


A Overexpression of MOAP - 1 downregulates of p62 bodies in ATG5 - depleted . HepG2 were infected with lentivirus encoding short hairpin for ATG5 ( shATG5 ) or scramble control ( shCtrl ) for 48 hours before being transfected with Myc - MOAP - 1 or empty vector ( EV ) control for 24 hours subjected to IF using anti - p62 ( in red ) anti - Myc ( in green ) antibodies . expressing Myc - MOAP - 1 were marked by dashed lines .

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

A Overexpression of MOAP-1 downregulates levels of p62 bodies in the ATG5-depleted cells. HepG2 cells were infected with lentivirus encoding short hairpin RNA for ATG5 (shATG5) or scramble control (shCtrl) for 48 hours before being transfected with Myc-MOAP-1 or empty vector (EV) control for 24 hours and subjected to IF analysis using anti-p62 (in red) and anti-Myc (in green) antibodies. Cells expressing Myc-MOAP-1 were marked by dashed lines.


C Western blotting of lysates from HepG2 infected with lentivirus encoding shATG5 or shCtrl transfected with Myc - MOAP - 1 .

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

C Western blotting of the lysates from HepG2 cells infected with lentivirus encoding shATG5 or shCtrl and transfected with Myc-MOAP-1.


D Inhibition of auto - lysosomal degradation pathway does not prevent MOAP - 1 - mediated reduction of p62 bodies . MOAP - 1 KO LO2 were pre - treated with auto - lysosome inhibitors chloroquine ( CQ ) ( 10 µM ) or BafA1 ( 1 µM ) for 3 hours , before being transfected with Myc - MOAP - 1 for 24 hours in presence of CQ or BafA1 subjected to immunofluorescence using anti - p62 ( in red ) anti - Myc ( in green ) antibodies . expressing Myc - MOAP - 1 were marked by dashed lines . E of p62 bodies in MOAP - 1 KO LO2 pre - treated with CQ or BafA1 before being transfected with Myc - MOAP - 1 or empty vector control . ( Upper panel ) number of p62 bodies per . n = 5 independently plated samples . ( Lower panel ) of p62 bodies . n = 362 ( Ctrl , BafA1 ) , 226 ( Myc - MOAP - 1 , BafA1 ) , 544 ( Ctrl , CQ ) 266 bodies ( Myc - MOAP - 1 , CQ ) , from three independently plated samples . Data information : nuclei were counterstained with DAPI ( blue ) ,

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D Inhibition of the auto-lysosomal degradation pathway does not prevent MOAP-1-mediated reduction of the p62 bodies. MOAP-1 KO LO2 cells were pre-treated with auto-lysosome inhibitors chloroquine (CQ) (10 µM) or BafA1 (1 µM) for 3 hours, before being transfected with Myc-MOAP-1 for 24 hours in the presence of CQ or BafA1 and subjected to immunofluorescence using anti-p62 (in red) and anti-Myc (in green) antibodies. Cells expressing Myc-MOAP-1 were marked by dashed lines. E Quantification of the p62 bodies in the MOAP-1 KO LO2 cells pre-treated with CQ or BafA1 before being transfected with Myc-MOAP-1 or empty vector control. (Upper panel) number of p62 bodies per cell. n = 5 independently plated samples. (Lower panel) size of p62 bodies. n = 362 (Ctrl, BafA1), 226 (Myc-MOAP-1, BafA1), 544 (Ctrl, CQ) and 266 bodies (Myc-MOAP-1, CQ), from three independently plated samples. Data information: nuclei were counterstained with DAPI (blue),


F Western blotting of lysates from MOAP - 1 KO LO2 pre - treated with CQ or BafA1 transfected with Myc - MOAP - 1 .

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F Western blotting of lysates from MOAP-1 KO LO2 cells pre-treated with CQ or BafA1 and transfected with Myc-MOAP-1.


A Larger RFP - p62 bodies in MOAP - 1 deficient . WT MOAP - 1 KO LO2 were transfected with expression vector encoding RFP - tagged p62 . 16 hours later , were subjected to live imaging . Z - stacks of confocal were flattened as maximal projections . Scale bar : 5 µm . B of of RFP - p62 bodies in WT MOAP - 1 KO LO2 . n = 129 161 bodies in WT MOAP - 1 KO , respectively , from three independently plated samples . * * * P < 0 . 001 , student ' s t - .

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A Larger RFP-p62 bodies in the MOAP-1 deficient cells. WT and MOAP-1 KO LO2 cells were transfected with expression vector encoding RFP-tagged p62. 16 hours later, cells were subjected to live imaging analysis. Z-stacks of the confocal images were flattened as maximal intensity projections. Scale bar: 5 µm. B Quantification of size of RFP-p62 bodies in WT and MOAP-1 KO LO2 cells. n = 129 and 161 bodies in the WT and MOAP-1 KO cells, respectively, from three independently plated samples. ***P<0.001, student's t-test.


A Larger RFP - p62 bodies in MOAP - 1 deficient . WT MOAP - 1 KO LO2 were transfected with expression vector encoding RFP - tagged p62 . 16 hours later , were subjected to live imaging . Z - stacks of confocal were flattened as maximal projections . Scale bar : 5 µm .

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A Larger RFP-p62 bodies in the MOAP-1 deficient cells. WT and MOAP-1 KO LO2 cells were transfected with expression vector encoding RFP-tagged p62. 16 hours later, cells were subjected to live imaging analysis. Z-stacks of the confocal images were flattened as maximal intensity projections. Scale bar: 5 µm.


C Western of lysates of WT MOAP - 1 KO LO2 transfected with expression vector encoding RFP - tagged p62 as in ( A ) .

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C Western analysis of lysates of the WT and MOAP-1 KO LO2 cells transfected with expression vector encoding RFP-tagged p62 as in (A).


D , E RFP - p62 bodies exhibited fusion ( D ) fission ( E ) characteristics of liquid droplets . WT MOAP - 1 KO LO2 were transfected with expression vector encoding RFP - tagged p62 14 hours later , were analyzed by live - imaging to visualize dynamics of RFP - p62 bodies . Scale bar : 2 µm

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D, E RFP-p62 bodies exhibited fusion (D) and fission (E) characteristics of liquid droplets. WT and MOAP-1 KO LO2 cells were transfected with expression vector encoding RFP-tagged p62 and 14 hours later, cells were analyzed by live-imaging to visualize the dynamics of RFP-p62 bodies. Scale bar: 2 µm


D , E RFP - p62 bodies exhibited fusion ( D ) fission ( E ) characteristics of liquid droplets . WT MOAP - 1 KO LO2 were transfected with expression vector encoding RFP - tagged p62 14 hours later , were analyzed by live - imaging to visualize dynamics of RFP - p62 bodies . Scale bar : 2

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F Loss of MOAP - 1 leads to slower exchange of p62 in RFP - p62 bodies . WT MOAP - 1 KO LO2 transfected with expression vector encoding RFP - tagged p62 . 14 hours later , were subjected to recovery after photobleaching ( FRAP ) . Scale bar : 2 µm . G of rate of recovery of RFP - p62 in WT MOAP - 1 KO . n = 5 samples .

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F Loss of MOAP-1 leads to slower exchange of p62 in the RFP-p62 bodies. WT and MOAP-1 KO LO2 cells transfected with expression vector encoding RFP-tagged p62. 14 hours later, cells were subjected to the fluorescence recovery after photobleaching (FRAP) assay. Scale bar: 2 µm. G Quantification of the rate of fluorescence recovery of RFP-p62 in WT and MOAP-1 KO cells. n = 5 samples.


F Loss of MOAP - 1 leads to slower exchange of p62 in RFP - p62 bodies . WT MOAP - 1 KO LO2 transfected with expression vector encoding RFP - tagged p62 . 14 hours later , were subjected to recovery after photobleaching ( FRAP ) . Scale bar : 2 µm .

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F Loss of MOAP-1 leads to slower exchange of p62 in the RFP-p62 bodies. WT and MOAP-1 KO LO2 cells transfected with expression vector encoding RFP-tagged p62. 14 hours later, cells were subjected to the fluorescence recovery after photobleaching (FRAP) assay. Scale bar: 2 µm.


H Overexpression of MOAP - 1 but not GFP leads to increased exchange of p62 in RFP - p62 bodies . HepG2 were transfected with expression vector encoding RFP - tagged p62 GFP - tagged MOAP - 1 or GFP only control 14 hours later , were subjected to FRAP . Scale bar : 2 µm . I of rate of recovery of RFP - p62 in HepG2 co - expressing GFP - MOAP - 1 or GFP . n = 5 samples .

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H Overexpression of MOAP-1 but not GFP leads to increased exchange of p62 in the RFP-p62 bodies. HepG2 cells were transfected with expression vector encoding RFP-tagged p62 and GFP-tagged MOAP-1 or GFP only control and 14 hours later, cells were subjected to FRAP assay. Scale bar: 2 µm. I Quantification of the rate of fluorescence recovery of RFP-p62 in HepG2 cells co-expressing GFP-MOAP-1 or GFP. n = 5 samples.


H Overexpression of MOAP - 1 but not GFP leads to increased exchange of p62 in RFP - p62 bodies . HepG2 were transfected with expression vector encoding RFP - tagged p62 GFP - tagged MOAP - 1 or GFP only control 14 hours later , were subjected to FRAP . Scale bar : 2 µm .

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H Overexpression of MOAP-1 but not GFP leads to increased exchange of p62 in the RFP-p62 bodies. HepG2 cells were transfected with expression vector encoding RFP-tagged p62 and GFP-tagged MOAP-1 or GFP only control and 14 hours later, cells were subjected to FRAP assay. Scale bar: 2 µm.


J Re - introduction of MOAP - 1 disrupts spherical structure of p62 bodies in MOAP - 1 deficient LO2 . MOAP - 1 KO LO2 were transfected with expression vector encoding Myc - MOAP - 1 harvested at 14 - or 24 - hours post - transfection for IF using anti - p62 ( in red ) anti - Myc ( in green ) antibodies . Insets were enlarged in lower panels . Scale bar : 2 µm .

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J Re-introduction of MOAP-1 disrupts the spherical structure of p62 bodies in the MOAP-1 deficient LO2 cells. MOAP-1 KO LO2 cells were transfected with expression vector encoding Myc-MOAP-1 and harvested at 14- or 24-hours post-transfection for IF analysis using anti-p62 (in red) and anti-Myc (in green) antibodies. Insets were enlarged in the lower panels. Scale bar: 2 µm.


A MOAP - 1 associates with p62 their interaction is enhanced by DEN - mediated stress . LO2 transfected with Myc - MOAP - 1 were treated with or without diethylnitrosamine ( DEN ) ( 200 µM ) for 12 h subjected to Duolink proximity ligation using anti - p62 anti - Myc antibodies . Red dots represent MOAP - 1 / p62 interaction .

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A MOAP-1 associates with p62 and their interaction is enhanced by DEN-mediated stress. LO2 cells transfected with Myc-MOAP-1 were treated with or without diethylnitrosamine (DEN) (200 µM) for 12 h and subjected to Duolink proximity ligation assay using anti-p62 and anti-Myc antibodies. Red dots represent MOAP-1/p62 interaction.


B Alanine substitution of positively charged residues in KYKKLR of MOAP - 1 abolishes its interaction with p62 . Replacement of positively charged residues in KYKKLR of MOAP - 1 with alanine ( referred to as M3 mutant ) disrupts MOAP - 1 / p62 interaction , whereas alanine substitution in neighboring region of positively charged KRRR ( M1 mutant ) negatively charged EEE ( M2 mutant ) has no effect . LO2 were transfected with plasmid encoding Myc - MOAP - 1 or indicated alanine mutants together with plasmid encoding Flag - p62 . were then subjected to co - IP with anti - Myc antibody .

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B Alanine substitution of positively charged residues in the KYKKLR sequence of MOAP-1 abolishes its interaction with p62. Replacement of the positively charged residues in the KYKKLR sequence of MOAP-1 with alanine (referred to as the M3 mutant) disrupts MOAP-1/p62 interaction, whereas alanine substitution in the neighboring region of positively charged KRRR (M1 mutant) and negatively charged EEE (M2 mutant) has no effect. LO2 cells were transfected with plasmid encoding Myc-MOAP-1 or the indicated alanine mutants together with plasmid encoding Flag-p62. The cells were then subjected to co-IP assay with anti-Myc antibody.


D MOAP - 1 , but not its p62 - binding defective mutant , MOAP - 1 - M3 , localizes at p62 bodies . MOAP - 1 KO LO2 were transfected with plasmid encoding Myc - MOAP - 1 or Myc - MOAP - 1 - M3 mutant . transfected were harvested at 14 hours post - transfection , which is time point that localization of MOAP - 1 at p62 bodies is readily detected . IF was performed on these using anti - p62 ( red ) anti - Myc ( green ) antibodies . Arrowheads indicate colocalization between Myc - MOAP - 1 p62 bodies .

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D MOAP-1, but not its p62-binding defective mutant, MOAP-1-M3, localizes at the p62 bodies. MOAP-1 KO LO2 cells were transfected with plasmid encoding Myc-MOAP-1 or Myc-MOAP-1-M3 mutant. The transfected cells were harvested at 14 hours post-transfection, which is the time point that localization of MOAP-1 at the p62 bodies is readily detected. IF analysis was performed on these cells using anti-p62 (red) and anti-Myc (green) antibodies. Arrowheads indicate colocalization between Myc-MOAP-1 and p62 bodies.


E MOAP - 1 but not MOAP - 1 - M3 - mutant promotes exchange of RFP - p62 in p62 bodies . WT LO2 transfected with expression vector encoding RFP - tagged p62 GFP - tagged MOAP - 1 or MOAP - 1 - M3 . At 14 hours post - transfection , were subjected to FRAP . Scale bar : 2 µm .

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E MOAP-1 but not the MOAP-1-M3-mutant promotes the exchange of RFP-p62 in the p62 bodies. WT LO2 cells transfected with expression vector encoding RFP-tagged p62 and GFP-tagged MOAP-1 or MOAP-1-M3. At 14 hours post-transfection, cells were subjected to FRAP assay. Scale bar: 2 µm.


F of rate of recovery of RFP - p62 in co - expressing GFP - MOAP - 1 or GFP - MOAP - 1 - M3 mutant . n = 6 samples . Error bars represent S . E . M . * P < 0 . 05 , * * P < 0 . 01 , ns , not significant , two - way ANOVA .

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F Quantification of the rate of fluorescence recovery of RFP-p62 in cells co-expressing GFP-MOAP-1 or GFP-MOAP-1-M3 mutant. n = 6 samples. Error bars represent S.E.M. *P<0.05, **P<0.01, ns, not significant, two-way ANOVA.


G Western of lysates of WT LO2 transfected with expression vector encoding RFP - tagged p62 GFP - tagged MOAP - 1 or MOAP - 1 - M3 as described in ( E ) .

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G Western analysis of lysates of the WT LO2 cells transfected with expression vector encoding RFP-tagged p62 and GFP-tagged MOAP-1 or MOAP-1-M3 as described in (E).


H Re - expression of MOAP - 1 , but not MOAP - 1 - M3 mutant , reduces of p62 bodies . MOAP - 1 KO LO2 were transfected with plasmid encoding Myc - MOAP - 1 or Myc - MOAP - 1 - M3 . transfected were harvested at 24 hours post - transfection , which is time point that MOAP - 1 was found to effectively downregulate p62 bodies , followed by IF as in ( D ) . expressing Myc - MOAP - 1 or Myc - MOAP - 1 - M3 were marked by dashed lines . I of p62 bodies in MOAP - 1 KO LO2 expressing Myc - MOAP - 1 Myc - MOAP - 1 - M3 mutant . ( Left panel ) number of p62 bodies per . n = 7 independently plated samples . ( Right panel ) of p62 bodies . n = 107 267 bodies detected in MOAP - 1 KO LO2 expressing Myc - MOAP - 1 Myc - MOAP - 1 - M3 mutant , respectively , from three independently plated samples . Error bars represent S . E . M . * * * P < 0 . 001 , Student ' s t - .

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H Re-expression of MOAP-1, but not the MOAP-1-M3 mutant, reduces the levels of p62 bodies. MOAP-1 KO LO2 cells were transfected with plasmid encoding Myc-MOAP-1 or Myc-MOAP-1-M3. The transfected cells were harvested at 24 hours post-transfection, which is the time point that MOAP-1 was found to effectively downregulate p62 bodies, followed by IF analysis as in (D). Cells expressing Myc-MOAP-1 or Myc-MOAP-1-M3 were marked by dashed lines. I Quantification of the p62 bodies in the MOAP-1 KO LO2 cells expressing Myc-MOAP-1 and Myc-MOAP-1-M3 mutant. (Left panel) number of p62 bodies per cell. n = 7 independently plated samples. (Right panel) size of p62 bodies. n = 107 and 267 bodies detected in the MOAP-1 KO LO2 cells expressing Myc-MOAP-1 and Myc-MOAP-1-M3 mutant, respectively, from three independently plated samples. Error bars represent S.E.M. ***P<0.001, Student's t-test.


H Re - expression of MOAP - 1 , but not MOAP - 1 - M3 mutant , reduces of p62 bodies . MOAP - 1 KO LO2 were transfected with plasmid encoding Myc - MOAP - 1 or Myc - MOAP - 1 - M3 . transfected were harvested at 24 hours post - transfection , which is time point that MOAP - 1 was found to effectively downregulate p62 bodies , followed by IF as in ( D ) . expressing Myc - MOAP - 1 or Myc - MOAP - 1 - M3 were marked by dashed lines .

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H Re-expression of MOAP-1, but not the MOAP-1-M3 mutant, reduces the levels of p62 bodies. MOAP-1 KO LO2 cells were transfected with plasmid encoding Myc-MOAP-1 or Myc-MOAP-1-M3. The transfected cells were harvested at 24 hours post-transfection, which is the time point that MOAP-1 was found to effectively downregulate p62 bodies, followed by IF analysis as in (D). Cells expressing Myc-MOAP-1 or Myc-MOAP-1-M3 were marked by dashed lines.


J Western blotting of MOAP - 1 KO LO2 transfected with plasmid encoding Myc - MOAP - 1 or Myc - MOAP - 1 - M3 mutant as described in ( H ) .

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J Western blotting of MOAP-1 KO LO2 cells transfected with plasmid encoding Myc-MOAP-1 or Myc-MOAP-1-M3 mutant as described in (H).


A Overexpression of MOAP - 1 inhibits dimerization of p62 . p62 KO LO2 hepatocytes were transfected with indicated plasmids . 24 hours later , lysates were subjected to co - IP with anti - Myc antibody . PB1 deletion ( ΔPB1 ) mutant , which is impaired in p62 oligomerization , is included as a control . TCL , total lysates .

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A Overexpression of MOAP-1 inhibits dimerization of p62. p62 KO LO2 hepatocytes were transfected with the indicated plasmids. 24 hours later, cell lysates were subjected to co-IP assay with anti-Myc antibody. PB1 deletion (ΔPB1) mutant, which is impaired in p62 oligomerization, is included as a control. TCL, total cell lysates.


B Dimerization of p62 is enhanced in MOAP - 1 deficient . WT MOAP - 1 KO LO2 were transfected with plasmids encoding Flag - tagged Myc - tagged p62 or empty vector . 24 h post - transfection , were harvested , lysed lysates were subjected to co - IP with anti - Myc antibody . C of of Flag - p62 bound to Myc - p62 in co - IP experiment as described in ( B ) by densitometric .

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

B Dimerization of p62 is enhanced in the MOAP-1 deficient cells. WT and MOAP-1 KO LO2 cells were transfected with plasmids encoding Flag-tagged and Myc-tagged p62 or empty vector. 24 h post-transfection, cells were harvested, lysed and the cell lysates were subjected to co-IP assay with anti-Myc antibody. C Quantification of the levels of Flag-p62 bound to Myc-p62 in the co-IP experiment as described in (B) by densitometric analysis.


B Dimerization of p62 is enhanced in MOAP - 1 deficient . WT MOAP - 1 KO LO2 were transfected with plasmids encoding Flag - tagged Myc - tagged p62 or empty vector . 24 h post - transfection , were harvested , lysed lysates were subjected to co - IP with anti - Myc antibody .

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

B Dimerization of p62 is enhanced in the MOAP-1 deficient cells. WT and MOAP-1 KO LO2 cells were transfected with plasmids encoding Flag-tagged and Myc-tagged p62 or empty vector. 24 h post-transfection, cells were harvested, lysed and the cell lysates were subjected to co-IP assay with anti-Myc antibody.


D Re - expression of MOAP - 1 , but not p62 - binding defective mutant , MOAP - 1 - M3 , reduces p62 oligomerization in MOAP - 1 KO LO2 hepatocytes . WT MOAP - 1 KO LO2 were transfected with plasmids encoding His - tagged p62 Myc - MOAP - 1 or Myc - MOAP - 1 - M3 24 h later , subjected to His - pull down cross - linking using DSSO . E of of p62 oligomer depicted in ( D ) by densitometric .

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

D Re-expression of MOAP-1, but not the p62-binding defective mutant, MOAP-1-M3, reduces p62 oligomerization in the MOAP-1 KO LO2 hepatocytes. WT and MOAP-1 KO LO2 cells were transfected with plasmids encoding His-tagged p62 and Myc-MOAP-1 or Myc-MOAP-1-M3 and 24 h later, subjected to His-pull down and cross-linking using DSSO. E Quantification of the levels of p62 oligomer depicted in (D) by densitometric analysis.


D Re - expression of MOAP - 1 , but not p62 - binding defective mutant , MOAP - 1 - M3 , reduces p62 oligomerization in MOAP - 1 KO LO2 hepatocytes . WT MOAP - 1 KO LO2 were transfected with plasmids encoding His - tagged p62 Myc - MOAP - 1 or Myc - MOAP - 1 - M3 24 h later , subjected to His - pull down cross - linking using DSSO .

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

D Re-expression of MOAP-1, but not the p62-binding defective mutant, MOAP-1-M3, reduces p62 oligomerization in the MOAP-1 KO LO2 hepatocytes. WT and MOAP-1 KO LO2 cells were transfected with plasmids encoding His-tagged p62 and Myc-MOAP-1 or Myc-MOAP-1-M3 and 24 h later, subjected to His-pull down and cross-linking using DSSO.


F region of p62 spanning PB1 ZZ domains are required for mediating its interaction with MOAP - 1 . p62 KO LO2 were used in . were transfected with plasmid encoding Myc - p62 or indicated deletion mutants Flag - MOAP - 1 for 24 hours . transfected were subjected to co - IP with anti - Myc antibody . ( Lower panel ) Schematics depicting domains in p62 . PB1 , Phox Bem1p ; ZZ , Zinc Finger ; TB , TRAF6 - binding domain ; KIR , Keap1 interacting region ; LIR , LC3 interacting region ; UBA , ubiquitin - associated domain .

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

F The region of p62 spanning the PB1 and ZZ domains are required for mediating its interaction with MOAP-1. p62 KO LO2 cells were used in the analysis. Cells were transfected with plasmid encoding Myc-p62 or the indicated deletion mutants and Flag-MOAP-1 for 24 hours. The transfected cells were subjected to co-IP assay with anti-Myc antibody. (Lower panel) Schematics depicting the domains in p62. PB1, Phox and Bem1p; ZZ, Zinc Finger; TB, TRAF6-binding domain; KIR, Keap1 interacting region; LIR, LC3 interacting region; UBA, ubiquitin-associated domain.


G fragment encompassing PB1 ZZ domains of p62 is sufficient for mediating its interaction with MOAP - 1 . p62 KO LO2 were transfected with plasmid encoding Myc - p62 or indicated fragments Flag - MOAP - 1 . 24 hours later , transfected were subjected to co - IP with anti - Myc antibody .

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

G The fragment encompassing the PB1 and ZZ domains of p62 is sufficient for mediating its interaction with MOAP-1. p62 KO LO2 cells were transfected with plasmid encoding Myc-p62 or the indicated fragments and Flag-MOAP-1. 24 hours later, the transfected cells were subjected to co-IP assay with anti-Myc antibody.


H MOAP - 1 binds inhibits homodimerization of PB1 - ZZ fragment . p62 KO LO2 were transfected with plasmid encoding Myc Flag - tagged PB1 - ZZ in absence of presence of HA - tagged MOAP - 1 for 24 h subjected to co - IP with anti - Myc antibody .

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

H MOAP-1 binds and inhibits homodimerization of the PB1-ZZ fragment. p62 KO LO2 cells were transfected with plasmid encoding Myc and Flag-tagged PB1-ZZ in the absence of presence of HA-tagged MOAP-1 for 24 h and subjected to co-IP assay with anti-Myc antibody.


A Increased recruitment of Keap1 to p62 bodies in MOAP - 1 deficient LO2 . WT MOAP - 1 KO LO2 were subjected to IF with anti - Keap1 ( green ) anti - p62 ( red ) antibodies . Insets in upper panels are enlarged in lower panels . Nuclei were counterstained with DAPI ( in blue ) . Scale bar : 5 µm .

O O O O B-MEASURED_VAR O B-MEASURED_VAR O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O B-MEASURED_VAR O O O O O B-MEASURED_VAR O O O O O O O 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 Increased recruitment of Keap1 to p62 bodies in the MOAP-1 deficient LO2 cells. WT and MOAP-1 KO LO2 cells were subjected to IF analysis with anti-Keap1 (green) and anti-p62 (red) antibodies. Insets in the upper panels are enlarged in the lower panels. Nuclei were counterstained with DAPI (in blue). Scale bar: 5 µm.


B MOAP - 1 deficiency promotes p62 / Keap1 interaction . WT , MOAP - 1 KO p62 KO LO2 were subjected to co - IP using anti - p62 antibody . TCL , total lysates . C of of Keap1 bound to p62 in co - IP experiment as described in ( B ) by densitometric .

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

B MOAP-1 deficiency promotes p62/Keap1 interaction. WT, MOAP-1 KO and p62 KO LO2 cells were subjected to co-IP assay using anti-p62 antibody. TCL, total cell lysates. C Quantification of the levels of Keap1 bound to p62 in the co-IP experiment as described in (B) by densitometric analysis.


B MOAP - 1 deficiency promotes p62 / Keap1 interaction . WT , MOAP - 1 KO p62 KO LO2 were subjected to co - IP using anti - p62 antibody . TCL , total lysates .

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

B MOAP-1 deficiency promotes p62/Keap1 interaction. WT, MOAP-1 KO and p62 KO LO2 cells were subjected to co-IP assay using anti-p62 antibody. TCL, total cell lysates.


D Diminished Keap1 / Nrf2 interaction in absence of MOAP - 1 . WT MOAP - 1 KO LO2 were subjected to Duolink PLA using anti - Keap1 anti - Nrf2 antibodies . Red dots represent Keap1 / Nrf2 interaction . Nuclei were counterstained with DAPI ( in blue ) . Scale bar : 10 µm . E of Duolink signals depicted in ( D ) . Duolink signals , which appear as red dots , were quantified by ImageJ normalized to number of nuclei .

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

D Diminished Keap1/Nrf2 interaction in the absence of MOAP-1. WT and MOAP-1 KO LO2 cells were subjected to Duolink PLA using anti-Keap1 and anti-Nrf2 antibodies. Red dots represent Keap1/Nrf2 interaction. Nuclei were counterstained with DAPI (in blue). Scale bar: 10 µm. E Quantification of the Duolink signals depicted in (D). Duolink signals, which appear as red dots, were quantified by ImageJ analysis and normalized to the number of nuclei.


D Diminished Keap1 / Nrf2 interaction in absence of MOAP - 1 . WT MOAP - 1 KO LO2 were subjected to Duolink PLA using anti - Keap1 anti - Nrf2 antibodies . Red dots represent Keap1 / Nrf2 interaction . Nuclei were counterstained with DAPI ( in blue ) . Scale bar : 10 µm .

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

D Diminished Keap1/Nrf2 interaction in the absence of MOAP-1. WT and MOAP-1 KO LO2 cells were subjected to Duolink PLA using anti-Keap1 and anti-Nrf2 antibodies. Red dots represent Keap1/Nrf2 interaction. Nuclei were counterstained with DAPI (in blue). Scale bar: 10 µm.


F Loss of MOAP - 1 results in marked elevation of Nrf2 . WT MOAP - 1 KO LO2 were subjected to IF with anti - Nrf2 ( in green ) anti - p62 ( in red ) antibodies . Scale bar : 10 µm . G of of Nrf2 in ( F ) . mean gray values of Nrf2 signals were quantified by ImageJ .

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

F Loss of MOAP-1 results in marked elevation of nuclear Nrf2. WT and MOAP-1 KO LO2 cells were subjected to IF analysis with anti-Nrf2 (in green) and anti-p62 (in red) antibodies. Scale bar: 10 µm. G Quantification of nuclear levels of Nrf2 in (F). The mean gray values of Nrf2 fluorescence signals were quantified by ImageJ analysis.


F Loss of MOAP - 1 results in marked elevation of Nrf2 . WT MOAP - 1 KO LO2 were subjected to IF with anti - Nrf2 ( in green ) anti - p62 ( in red ) antibodies . Scale bar : 10 µm .

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

F Loss of MOAP-1 results in marked elevation of nuclear Nrf2. WT and MOAP-1 KO LO2 cells were subjected to IF analysis with anti-Nrf2 (in green) and anti-p62 (in red) antibodies. Scale bar: 10 µm.


H MOAP - 1 deficiency promotes activation of Nrf2 signaling . Transcript of downstream targets of Nrf2 ( i . e . HMOX - 1 , NQO1 , SLC7A11 , GCLC G6PD ) in WT MOAP - 1 KO LO2 were determined by RT - PCR normalized to GAPDH .

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

H MOAP-1 deficiency promotes activation of Nrf2 signaling. Transcript levels of downstream targets of Nrf2 (i.e. HMOX-1, NQO1, SLC7A11, GCLC and G6PD) in WT and MOAP-1 KO LO2 cells were determined by RT-PCR and normalized to GAPDH.


I Elevated transcriptional activity of Nrf2 in MOAP - 1 deficient is mediated through p62 . Transcript of Nrf2 target genes ( HMOX - 1 NQO1 ) in WT , MOAP - 1 KO MOAP - 1 / p62 DKO LO2 were determined by RT - PCR normalized to GAPDH .

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

I Elevated transcriptional activity of Nrf2 in the MOAP-1 deficient cells is mediated through p62. Transcript levels of Nrf2 target genes (HMOX-1 and NQO1) in WT, MOAP-1 KO and MOAP-1/p62 DKO LO2 cells were determined by RT-PCR and normalized to GAPDH.


J Re - expression of MOAP - 1 MOAP - 1 - L120E , but not p62 binding defective mutant of MOAP - 1 , MOAP - 1 - M3 , inhibits p62 / Keap1 interaction in MOAP - 1 deficient . MOAP - 1 KO LO2 were transfected with plasmid encoding Myc - tagged MOAP - 1 , L120E or M3 . transfected were then subjected to co - IP with anti - p62 antibody .

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

J Re-expression of MOAP-1 and MOAP-1-L120E, but not the p62 binding defective mutant of MOAP-1, MOAP-1-M3, inhibits p62/Keap1 interaction in the MOAP-1 deficient cells. MOAP-1 KO LO2 cells were transfected with plasmid encoding Myc-tagged MOAP-1, L120E or M3. The transfected cells were then subjected to co-IP assay with anti-p62 antibody.


K Re - introduction of MOAP - 1 MOAP - 1 - L120E , but not p62 binding defective MOAP - 1 - M3 , reduces Nrf2 transcriptional activity in MOAP - 1 KO . MOAP - 1 KO LO2 were transfected with Myc - tagged MOAP - 1 , MOAP - 1 - L120E or MOAP - 1 - M3 ARE - Nrf2 - firely renilla luciferase reporter constructs for overnight subjected to luciferase .

K Re-introduction of MOAP-1 and MOAP-1-L120E, but not the p62 binding defective MOAP-1-M3, reduces Nrf2 transcriptional activity in the MOAP-1 KO cells. MOAP-1 KO LO2 cells were transfected with Myc-tagged MOAP-1, MOAP-1-L120E or MOAP-1-M3 and ARE-Nrf2-firely and renilla luciferase reporter constructs for overnight and subjected to luciferase assay.


A High level of p62 bodies in liver of MOAP - 1 KO mice subjected to acute diethylnitrosamine ( DEN ) treatment . Eight - week - old WT MOAP - 1 KO mice were injected with an acute dose of DEN ( 100 µg / g ) . Mice were sacrificed at 48 hours post - injection . Livers were harvested for immunohistochemistry ( ) with anti - p62 antibody ( in red ) . Nuclei were counterstained with DAPI ( in blue ) . Insets in upper panels are enlarged in lower panels . Scale bar : 20 µm .

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

A High level of p62 bodies in the liver of the MOAP-1 KO mice subjected to acute diethylnitrosamine (DEN) treatment. Eight-week-old male WT and MOAP-1 KO mice were injected with an acute dose of DEN (100 µg/g body weight). Mice were sacrificed at 48 hours post-injection. Livers were harvested for immunohistochemistry (IHC) analysis with anti-p62 antibody (in red). Nuclei were counterstained with DAPI (in blue). Insets in the upper panels are enlarged in the lower panels. Scale bar: 20 µm.


B Loss of MOAP - 1 results in higher of Nrf2 its downstream target , HMOX - 1 , in liver upon acute DEN treatment . Lysates of livers harvested from mice subjected to acute DEN treatment as described in ( A ) were prepared in RIPA lysis buffer containing 1 % SDS analyzed by Western blotting . Actin as loading control .

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

B Loss of MOAP-1 results in higher levels of Nrf2 and its downstream target protein, HMOX-1, in liver upon acute DEN treatment. Lysates of the livers harvested from mice subjected to acute DEN treatment as described in (A) were prepared in RIPA lysis buffer containing 1% SDS and analyzed by Western blotting. Actin as loading control.


C MOAP - 1 deficiency elevates of p62 in detergent - insoluble fractions of liver lysates from mice subjected to acute DEN treatment . Livers from mice injected as described in ( A ) were lysed in 1 % Triton - X lysis buffer , separated into detergent - soluble ( S ) insoluble ( I ) fractions analyzed by Western blotting .

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C MOAP-1 deficiency elevates levels of p62 protein in the detergent-insoluble fractions of the liver lysates from mice subjected to acute DEN treatment. Livers from mice injected as described in (A) were lysed in 1% Triton-X lysis buffer, separated into detergent-soluble (S) and insoluble (I) fractions and analyzed by Western blotting.


D High of p62 bodies detected in tumors of MOAP - 1 KO mice . sections from livers dissected from 8 . 5 - month - old WT MOAP - 1 KO mice injected with a single dose of DEN ( 25 µg / g ) at 15 - day - old were subjected to with anti - p62 antibody ( in red ) . were stained with DAPI ( in blue ) . Insets were enlarged in bottom right . Scale bar : 20 µm . E of p62 bodies in WT MOAP - 1 KO tumors . ( Left panel ) number of p62 bodies per . n = 7 tumors from livers of WT MOAP - 1 KO mice . ( Right panel ) of p62 bodies . n = 456 1211 bodies detected in WT MOAP - 1 KO tumors , respectively , from three independent samples . Error bar represents S . E . M . * P < 0 . 05 , * * * P < 0 . 001 , student ' s t - .

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

D High levels of p62 bodies detected in the tumors of the MOAP-1 KO mice. Tumor sections from livers dissected from the 8.5-month-old male WT and MOAP-1 KO mice injected with a single dose of DEN (25 µg/g body weight) at 15-day-old were subjected to IHC analysis with anti-p62 antibody (in red). Nuclear were stained with DAPI (in blue). Insets were enlarged in the bottom right. Scale bar: 20 µm. E Quantification of the p62 bodies in the WT and MOAP-1 KO tumors. (Left panel) number of p62 bodies per cell. n = 7 tumors from the livers of WT and MOAP-1 KO mice. (Right panel) size of p62 bodies. n = 456 and 1211 bodies detected in the WT and MOAP-1 KO tumors, respectively, from three independent samples. Error bar represents S.E.M. *P<0.05, ***P<0.001, student's t-test.


D High of p62 bodies detected in tumors of MOAP - 1 KO mice . sections from livers dissected from 8 . 5 - month - old WT MOAP - 1 KO mice injected with a single dose of DEN ( 25 µg / g ) at 15 - day - old were subjected to with anti - p62 antibody ( in red ) . were stained with DAPI ( in blue ) . Insets were enlarged in bottom right . Scale bar : 20 µm .

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

D High levels of p62 bodies detected in the tumors of the MOAP-1 KO mice. Tumor sections from livers dissected from the 8.5-month-old male WT and MOAP-1 KO mice injected with a single dose of DEN (25 µg/g body weight) at 15-day-old were subjected to IHC analysis with anti-p62 antibody (in red). Nuclear were stained with DAPI (in blue). Insets were enlarged in the bottom right. Scale bar: 20 µm.


F Increased of Nrf2 its downstream target , HMOX - 1 , in MOAP - 1 deficient tumors . While p62 is upregulated in tumors compared to adjacent non - tissues in both WT MOAP - 1 KO livers , no significant difference is observed in p62 between WT MOAP - 1 KO tumors . Lysates of tumors non - parts of livers from mice injected with DEN as described in ( D ) were prepared in RIPA lysis buffer containing 1 % SDS subjected to Western blotting . Actin as loading control . P , PBS injected control ; N , non - ; T , .

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

F Increased levels of Nrf2 and its downstream target protein, HMOX-1, in the MOAP-1 deficient tumors. While p62 protein is upregulated in the tumors compared to the adjacent non-tumor tissues in both WT and MOAP-1 KO livers, no significant difference is observed in the p62 protein levels between the WT and MOAP-1 KO tumors. Lysates of the tumors and non-tumor parts of the livers from mice injected with DEN as described in (D) were prepared in RIPA lysis buffer containing 1% SDS and subjected to Western blotting. Actin as loading control. P, PBS injected control; N, non-tumor; T, tumor.


G Upregulation of mRNA of HMOX - 1 NQO1 in MOAP - 1 deficient tumors . Livers harvested from mice injected with PBS or DEN as described in ( D ) were subjected to RT - PCR . n = 3 WT 3 MOAP - 1 KO mice injected with PBS control , 8 WT 9 MOAP - 1 KO mice injected with DEN . Error bar represents S . E . M . * * P < 0 . 01 , student ' s t - .

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

G Upregulation of mRNA levels of HMOX-1 and NQO1 in the MOAP-1 deficient tumors. Livers harvested from the mice injected with PBS or DEN as described in (D) were subjected to RT-PCR analysis. n = 3 WT and 3 MOAP-1 KO mice injected with the PBS control, and 8 WT and 9 MOAP-1 KO mice injected with DEN. Error bar represents S.E.M. **P<0.01, student's t-test.


H MOAP - 1 deficient mice bear higher burden in DEN - mediated liver cancer model . Representative of liver tumors dissected from 8 . 5 - month - old WT MOAP - 1 KO mice injected with a single dose of DEN ( 25 µg / g ) at 15 - day - old . Scale bar : 5 mm . I Measurements of number , maximum diameter of nodules total liver from WT MOAP - 1 KO mice ( n = 13 14 respectively ) injected with DEN as described in ( H ) . Error bar represents S . E . M . * * P < 0 . 01 , * * * P < 0 . 001 , student ' s t - .

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

H MOAP-1 deficient mice bear higher tumor burden in the DEN-mediated liver cancer model. Representative images of liver tumors dissected from the 8.5-month-old male WT and MOAP-1 KO mice injected with a single dose of DEN (25 µg/g body weight) at 15-day-old. Scale bar: 5 mm. I Measurements of the number, maximum diameter of the tumor nodules and total liver weight from WT and MOAP-1 KO mice (n=13 and 14 respectively) injected with DEN as described in (H). Error bar represents S.E.M. **P<0.01, ***P<0.001, student's t-test.


H MOAP - 1 deficient mice bear higher burden in DEN - mediated liver cancer model . Representative of liver tumors dissected from 8 . 5 - month - old WT MOAP - 1 KO mice injected with a single dose of DEN ( 25 µg / g ) at 15 - day - old . Scale bar : 5 mm .

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

H MOAP-1 deficient mice bear higher tumor burden in the DEN-mediated liver cancer model. Representative images of liver tumors dissected from the 8.5-month-old male WT and MOAP-1 KO mice injected with a single dose of DEN (25 µg/g body weight) at 15-day-old. Scale bar: 5 mm.