( A - B ) Relative Pseudomonas abundance in rhizosphere samples after ( A ) , Pi - stress ( LP ) or ( B ) treatment with B . subtilis expressing RALF23 ( RALF23 ) . data shown indicate means ± SDs ( n = 3 , n refers to biological replicates ) . * * , p < 0 . 01 ( Student ' s t ) . ( C - D ) Relative Bacillus abundance in rhizosphere samples after ( C ) , Pi - stress ( LP ) or ( D ) treatment with B . subtilis expressing RALF23 ( RALF23 ) . data shown indicate means ± SDs ( n = 3 , n refers to biological replicates ) . * , p < 0 . 05 ; * * , p < 0 . 01 ( Student ' s t ) . ( E - F ) Relative Geodermatophilus abundance in rhizosphere samples after ( E ) , Pi - stress ( LP ) or ( F ) treatment with B . subtilis expressing RALF23 ( RALF23 ) . data shown indicate means ± SDs ( n = 3 , n refers to biological replicates ) . * , p < 0 . 05 ; * * , p < 0 . 01 ( Student ' s t ) . ( G - H ) Relative Methylobacillus abundance in rhizosphere samples after ( G ) , Pi - stress ( LP ) or ( H ) treatment with B . subtilis expressing RALF23 ( RALF23 ) . data shown indicate means ± SDs ( n = 3 , n refers to biological replicates ) . * * , p < 0 . 01 ( Student ' s t ) .

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

(A-B) Relative Pseudomonas abundance in rhizosphere samples after (A), Pi-stress (LP) or (B) treatment with B. subtilis expressing RALF23 (RALF23). The data shown indicate the means ± SDs (n = 3, n refers to biological replicates). **, p < 0.01 (Student's t test). (C-D) Relative Bacillus abundance in rhizosphere samples after (C), Pi-stress (LP) or (D) treatment with B. subtilis expressing RALF23 (RALF23). The data shown indicate the means ± SDs (n = 3, n refers to biological replicates). *, p < 0.05; **, p < 0.01 (Student's t test). (E-F) Relative Geodermatophilus abundance in rhizosphere samples after (E), Pi-stress (LP) or (F) treatment with B. subtilis expressing RALF23 (RALF23). The data shown indicate the means ± SDs (n = 3, n refers to biological replicates). *, p < 0.05; **, p < 0.01 (Student's t test). (G-H) Relative Methylobacillus abundance in rhizosphere samples after (G), Pi-stress (LP) or (H) treatment with B. subtilis expressing RALF23 (RALF23). The data shown indicate the means ± SDs (n = 3, n refers to biological replicates). **, p < 0.01 (Student's t test).


inoculation promotes growth alleviates LP stress . Arabidopsis seeds were germinated on 1 / 2 - strength MS media for 3 days then transplanted to either HP or LP media for another 5 days . seedlings were then transferred to vermiculite inoculated with Pto DC3000 or B . subtilis . ( A ) , were taken 4 after inoculation . Data information : experiments were repeated three times with similar results .

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Bacterial inoculation promotes plant growth and alleviates LP stress. Arabidopsis seeds were germinated on 1/2-strength MS media for 3 days and then transplanted to either HP or LP media for another 5 days. The seedlings were then transferred to vermiculite and inoculated with Pto DC3000 or B. subtilis. (A), Images were taken 4 weeks after inoculation. Data information: All experiments were repeated three times with similar results.


( C ) Pto DC3000 B . subtilis upregulate phosphate absorption gene expression . Col - 0 , phr1 fer - 4 mutant roots were harvested 48 hours after inoculation with Pto DC3000 or B . subtilis in Pi - sufficient or Pi - deficient condition , relative expression of Pi - absorbing genes was quantified via RT - qPCR . data shown indicate means ± SDs ( n = 3 , n refers to technical replicates ) ; * , p < 0 . 05 ; * * , p < 0 . 01 ( Student ' s t ) . Data information : experiments were repeated three times with similar results .

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(C) Pto DC3000 and B. subtilis upregulate phosphate absorption gene expression. Col-0, phr1 and fer-4 mutant roots were harvested 48 hours after inoculation with Pto DC3000 or B. subtilis in Pi-sufficient or Pi-deficient condition, and the relative expression of Pi-absorbing genes was quantified via RT-qPCR. The data shown indicate the means ± SDs (n = 3, n refers to technical replicates); *, p < 0.05; **, p < 0.01 (Student's t test). Data information: All experiments were repeated three times with similar results.


fer - 4 rhizosphere microbiome promotes growth alleviates LP stress . Arabidopsis seeds were germinated on 1 / 2 - strength MS media for 3 days then transplanted to either HP or LP media for another 5 days . seedlings were then transferred to vermiculite inoculated with Col - 0 or fer - 4 rhizosphere microbiomes . ( B ) , were taken 4 after inoculation . Data information : experiments were repeated three times with similar results .

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

The fer-4 rhizosphere microbiome promotes plant growth and alleviates LP stress. Arabidopsis seeds were germinated on 1/2-strength MS media for 3 days and then transplanted to either HP or LP media for another 5 days. The seedlings were then transferred to vermiculite and inoculated with Col-0 or fer-4 rhizosphere microbiomes. (B), Images were taken 4 weeks after inoculation. Data information: All experiments were repeated three times with similar results.


( D ) fer - 4 rhizosphere microbiome upregulated phosphate absorption gene expression . roots of Col - 0 were harvested after 4 of inoculation with Col - 0 or fer - 4 rhizosphere microbiome under LP condition , relative expression of Pi - absorbing genes was quantified via RT - qPCR . data shown indicate means ± SDs ( n = 3 , n refers to technical replicates ) ; n . s . , not significant ; * , p < 0 . 05 ; * * , p < 0 . 01 ( Student ' s t ) . Data information : experiments were repeated three times with similar results .

(D) The fer-4 rhizosphere microbiome upregulated phosphate absorption gene expression. The roots of Col-0 were harvested after 4 weeks of inoculation with the Col-0 or fer-4 rhizosphere microbiome under LP condition, and the relative expression of Pi-absorbing genes was quantified via RT-qPCR. The data shown indicate the means ± SDs (n = 3, n refers to technical replicates); n.s., not significant; *, p < 0.05; **, p < 0.01 (Student's t test). Data information: All experiments were repeated three times with similar results.


( A ) Left quadriceps muscle biopsy performed at 10 months ( i - iii ) demonstrating a dystrophic process with variation in fiber ( black arrow ) , increased connective tissue ( blue arrow ) rare internalized nuclei ( arrow ) on hematoxylin eosin ( H & E ) ( i ) Gömöritrichrome ( ii ) type 1 fiber atrophy on nicotinamide adenine dinucleotide ( NADH ) ( iii ) . Muscle biopsy in an unaffected ( control ) ( iv - vi ) . Scale bar : 100μm .

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(A) Left quadriceps muscle biopsy performed at age 10 months (i-iii) demonstrating a dystrophic process with variation in fiber size (black arrow), increased connective tissue (blue arrow) and rare internalized nuclei (white arrow) on hematoxylin and eosin (H&E) staining (i) and Gömöritrichrome staining (ii) and type 1 fiber atrophy on nicotinamide adenine dinucleotide (NADH) staining (iii). Muscle biopsy in an unaffected (control) individual (iv-vi). Scale bar: 100μm.


( B ) Western blot data ( n = 1 ) with demonstrated reduced α - Dystroglycan ( α - DG ) in P1 ' s muscle compared to control . ( C ) Western blot data with demonstrated reduced α - Dystroglycan in fibroblasts from P1 P2 compared to control ( c ) , although not to extent of reduction observed in fibroblasts from a with a confirmed α - dystroglycanopathy ( α - DG pt ) due to biallelic pathogenic variants in LARGE ( n = 1 ) .

(B) Western blot data (n=1) with quantification demonstrated reduced α-Dystroglycan (α-DG) in P1's muscle compared to control. (C) Western blot data with quantification demonstrated reduced α-Dystroglycan in fibroblasts from P1 and P2 compared to control (c), although not to the extent of the reduction observed in fibroblasts from a patient with a confirmed α-dystroglycanopathy (α-DG pt) due to biallelic pathogenic variants in LARGE (n=1).


( D ) Brain MRI findings in P1 at 28 months of ( top ) revealing normal appearing cortex , cerebellum pons on T1 - weighted T2 - weighted image . There is evidence of mild thinning of corpus callosum ( arrow ) . Brain MRI in an unaffected ( control ) at 30 months of ( bottom ) demonstrating normal sized corpus callosum .

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(D) Brain MRI findings in P1 at 28 months of age (top) revealing normal appearing cortex, cerebellum and pons on T1-weighted images and T2-weighted image. There is evidence of mild thinning of the corpus callosum (arrow). Brain MRI in an unaffected (control) individual at 30 months of age (bottom) demonstrating normal sized corpus callosum.


( B ) Sashimi plots comparing muscle sequencing reads in P1 ( red ) two control muscle samples ( blue green ) at exons 3 - 5 of BET1 .

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(B) Sashimi plots comparing muscle RNA sequencing reads in P1 (red) and two control muscle samples (blue and green) at the exons 3-5 of BET1.


( A ) Immunoblot of fibroblast lines ( P1 P2 ) three independent controls ( C1 - 3 ) . Immunoblots were probed with indicated antibodies . of BET1 normalized to GAPDH . Data represented are mean ± SD of three independent biological experiments . Significant differences of P1 BET1 - were checked compared to each control ( * * * = p < 0 . 001 , one - way - ANOVA followed by Tukey ` s post - hoc ) .

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(A) Immunoblot of patient fibroblast cell lines (P1 and P2) and three independent controls (C1-3). Immunoblots were probed with the indicated primary antibodies. Quantification of BET1 protein levels normalized to GAPDH. Data represented are mean ± SD of three independent biological experiments. Significant differences of P1 BET1-levels were checked compared to each control (***=p<0.001, one-way-ANOVA followed by Tukey`s post-hoc test).


( B ) Subcellular localization of BET1 in control fibroblasts by indirect immunofluorescent confocal microscopy . Representative immunofluorescence of BET1 ( red ) cis - Golgi marker GM130 ( green ) . Scale bar : 20µm . Colocalization of BET1 with GM130 was determined using Pearson ' s correlation coefficient ( PCC ) measured per ( right panel ; C1 : n = 63 , C2 : n = 50 , C3 : n = 49 P2 : n = 33 ) ( * * * = p ≤ 0 . 001 , one - way ANOVA was followed by Tukey ' s post - hoc ) . Means are shown ± . ( C ) Representative immunofluorescence of BET1 ( red ) ER marker PDI ( green ) . Scale bar : 20 µm . Right panel shows PCCs for BET1 versus PDI ( C1 : n = 61 , C2 : n = 42 , C3 : n = 59 P2 : n = 32 of two biological replicates ) . ( * * * = p ≤ 0 . 001 , one - way ANOVA was followed by Tukey ' s post - hoc ) . Means are shown ± .

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(B) Subcellular localization of BET1 in control and patient fibroblasts by indirect immunofluorescent confocal microscopy. Representative immunofluorescence images of BET1 (red) and cis-Golgi marker GM130 (green). Scale bar: 20µm.Colocalization of BET1 with GM130 was determined using Pearson's correlation coefficient (PCC) measured per cell (right panel; C1: n=63, C2: n=50, C3: n=49 and P2: n=33) (***=p≤0.001, one-way ANOVA was followed by Tukey's post-hoc test). Means are shown ± SEM. (C) Representative immunofluorescence images of BET1 (red) and ER marker PDI (green). Scale bar: 20 µm. Right panel shows PCCs for BET1 versus PDI (C1: n=61, C2: n=42, C3: n=59 and P2: n=32 of two biological replicates). (***=p≤0.001, one-way ANOVA was followed by Tukey's post-hoc test). Means are shown ± SEM.


( D ) HeLa - stably expressing pC4 - reporter were transfected with control siRNA ( ctrl ) or two independent siRNAs ( # 1 # 2 ) directed against BET1 ( siRNA # 1 is shown in figure EV 2B ) . 2d post - transfection pC4 - secretion was performed for 0 10 min post induction of secretion . Representative are shown ( upper panel ctrl lower panel # 2 siRNA , respectively . Scale bar : 20 µm . Right panels represent magnifications of dashed areas .

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(D) HeLa-cells stably expressing the pC4-reporter were transfected with control siRNA (ctrl) or two independent siRNAs (#1 and #2) directed against BET1 (siRNA #1 is shown in figure EV 2B). 2d post-transfection the pC4-secretion assay was performed for 0 and 10 min post induction of secretion. Representative images are shown (upper panel ctrl and lower panel #2 siRNA, respectively. Scale bar: 20 µm. Right panels represent magnifications of dashed areas.


( B ) Stability of hydrogen bonds between BET1Asp68 BET1Asp72 respectively was estimated using molecular dynamics simulation in three replicas . Parameter occupancy could be more than 100 % if several stable hydrogen bonds could be established by residue . Bars represent average fraction of simulated time residue is involved in a hydrogen bond ( s ) . Data represented are mean ± SD .

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

(B) Stability of hydrogen bonds between BET1Asp68 and BET1Asp72 respectively was estimated using molecular dynamics simulation in three replicas. Parameter occupancy could be more than 100% if several stable hydrogen bonds could be established by residue. Bars represent average fraction of the simulated time the residue is involved in a hydrogen bond(s). Data represented are mean ± SD.


( A ) Volcano plot of AP - MS data using HA - BET1 or Ile51Ser .

(A) Volcano plot of AP-MS data using HA-BET1 or Ile51Ser.


( B ) Co - IPs using HA - tagged constructs expressed in HEK293 . As baits HA - tagged were immunoprecipitated with anti - HA beads . IP samples lysates were subjected to immunoblotting with α - HA , α - ERGIC - 53 , α - BET1 , α - SEC22b , α - GOSR2 α - GAPDH antibodies , respectively .

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(B) Co-IPs using HA-tagged constructs expressed in HEK293 cells. As baits HA-tagged proteins were immunoprecipitated with anti-HA beads. IP samples and cell lysates were subjected to immunoblotting with α-HA, α-ERGIC-53, α-BET1, α-SEC22b, α-GOSR2 and α-GAPDH antibodies, respectively.


( E ) Subcellular localization of ERGIC - 53 in fibroblasts by indirect immunofluorescent confocal microscopy . Representative immunofluorescence of ERGIC - 53 ( green ) GM130 ( red ) in fibroblasts from P1 , P2 a control . Scale bar : 20 µm . PCC ( right panel ) for GM130 versus ERGIC - 53 ( C1 : n = 74 , C2 : n = 95 , C3 : n = 59 , P1 : n = 72 P2 : n = 76 ) . ( * * * = p ≤ 0 . 001 , one - way ANOVA was followed by Tukey ' s post - hoc ) . Shown are means ± . Immunofluorescence shown are one example of a total of two biological replicates .

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(E) Subcellular localization of ERGIC-53 in fibroblasts by indirect immunofluorescent confocal microscopy. Representative immunofluorescence images of ERGIC-53 (green) and GM130 (red) in fibroblasts from P1, P2 and a control cell line. Scale bar: 20 µm. PCC (right panel) for GM130 versus ERGIC-53 (C1: n=74, C2: n=95, C3: n=59, P1: n=72 and P2: n=76). (*** = p≤ 0.001, one-way ANOVA was followed by Tukey's post-hoc test). Shown are means ± SEM. Immunofluorescence images shown are one example of a total of two biological replicates.


( F ) Colocalization of ERGIC - 53 BET1 in control P2 - derived fibroblasts . Representative immunofluorescence of ERGIC - 53 ( green ) BET1 ( red ) in fibroblasts from P2 a control . Scale bar : 20 µm . PCC ( right panel ) for BET1 versus ERGIC - 53 ( C1 : n = 72 , C2 : n = 46 , C3 : n = 56 P2 : n = 37 ) . ( * * * = p ≤ 0 . 001 , one - way ANOVA was followed by Tukey ' s post - hoc ) . Data represented are mean ± of two independent experiments .

(F) Colocalization of ERGIC-53 and BET1 in control and P2-derived fibroblasts. Representative immunofluorescence images of ERGIC-53 (green) and BET1 (red) in fibroblasts from P2 and a control cell line. Scale bar: 20 µm. PCC (right panel) for BET1 versus ERGIC-53 (C1: n=72, C2: n=46, C3: n=56 and P2: n=37). (*** = p≤ 0.001, one-way ANOVA was followed by Tukey's post-hoc test). Data represented are mean ± SEM of two independent experiments.


( H ) number of with intact Golgi was divided by number of total per image plotted against time ( three biological replicates ) . Dots are means ± SD . For , a sigmoidal fit was performed with software GraphPad Prism 7 . Dashed lines indicate extrapolated plateaus . LogIC50 ± 95 % confidence interval : C1 = 59 . 38 ± 2 . 31 min , P1 = 72 . 89 ± 1 . 65 min , P2 = 68 . 57 ± 1 . 929 min . Significant differences were assumed when 95 % confidence interval do not overlap .

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(H) The number of cells with intact Golgi was divided by the number of total cells per image and plotted against time (three biological replicates). Dots are means ±SD. For quantification, a sigmoidal fit was performed with the software GraphPad Prism 7. Dashed lines indicate extrapolated plateaus. LogIC50 ±95% confidence interval: C1 = 59.38±2.31 min, P1 = 72.89±1.65 min, P2 = 68.57±1.929 min. Significant differences were assumed when 95% confidence interval do not overlap.


( A ) Heat map representation of TNF - α - up - regulated genes . Genes were classified on TNF - α - treated time points , using algorithm " HOPACH " .

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(A) Heat map representation of the TNF-α-up-regulated genes. Genes were classified based on the TNF-α-treated time points, using the algorithm "HOPACH".


( B ) Heat map showing down - regulated miRNAs at 4 hrs , but not 24 hrs , after TNF - α treatment . middle column indicates fold - change values ( control / TNF ) at 4 hrs . right column indicates relative expression level of each miRNA from FANTOM5 database ( Consortium et al , 2014 ) . Asterisks indicate top three most down - regulated miRNAs which were reported to be expressed in human endothelial ( ECs ) according to FANTOM5 database .

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(B) Heat map showing down-regulated miRNAs at 4 hrs, but not 24 hrs, after TNF-α treatment. The middle column indicates the fold-change values (control/TNF) at 4 hrs. The right column indicates the relative expression level of each miRNA from the FANTOM5 database (Consortium et al, 2014). Asterisks indicate the top three most down-regulated miRNAs which were reported to be expressed in human endothelial cells (ECs) according to the FANTOM5 database.


( C ) Bar plots showing mean mRNA of vascular adhesion molecule - 1 ( VCAM1 ) , intracellular adhesion molecule - 1 ( ICAM1 ) , E - selectin ( SELE ) , measured 4 hrs after stimulation of human ECs with or without TNF - α ± miR - 3679 - 5p . Data are shown as means ± SE ( n = 3 ) . * P < 0 . 05 compared to TNF - α ( - ) mimics ( - ) . † P < 0 . 05 ‡ P = 0 . 203 compared to TNF - α ( + ) mimics ( - ) . Statistical differences were analyzed by Tukey - Kramer .

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(C) Bar plots showing mean mRNA levels of vascular cell adhesion molecule-1 (VCAM1), intracellular adhesion molecule-1 (ICAM1), and E-selectin (SELE), measured 4 hrs after stimulation of human ECs with or without TNF-α ± miR-3679-5p. Data are shown as means ± SE (n=3). *P < 0.05 compared to TNF-α (-) mimics (-). † P < 0.05 and ‡ P = 0.203 compared to TNF-α (+) mimics (-). Statistical differences were analyzed by the Tukey-Kramer test.


( D ) Western blots for VCAM1 , ICAM , SELE , β - actin ( ACTB ) in lysates from ECs treated with or without TNF - α ( 4 hrs ) ± miR - 3679 - 5p .

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(D) Western blots for VCAM1, ICAM, SELE, and β-actin (ACTB) in lysates from ECs treated with or without TNF-α (4 hrs) ± miR-3679-5p.


( E ) Representative ( top ) bar plot ( bottom ) showing adhesion of calcein - labeled U937 monocytes to ECs treated with or without TNF - α ± miR - 3679 - 5p . Scale bar represents 250 μm . Graphs are representative of three independent experiments . Data are shown as means ± SD . * P < 0 . 05 compared to TNF - α ( - ) mimics ( - ) . † P < 0 . 05 compared to TNF - α ( + ) mimics ( - ) . Statistical differences were analyzed by Tukey - Kramer .

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(E) Representative images (top) and bar plot quantification (bottom) showing the adhesion of calcein-labeled U937 monocytes to ECs treated with or without TNF-α ± miR-3679-5p. Scale bar represents 250 μm. Graphs are representative of three independent experiments. Data are shown as means ± SD. *P < 0.05 compared to TNF-α (-) mimics (-). † P < 0.05 compared to TNF-α (+) mimics (-). Statistical differences were analyzed by the Tukey-Kramer test.


( F ) scatter plot shows correlation of immunoprecipitation ( RIP ) followed by microarray data from two replicate screens for identification of miR - 3679 - 5p target genes . Genes up - down - regulated by miR - 3679 - 5p treatment ( | average Log2FC | > 1 ) are indicated in red blue , respectively .

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(F) The scatter plot shows the correlation of RNA immunoprecipitation (RIP)　followed by microarray data from two replicate screens for the identification of miR-3679-5p target genes. Genes up- and down-regulated by miR-3679-5p treatment (|average Log2FC|>1) are indicated in red and blue, respectively.


( J K ) Effect of miR - 3679 - 5p mimics ( J ) or miR - 3679 - 5p inhibitor ( K ) on luciferase activity in pmirGLO - transfected ECs expressing 3 ′ UTR of KDM7A . Data are shown as means ± SE ( n = 3 ) . * P < 0 . 05 compared to control . Statistical differences were analyzed by Student ' s t .

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(J and K) Effect of miR-3679-5p mimics (J) or miR-3679-5p inhibitor (K) on luciferase activity in pmirGLO-transfected ECs expressing the 3′ UTR of KDM7A. Data are shown as means ± SE (n=3). *P < 0.05 compared to control. Statistical differences were analyzed by the Student's t test.


( M N ) effect of miR - 3679 - 5p mimics ( M ) or miR - 3679 - 5p inhibitor ( N ) on luciferase activity in pmirGLO - transfected ECs expressing 3 ′ UTR of UTX was measured . Data are shown as means ± SE ( n = 3 ) . * P < 0 . 05 compared to control . Statistical differences were analyzed by Student ' s t .

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(M and N) The effect of miR-3679-5p mimics (M) or miR-3679-5p inhibitor (N) on luciferase activity in pmirGLO-transfected ECs expressing the 3′ UTR of UTX was measured. Data are shown as means ± SE (n=3). *P < 0.05 compared to control. Statistical differences were analyzed by the Student's t test.


( A ) Heat map representation of - seq results . p65 - dependent genes ( total 123 ; defined as genes up - regulated more than 2 - fold via TNF - α treatment down - regulated more than 0 . 75 - fold via BAY 11 - 7082 treatment ) were classified on alteration patterns by siRNA knockdown of KDM7A , UTX , both , using algorithm " HOPACH " .

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(A) Heat map representation of RNA-seq results. The p65-dependent genes (total 123; defined as genes up-regulated more than 2-fold via TNF-α treatment and down-regulated more than 0.75-fold via BAY 11-7082 treatment) were classified based on the alteration patterns by the siRNA knockdown of KDM7A, UTX, and both, using the algorithm "HOPACH".


( C - E ) Bar plot showing mean mRNA of VCAM1 , ICAM1 , SELE measured 4 hrs after stimulation of ECs with or without TNF - α ± ( siKDM7A + siUTX ) . Data are shown as means ± SE ( n = 3 ) . * P < 0 . 05 compared to TNF - α ( - ) siKDM7A + siUTX ( - ) . † P < 0 . 05 compared to TNF - α ( + ) siKDM7A + siUTX ( - ) . Statistical differences were analyzed by Tukey - Kramer .

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(C-E) Bar plot showing mean mRNA levels of VCAM1, ICAM1, and SELE measured 4 hrs after stimulation of ECs with or without TNF-α ± (siKDM7A+siUTX). Data are shown as means ± SE (n=3). *P < 0.05 compared to TNF-α (-) siKDM7A+siUTX (-). † P < 0.05 compared to TNF-α (+) siKDM7A+siUTX (-). Statistical differences were analyzed by the Tukey-Kramer test.


( F ) Western blots for VCAM1 , ICAM , SELE , ACTB in lysates from ECs treated with or without TNF - α ( 4 hrs ) ± ( siKDM7A + siUTX ) .

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(F) Western blots for VCAM1, ICAM, SELE, and ACTB in lysates from ECs treated with or without TNF-α (4 hrs) ± (siKDM7A+siUTX).


( G ) Representative ( top ) bar plot ( bottom ) showing adhesion of calcein - labeled U937 monocytes to ECs treated with or without TNF - α ± ( siKDM7A + siUTX ) . Scale bar represents 250 μm . Graphs are representative of three independent experiments . Data are shown as means ± SD . * P < 0 . 05 compared to TNF - α ( - ) siKDM7A + siUTX ( - ) . † P < 0 . 05 compared to TNF - α ( + ) siKDM7A + siUTX ( - ) . Statistical differences were analyzed by Tukey - Kramer .

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(G) Representative images (top) and bar plot quantification (bottom) showing the adhesion of calcein-labeled U937 monocytes to ECs treated with or without TNF-α ± (siKDM7A+siUTX). Scale bar represents 250 μm. Graphs are representative of three independent experiments. Data are shown as means ± SD. *P < 0.05 compared to TNF-α (-) siKDM7A+siUTX (-). † P < 0.05 compared to TNF-α (+) siKDM7A+siUTX (-). Statistical differences were analyzed by the Tukey-Kramer test.


( A B ) Scatter plots showing connection between KDM7A or UTX binding expression in TNF - α - stimulated ECs . X Y axes indicate log2 of fragments per kilobase of exon per million mapped reads ( FPKM ) values ChIP - seq signals ( TNF - α 60min / 0min ) for KDM7A ( A ) UTX ( B ) , respectively . Genes with more than a 1 . 5 - fold increase in FPKM values ( red dots ) satisfying a 0 . 5 - fold increase in ChIP - seq signals are indicated as red dots . Genes with a 1 . 5 - fold decrease in FPKM values ( blue dots ) satisfying a 0 . 5 - fold decrease in ChIP - seq signals are indicated as blue dots . These genes are defined as target genes for KDM7A ( A ) UTX ( B ) , respectively . names of representative genes are shown in graphs .

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(A and B) Scatter plots showing the connection between KDM7A or UTX binding and RNA expression in TNF-α-stimulated ECs. The X and Y axes indicate the log2 ratio of fragments per kilobase of exon per million mapped sequence reads (FPKM) values and the ChIP-seq signals (TNF-α 60min/0min) for KDM7A (A) and UTX (B), respectively. Genes with more than a 1.5-fold increase in FPKM values (light red dots) satisfying a 0.5-fold increase in ChIP-seq signals are indicated as dark red dots. Genes with a 1.5-fold decrease in FPKM values (light blue dots) satisfying a 0.5-fold decrease in ChIP-seq signals are indicated as dark blue dots. These genes are defined as the target genes for KDM7A (A) and UTX (B), respectively. The names of representative genes are shown in the graphs.


( C D ) Metagene representations of KDM7A ( C ) UTX ( D ) ChIP - seq signals in units of read per million mapped reads at a meta composite of genomic regions around TNF - α - up - regulated genes .

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(C and D) Metagene representations of KDM7A (C) and UTX (D) ChIP-seq signals in units of read count per million mapped reads at a meta composite of the genomic regions around the TNF-α-up-regulated genes.


( F ) Heat map of H3K27ac ( green ) , p65 ( yellow ) , KDM7A ( red ) , UTX ( purple ) in resting TNF - α - stimulated ECs . Each row shows ± 10 kb centered on KDM7A ( top ) or UTX ( bottom ) peak . Rows are ordered by max KDM7A or UTX signal in each region . ChIP - seq signal is depicted by color scaled intensities .

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(F) Heat map of H3K27ac (green), p65 (yellow), KDM7A (red), and UTX (purple) levels in resting and TNF-α-stimulated ECs. Each row shows ± 10 kb centered on the KDM7A (top) or UTX (bottom) peak. Rows are ordered by max KDM7A or UTX signal in each region. The ChIP-seq signal is depicted by color scaled intensities.


( G H ) Gene tracks of ChIP - seq signals for H3K27Ac , p65 , KDM7A , UTX , - seq signals around VCAM1 ( G ) SELE ( H ) loci in TNF - α ( - ) TNF - α ( + ) ECs . ChIP - Seq - seq signals are visualized by Integrated Genome Viewer ( http : / / software . broadinstitute . org / software / igv / ) .

(G and H) Gene tracks of ChIP-seq signals for H3K27Ac, p65, KDM7A, and UTX, and RNA-seq signals around the VCAM1 (G) and SELE (H) loci in TNF-α (-) and TNF-α (+) ECs. ChIP-Seq and RNA-seq signals are visualized by Integrated Genome Viewer (http://software.broadinstitute.org/software/igv/).


( I J ) ChIP - qPCR of p65 ( I ) KDM7A ( J ) at VCAM1 TSS , normalized to input . Graphs are representative of three independent experiments . Data are shown as means ± SD . * P < 0 . 05 compared to TNF - α ( - ) . † P < 0 . 05 compared to TNF - α ( + ) siKDM7A ( - ) . Statistical differences were analyzed by Tukey - Kramer .

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(I and J) ChIP-qPCR of p65 (I) and KDM7A (J) at the VCAM1 TSS, normalized to input. Graphs are representative of three independent experiments. Data are shown as means ± SD. *P < 0.05 compared to TNF-α (-). † P < 0.05 compared to TNF-α (+) siKDM7A (-). Statistical differences were analyzed by the Tukey-Kramer test.


( K L ) ChIP - qPCR of p65 ( K ) UTX ( L ) at SELE TSS , normalized to input . Graphs are representative of three independent experiments . Data are shown as means ± SD . * P < 0 . 05 compared to TNF - α ( - ) . Statistical differences were analyzed by Tukey - Kramer .

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(K and L) ChIP-qPCR of p65 (K) and UTX (L) at the SELE TSS, normalized to input. Graphs are representative of three independent experiments. Data are shown as means ± SD. *P < 0.05 compared to TNF-α (-). Statistical differences were analyzed by the Tukey-Kramer test.


( A B ) Boxplots of log2 fold change of ChIP - seq enrichment of H3K9me2 in KDM7A target genes ( A ) that of H3K27me3 in UTX target genes ( B ) with TNF - α treatment ( TNF + ) control ( TNF - ) . horizontal within box represents median sample value . ends of box represent 3rd 1st quantiles . whiskers extend from ends of box to outermost data point that falls within distances computed as follows : 3rd quartile + 1 . 5 * ( interquartile range ) 1st quartile - 1 . 5 * ( interquartile range ) . Each dot in box plot shows data for one single target gene . Genes colored red represent top five downregulated target genes with TNF - α - treatment , as compared to control . Graphs are representative of two independent experiments .

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(A and B) Boxplots of the log2 fold change of ChIP-seq enrichment of H3K9me2 in KDM7A target genes (A) and that of H3K27me3 in UTX target genes (B) with TNF-α treatment (TNF+) and control (TNF-). The horizontal line within the box represents the median sample value. The ends of the box represent the 3rd and 1st quantiles. The whiskers extend from the ends of the box to the outermost data point that falls within the distances computed as follows: 3rd quartile + 1.5*(interquartile range) and 1st quartile - 1.5*(interquartile range). Each dot in the box plot shows data for one single target gene. Genes colored red represent the top five downregulated target genes with TNF-α-treatment, as compared to control. Graphs are representative of two independent experiments.


( C ) Gene tracks of ChIP - seq signals for H3K9me2 H3K27me3 around VCAM1 locus in TNF - α ( - ) TNF - α ( + ) ECs .

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(C) Gene tracks of ChIP-seq signals for H3K9me2 and H3K27me3 around the VCAM1 locus in TNF-α (-) and TNF-α (+) ECs.


( D E ) ChIP - qPCR showing enrichment ( input ) of H3K9me2 ( D ) H3K27me3 ( E ) around VCAM1 locus . Primer pairs targeting distinct regions are listed on X axis . Graphs are representative of three independent experiments . Data are shown as means ± SD . * P < 0 . 05 compared to TNF - α ( - ) . Statistical differences were analyzed by Student ' s t .

(D and E) ChIP-qPCR showing enrichment (percent input) of H3K9me2 (D) and H3K27me3 (E) around the VCAM1 locus. Primer pairs targeting distinct regions are listed on the X axis. Graphs are representative of three independent experiments. Data are shown as means ± SD. *P < 0.05 compared to TNF-α (-). Statistical differences were analyzed by the Student's t test.


( F ) Gene tracks of ChIP - seq signals for H3K9me2 H3K27me3 around SELE locus in TNF - α ( - ) TNF - α ( + ) ECs .

(F) Gene tracks of ChIP-seq signals for H3K9me2 and H3K27me3 around the SELE locus in TNF-α (-) and TNF-α (+) ECs.


( G H ) ChIP - qPCR showing enrichment ( input ) of H3K9me2 ( G ) H3K27me3 ( H ) around SELE locus . Primer pairs targeting distinct regions are listed on X axis . Graphs are representative of three independent experiments . Data are shown as means ± SD . * P < 0 . 05 compared to TNF - α ( - ) . Statistical differences were analyzed by Student ' s t .

(G and H) ChIP-qPCR showing enrichment (percent input) of H3K9me2 (G) and H3K27me3 (H) around the SELE locus. Primer pairs targeting distinct regions are listed on the X axis. Graphs are representative of three independent experiments. Data are shown as means ± SD. *P < 0.05 compared to TNF-α (-). Statistical differences were analyzed by the Student's t test.


( I J ) ChIP - qPCR showing enrichment ( input ) of H3K9me2 H3K27me3 around HOXA13 ( I ) GATA4 ( J ) loci . Graphs are representative of three independent experiments . Data are shown as means ± SD . * P < 0 . 05 compared to TNF - α ( - ) . Statistical differences were analyzed by Student ' s t .

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(I and J) ChIP-qPCR showing enrichment (percent input) of H3K9me2 and H3K27me3 around the HOXA13 (I) and GATA4 (J) loci. Graphs are representative of three independent experiments. Data are shown as means ± SD. *P < 0.05 compared to TNF-α (-). Statistical differences were analyzed by the Student's t test.


( K L ) ChIP - qPCR showing enrichment ( input ) of H3K9me2 around VCAM1 - 11kb ( K ) H3K27me3 around SELE - 12kb ( L ) . Graphs are representative of three independent experiments . Data are shown as means ± SD . * P < 0 . 05 compared to TNF - α ( - ) . † P < 0 . 05 compared to TNF - α ( + ) siKDM7A ( - ) or TNF - α ( + ) siUTX ( - ) . Statistical differences were analyzed by Tukey - Kramer .

(K and L) ChIP-qPCR showing enrichment (percent input) of H3K9me2 around VCAM1 -11kb (K) and H3K27me3 around SELE -12kb (L). Graphs are representative of three independent experiments. Data are shown as means ± SD. *P < 0.05 compared to TNF-α (-). † P < 0.05 compared to TNF-α (+) siKDM7A (-) or TNF-α (+) siUTX (-). Statistical differences were analyzed by the Tukey-Kramer test.


( A ) Plot of enhancers defined in TNF - α - treated ECs , ranked by increasing bromodomain - containing 4 ( BRD4 ) signal . Super enhancers ( SEs ) were defined by ROSE ( Loven et al , 2013 , Whyte et al , 2013 ) , on ChIP - Seq signals for BRD4 ( Brown et al , 2014 ) . SEs are indicated by dashed lines are colored yellow . numbers show total SEs their classification on KDM7A UTX binding . Representative SE - related genes are indicated in graph .

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(A) Plot of enhancers defined in TNF-α-treated ECs, ranked by increasing bromodomain-containing protein 4 (BRD4) signal. Super enhancers (SEs) were defined by ROSE (Loven et al, 2013, Whyte et al, 2013), based on the ChIP-Seq signals for BRD4 (Brown et al, 2014). SEs are indicated by dashed lines and are colored yellow. The numbers show the total SEs and their classification based on KDM7A and UTX binding. Representative SE-related genes are indicated in the graph.


( B C ) Metagene representations of KDM7A ( B ) UTX ( C ) ChIP - seq signals in units of read per million mapped reads , at a meta composite of SEs typical enhancers ( TEs ) . TEs were obtained from pre - compiled HUVEC ngsplotdb ( https : / / github . com / shenlab - sinai / ngsplot ) .

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(B and C) Metagene representations of KDM7A (B) and UTX (C) ChIP-seq signals in units of read count per million mapped reads, at a meta composite of SEs and typical enhancers (TEs). TEs were obtained from the pre-compiled HUVEC ngsplotdb (https://github.com/shenlab-sinai/ngsplot).


( D ) Pirate plots showing ChIA - PET interaction changes at control - specific , common , TNF - α - specific SEs . Y axis indicates log2 of ChIA - PET signals ( TNF - α 60 min / 0 min ) . central bar indicates average .

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(D) Pirate plots showing ChIA-PET interaction changes at control-specific, common, and TNF-α-specific SEs. The Y axis indicates the log2 ratio of ChIA-PET signals (TNF-α 60 min/0 min). The central bar indicates the average.


( E F ) Bean plots showing ChIA - PET interaction changes at KDM7A ( E ) UTX ( F ) binding sites . Y axis indicates log2 of ChIA - PET signals ( TNF - α 60 min / 0 min ) . central bar indicates median .

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(E and F) Bean plots showing ChIA-PET interaction changes at KDM7A (E) and UTX (F) binding sites. The Y axis indicates the log2 ratio of ChIA-PET signals (TNF-α 60 min/0 min). The central bar indicates the median.


( G H ) ChIA - PET interactions of active pol II around VCAM1 ( G ) IL8 ( H ) loci integrated with ChIP - seq profiles of KDM7A , UTX , BRD4 , - seq profiles in TNF - α ( - ) TNF - α ( + ) ECs . ChIA - PET interactions were visualized by WashU Epigenome Browser ( http : / / epigenomegateway . wustl . edu / browser / ) . Interactions detected by ChIA - PET are depicted with purple lines . Red bars show TNF - α - specific SEs .

(G and H) ChIA-PET interactions of active RNA pol II around the VCAM1 (G) and IL8 (H) loci integrated with the ChIP-seq profiles of KDM7A, UTX, and BRD4, and RNA-seq profiles in TNF-α (-) and TNF-α (+) ECs. ChIA-PET interactions were visualized by the WashU Epigenome Browser (http://epigenomegateway.wustl.edu/browser/). Interactions detected by ChIA-PET are depicted with purple lines. Red bars show TNF-α-specific SEs.


( I - K ) Gene tracks of ChIP - seq signals for H3K9me2 H3K27me3 around VCAM1 ( I ) , IL8 ( J ) , SELE ( K ) in TNF - α ( - ) TNF - α ( + ) ECs . Red bars show TNF - α - specific SEs .

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(I-K) Gene tracks of ChIP-seq signals for H3K9me2 and H3K27me3 around VCAM1 (I), IL8 (J), and SELE (K) in TNF-α (-) and TNF-α (+) ECs. Red bars show TNF-α-specific SEs.


( B ) Representative snapshot from IVM of leukocyte adhesive interactions in femoral arteries . Margins of vessels are indicated with dashed lines . spots represent fluorescently labeled leukocytes visualized by intravenous injection of rhodamine 6G .

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(B) Representative snapshot from the IVM analysis of leukocyte adhesive interactions in the femoral arteries. Margins of vessels are indicated with dashed lines. White spots represent fluorescently labeled leukocytes visualized by the intravenous injection of rhodamine 6G.


( C ) Quantitative analyses of leukocyte adhesive interactions in femoral arteries . Data are shown as means ± SE ( n = 3 / group ) . * P < 0 . 05 compared to vehicle + TNF - α group . Statistical differences were analyzed by Student ' s t . See also Movies EV1 2 .

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(C) Quantitative analyses of leukocyte adhesive interactions in the femoral arteries. Data are shown as means ± SE (n=3/group). *P < 0.05 compared to the vehicle + TNF-α group. Statistical differences were analyzed by the Student's t test. See also Movies EV1 and 2.


RT - qPCR of METTL3 METTL14 expression ( A ) in shRNA knockdown METTL3 .

RT-qPCR analysis of METTL3 and METTL14 expression (A) in shRNA knockdown METTL3 cells.


I HA - METTL14 Co - IP with FLAG - tagged METTL3 domain - deletion mutants . β - actin was used as loading control .

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I HA-METTL14 Co-IP with FLAG-tagged METTL3 domain-deletion mutants. β-actin was used as the loading control.


F Immunoblot of FLAG - METTL14 , LC3 - II , β - actin in HEK293T exposed to autophagy inhibitor Baf - A1 activator Torin 1 , respectively . FLAG - METTL14 or LC3 - II / β - actin densitometric was recorded by ImageJ .

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F Immunoblot analysis of FLAG-METTL14, LC3-II, and β-actin in HEK293T cells exposed to the autophagy inhibitor Baf-A1 and activator Torin 1, respectively. The FLAG-METTL14 or LC3-II /β-actin densitometric ratio was recorded by ImageJ.


G Dot blots of m6A in presence of MG132 in 293T , RBE , SK - Cha - 1 , SK - N - SH .

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G Dot blots of m6A levels in the presence of MG132 in 293T, RBE, SK-Cha-1, and SK-N-SH cells.


G , H HA - METTL14 Co - IP with FLAG - tagged STUB1 - domain - deletion mutants .

G, H HA-METTL14 Co-IP with FLAG-tagged STUB1-domain-deletion mutants.


L Immunoblots showing CHX in HEK293T . Negative control ( NC ; no transfected ) STUB1 - FLAG plasmid - transfected HEK293T were exposed to 100 μg / ml CHX for 0 , 4 , 8 , or 12 hours . β - actin was used as loading control . METTL14 / β - actin densitometric was recorded by ImageJ .

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L Immunoblots showing CHX assay in HEK293T cells. Negative control (NC; no transfected cells) and STUB1-FLAG plasmid-transfected HEK293T cells were exposed to 100 μg/ml CHX for 0, 4, 8, or 12 hours. β-actin was used as the loading control. The METTL14/β-actin densitometric ratio was recorded by ImageJ.


M , N Dot blot assays showing that enforced expression of wild - type STUB1 , but not STUB1H260Q mutant , significantly decreases total m6A .

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M, N Dot blot assays showing that enforced expression of wild-type STUB1, but not the STUB1H260Q mutant, significantly decreases total m6A levels.


B Immunoblots showing abundance of METTL14 truncated mutants with or without 10 µM MG132 treatment . Histone H4 was used as loading control . HA - METTL14 / β - actin densitometric was recorded by ImageJ .

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B Immunoblots showing the abundance of METTL14 truncated mutants with or without 10 µM MG132 treatment. Histone H4 was used as the loading control. The HA-METTL14/β-actin densitometric ratio was recorded by ImageJ.


F Immunoblots showing of METTL14 after transfection with METTL14 - 3KR mutant plasmids with or without 10 µM MG132 . β - actin was used as loading control . HA - METTL14 / β - actin densitometric was recorded by ImageJ . G Immunoblots showing level of METTL14 after transfection with METTL14 - 3KR mutant plasmids under STUB1 knockdown . β - actin was used as loading control . HA - METTL14 / β - actin densitometric was recorded by ImageJ .

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F Immunoblots showing the levels of METTL14 protein after transfection with METTL14-3KR mutant plasmids with or without 10 µM MG132. β-actin was used as the loading control. The HA-METTL14/β-actin densitometric ratio was recorded by ImageJ. G Immunoblots showing the protein level of METTL14 after transfection with METTL14-3KR mutant plasmids under STUB1 knockdown. β-actin was used as the loading control. The HA-METTL14/β-actin densitometric ratio was recorded by ImageJ.


B METTL14 ubiquitination , as detected by IP of METTL3 truncated mutants with anti - HA antibody . accumulation of Ub METTL14 was confirmed in whole - lysates . β - actin was used as loading control .

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B METTL14 ubiquitination, as detected by IP of METTL3 truncated mutants with anti-HA antibody. The accumulation of Ub and METTL14 was confirmed in whole-cell lysates. β-actin was used as the loading control.


C Immunoblots showing METTL14 , as detected by IP in response to METTL3 truncated mutants STUB1 - FLAG . β - actin was used as loading control .

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C Immunoblots showing METTL14 protein levels, as detected by IP in response to METTL3 truncated mutants and STUB1-FLAG. β-actin was used as the loading control.


E - G Following subcutaneous inoculation of SK - Cha - 1 - NC ( left ) SK - Cha - 1 - STUB1 - FLAG ( right ) into flanks of NOD - SCID mice ( E ) , overexpression of STUB1 - FLAG inhibited malignant proliferation ( F ) reduced subsequent growth ( G ) in vivo . Data are means ± . Statistical significance was analyzed by two - way ANOVA ( F ) or two - unpaired t - ( G ) ( Five mice , * p < 0 . 05 ; * * p < 0 . 01 ) . H - J Following subcutaneous inoculation of SK - Cha - 1 - sh - NC ( left ) SK - Cha - 1 - sh - STUB1 - 1 ( right ) into flanks of nude mice ( H ) , STUB1 knockdown promoted malignant proliferation ( I ) increased subsequent growth ( J ) . Data are means ± . Statistical significance was analyzed by two - way ANOVA ( I ) or two - unpaired t - ( J ) . ( Six mice , * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 ) .

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E-G Following the subcutaneous inoculation of SK-Cha-1-NC (left) and SK-Cha-1-STUB1-FLAG (right) cells into the flanks of male NOD-SCID mice (E), overexpression of STUB1-FLAG inhibited malignant cell proliferation (F) and reduced subsequent tumor size and growth (G) in vivo. Data are means ± SEM. Statistical significance was analyzed by two-way ANOVA (F) or two-tailed unpaired t-test (G) (Five mice, *p < 0.05; **p < 0.01). H-J Following the subcutaneous inoculation of SK-Cha-1-sh-NC (left) and SK-Cha-1-sh-STUB1-1 (right) cells into the flanks of male nude mice (H), STUB1 knockdown promoted malignant cell proliferation (I) and increased subsequent tumor size and growth (J). Data are means ± SEM. Statistical significance was analyzed by two-way ANOVA (I) or two-tailed unpaired t-test (J). (Six mice, *p < 0.05; **p < 0.01; ***p < 0.001).


Immunoblots ( K ) showing METTL14 STUB1 - FLAG overexpression in five pairs of mouse tumors . β - actin was used as loading control . METTL14 or STUB1 - FLAG / β - actin densitometric was recorded by ImageJ .

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Immunoblots (K) showing METTL14 protein levels STUB1-FLAG overexpression in five pairs of mouse tumors. β-actin was used as the loading control. The METTL14 or STUB1-FLAG /β-actin densitometric ratio was recorded by ImageJ.


B Immunoblot showing STUB1 in eight pairs of CCA samples . N , adjacent non - tissue ; T , tissue . β - actin was used as loading control . STUB1 / β - actin densitometric was recorded by ImageJ .

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B Immunoblot analysis showing STUB1 protein levels in eight pairs of CCA patient samples. N, adjacent non-tumor tissue; T, tumor tissue. β-actin was used as the loading control. The STUB1/β-actin densitometric ratio was recorded by ImageJ.


C 5 - year disease - free survival of with high expression of METTL14 ( p = 0 . 0366 ) low expression of STUB1 ( p = 0 . 0803 ) in Fudan University intrahepatic cholangiocarcinoma ( FU - iCCA ) cohort among samples within 5 - 95 % ranges of STUB1 METTL14 .

C The 5-year disease-free survival of patients with high expression levels of METTL14 (p = 0.0366) and low expression levels of STUB1 (p = 0.0803) in the Fudan University intrahepatic cholangiocarcinoma (FU-iCCA) cohort among patient samples within the 5-95% ranges of STUB1 and METTL14 protein levels.


( A ) Immunoblotting against GFP or GAPDH of extracted from HEK293 transfected for 24 hours with 80 G4C2 repeats embedded in human sense C9ORF72 fused in three possible frames with GFP deleted of its ATG .

(A) Immunoblotting against the GFP or the GAPDH of proteins extracted from HEK293 cells transfected for 24 hours with 80 G4C2 repeats embedded in the human sense C9ORF72 and fused in all three possible frames with the GFP deleted of its ATG.


( B ) LC - MS / MS spectra of N - terminal part of GFP - immunoprecipitated LysC digested polyGA expressed as in ( A ) .

(B) LC-MS/MS spectra of the N-terminal part of the GFP-immunoprecipitated and LysC digested polyGA protein expressed as in (A).


( C , D ) Immunoblotting against GFP ( C ) or HA tag ( D ) or GAPDH of extracted from HEK293 transfected for 24 hours with 3 , 20 or 80 G4C2 repeats embedded in human sense C9ORF72 fused to GFP in GA frame .

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(C, D) Immunoblotting against the GFP (C) or the HA tag (D) or the GAPDH of proteins extracted from HEK293 cells transfected for 24 hours with 3, 20 or 80 G4C2 repeats embedded in the human sense C9ORF72 sequence fused to the GFP in the GA frame.


( E ) Immunoblotting against GFP or GAPDH of extracted from HEK293 transfected with either a wild type or mutant ( AGG into CGG ) construct containing 80 G4C2 repeats embedded in sense C9ORF72 fused to GFP in GR frame .

(E) Immunoblotting against the GFP or the GAPDH of proteins extracted from HEK293 cells transfected with either a wild type or mutant (AGG into CGG) construct containing 80 G4C2 repeats embedded in sense C9ORF72 fused to the GFP in the GR frame.


( A ) Immunoblotting against HA tag or GAPDH of extracted from HEK293 transfected for 24 hours with 100 C4G2 repeats embedded in human antisense C9ORF72 fused in three possible frames with a HA tag .

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(A) Immunoblotting against the HA tag or the GAPDH of proteins extracted from HEK293 cells transfected for 24 hours with 100 C4G2 repeats embedded in the human antisense C9ORF72 and fused in all three possible frames with a HA tag.


( B ) LC - MS / MS spectra of N - terminal part of HA - immunoprecipitated LysC digested polyPG expressed as in ( A ) .

(B) LC-MS/MS spectra of the N-terminal part of the HA-immunoprecipitated and LysC digested polyPG protein expressed as in (A).


( C , D ) Immunoblotting against GFP ( C ) or HA tag ( D ) or GAPDH of extracted from HEK293 transfected for 24 hours with 3 , 10 or 100 C4G2 repeats embedded in human antisense C9ORF72 fused to GFP in PG frame .

(C, D) Immunoblotting against the GFP (C) or the HA tag (D) or the GAPDH of proteins extracted from HEK293 cells transfected for 24 hours with 3, 10 or 100 C4G2 repeats embedded in the human antisense C9ORF72 sequence fused to the GFP in the PG frame.


( A ) viability ( TO - PRO - 3 FACS ) of GT1 - 7 neuronal transfected for 24 hours with either wild type or indicated mutant constructs containing either 80 G4C2 repeats or 100 C4G2 repeats embedded in sense or antisense C9ORF72 fused to GFP in GA , PG or GR frame .

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(A) Cell viability (TO-PRO-3 FACS staining) of GT1-7 neuronal cells transfected for 24 hours with either wild type or indicated mutant constructs containing either 80 G4C2 repeats or 100 C4G2 repeats embedded in sense or antisense C9ORF72 fused to the GFP in the GA, PG or GR frame.


( B ) Immunoblotting against GFP , endogenous C9ORF72 or GAPDH of extracted from Neuro2A transfected for 24 hours with 80 G4C2 repeats embedded in human sense C9ORF72 fused to GFP in GA frame with either a control siRNA or a siRNA targeting C9orf72 mRNA .

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(B) Immunoblotting against the GFP, endogenous C9ORF72 or the GAPDH of proteins extracted from Neuro2A cells transfected for 24 hours with 80 G4C2 repeats embedded in the human sense C9ORF72 sequence fused to the GFP in the GA frame and with either a control siRNA or a siRNA targeting C9orf72 mRNA.


( C ) Immunoblotting against HA tag , endogenous C9ORF72 or GAPDH of extracted from Neuro2A transfected for 24 hours with 100 C4G2 repeats embedded in human antisense C9ORF72 fused to a HA tag in PG frame with either a control siRNA or a siRNA targeting C9orf72 mRNA or treated with Bafilomycin A1 for 15 hours .

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(C) Immunoblotting against the HA tag, endogenous C9ORF72 or GAPDH of proteins extracted from Neuro2A cells transfected for 24 hours with 100 C4G2 repeats embedded in the human antisense C9ORF72 sequence fused to a HA tag in the PG frame and with either a control siRNA or a siRNA targeting C9orf72 mRNA or treated with Bafilomycin A1 for 15 hours.


( D ) Immunoblotting against HA tag , endogenous C9ORF72 or GAPDH of extracted from Neuro2A transfected for 24 hours with 80 G4C2 repeats embedded in human sense C9ORF72 fused to a HA tag in GR frame with either a control siRNA or a siRNA targeting C9orf72 mRNA or treated with Bafilomycin A1 for 15 hours .

(D) Immunoblotting against the HA tag, endogenous C9ORF72 or GAPDH of proteins extracted from Neuro2A cells transfected for 24 hours with 80 G4C2 repeats embedded in the human sense C9ORF72 sequence fused to a HA tag in the GR frame and with either a control siRNA or a siRNA targeting C9orf72 mRNA or treated with Bafilomycin A1 for 15 hours.


( E ) viability ( TO - PRO - 3 FACS ) of GT1 - 7 neuronal co - transfected for 24 hours with either a control siRNA or a siRNA targeting C9orf72 mRNA wild - type or mutants constructs containing either 80 G4C2 repeats or 100 C4G2 repeats embedded in sense or antisense C9ORF72 fused to GFP in GA , PG or GR frame .

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(E) Cell viability (TO-PRO-3 FACS staining) of GT1-7 neuronal cells co-transfected for 24 hours with either a control siRNA or a siRNA targeting C9orf72 mRNA and wild-type or mutants constructs containing either 80 G4C2 repeats or 100 C4G2 repeats embedded in sense or antisense C9ORF72 fused to the GFP in the GA, PG or GR frame.


( A ) Left panel , immunoblotting against HA tag or GAPDH of extracted from Neuro2A transfected for 24 hours with a construct expressing 80 G4C2 repeats embedded in human sense C9ORF72 fused to a HA in GA frame treated with 10 µM of indicated compound for 15 hours . Right panel , of polyGA expression relative to GAPDH .

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(A) Left panel, immunoblotting against the HA tag or the GAPDH of proteins extracted from Neuro2A cells transfected for 24 hours with a construct expressing 80 G4C2 repeats embedded in the human sense C9ORF72 sequence fused to a HA in the GA frame and treated with 10 µM of the indicated compound for 15 hours. Right panel, quantification of polyGA expression relative to the GAPDH.


( B ) Left panel , immunoblotting against HA tag or GAPDH of extracted from Neuro2A transfected for 24 hours with a construct expressing 100 C4G2 repeats embedded in human antisense C9ORF72 fused to a HA tag in PG frame treated with 10 µM of indicated compound for 15 hours . Right panel , of polyPG expression relative to GAPDH .

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(B) Left panel, immunoblotting against the HA tag or the GAPDH of proteins extracted from Neuro2A cells transfected for 24 hours with a construct expressing 100 C4G2 repeats embedded in the human antisense C9ORF72 sequence fused to a HA tag in the PG frame and treated with 10 µM of the indicated compound for 15 hours. Right panel, quantification of polyPG expression relative to the GAPDH.


( C ) viability ( TO - PRO - 3 FACS ) of GT1 - 7 neuronal treated with 1 , 3 or 10 µM of promethazine co - transfected for 24 hours with either a control siRNA or a siRNA targeting C9orf72 mRNA a construct expressing either 80 G4C2 repeats or 100 C4G2 repeats embedded in sense or antisense C9ORF72 fused to GFP in GA or PG frame .

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(C) Cell viability (TO-PRO-3 FACS staining) of GT1-7 neuronal cells treated with 1, 3 or 10 µM of promethazine and co-transfected for 24 hours with either a control siRNA or a siRNA targeting C9orf72 mRNA and a construct expressing either 80 G4C2 repeats or 100 C4G2 repeats embedded in sense or antisense C9ORF72 fused to the GFP in the GA or PG frame.


B Phos ‐ tag gel following complete conversion of Ub to phosphoUb , using GST ‐ PhPINK1 . A Ser65 mutant Ub is not modified .

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B Phos‐tag gel following complete conversion of Ub to phosphoUb, using GST‐PhPINK1. A Ser65 mutant Ub is not modified.


C ESI ‐ MS of pure Ub ( left , mass 8 , 565 . 65 Da , reference 8 , 565 . 80 Da ) purified phosphoUb ( right , mass 8 , 645 . 61 Da , reference 8 , 645 . 80 Da ) . difference of 80 Da corresponds to one phosphate group .

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C ESI‐MS analysis of pure Ub (left, mass 8,565.65 Da, reference 8,565.80 Da) and purified phosphoUb (right, mass 8,645.61 Da, reference 8,645.80 Da). The difference of 80 Da corresponds to one phosphate group.


D AQUA of PhPINK1 ‐ phosphorylated Lys63 ‐ linked hexaUb . Lys63 peptide ( K63 ) is modified by Ser65 phosphorylation during time course , as observed by rise of Lys63 / pSer65 peptide ( K63pS65 ) .

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D AQUA analysis of PhPINK1‐phosphorylated Lys63‐linked hexaUb. The Lys63 peptide (K63) is modified by Ser65 phosphorylation during the time course, as observed by rise of the Lys63/pSer65 peptide (K63pS65).


E AQUA after 180 min as in ( D ) with Lys6 ‐ , Lys11 ‐ , Lys33 ‐ , Lys48 ‐ , Lys63 ‐ , Met1 ‐ linked tetraUb ( K6 , K11 , K33 , K48 , K63 , M1 ) . For tetraUb chains , Ser65 is phosphorylated to greater than 80 % after incubation with PhPINK1 for 180 min .

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E AQUA analysis after 180 min as in (D) with Lys6‐, Lys11‐, Lys33‐, Lys48‐, Lys63‐, and Met1‐linked tetraUb (K6, K11, K33, K48, K63, M1). For all tetraUb chains, Ser65 is phosphorylated to greater than 80% after incubation with PhPINK1 for 180 min.


F Time course of autoubiquitination reactions with unphosphorylated full ‐ humanParkin ( hParkin ) Ub ( left ) , a 80 % Ub / 20 % phosphoUb mixture ( middle ) 100 % phosphoUb ( right ) .

F Time course analysis of autoubiquitination reactions with unphosphorylated full‐length humanParkin (hParkin) and Ub (left), a 80% Ub/20% phosphoUb mixture (middle) and 100% phosphoUb (right).


G of different ratios of Ub : phosphoUb in a 120 ‐ min ubiquitination reaction of full ‐ hParkin . Parkin is activated by a mixture of Ub phosphoUb , is much less active in a reaction with no phosphoUb as well as with 100 % phosphoUb .

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G Analysis of different ratios of Ub:phosphoUb in a 120‐min ubiquitination reaction of full‐length hParkin. Parkin is activated by a mixture of Ub and phosphoUb, and is much less active in a reaction with no phosphoUb as well as with 100% phosphoUb.


A 1H , 15N ‐ BEST ‐ TROSY spectrum of phosphoUb ( shades of blue ) overlaid with wild ‐ type ( wt ) Ub ( orange ) . phosphoUb spectrum contains 130 non ‐ sidechain resonances that have been colored in blue for major species blue for minor species on assignment of each species in Supplementary Fig S2B - D . B - D Weighted chemical shift perturbation ( CSP ) graphs for ( B ) wt Ub versus major phosphoUb species , ( C ) wt Ub versus minor phosphoUb species , ( D ) major versus minor phosphoUb species .

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A 1H, 15N‐BEST‐TROSY spectrum of phosphoUb (shades of blue) overlaid with wild‐type (wt) Ub (orange). The phosphoUb spectrum contains 130 non‐sidechain resonances that have been colored in dark blue for the major species and light blue for the minor species based on the assignment of each species in Supplementary Fig S2B-D.B-D Weighted chemical shift perturbation (CSP) graphs for (B) wt Ub versus major phosphoUb species, (C) wt Ub versus minor phosphoUb species, (D) major versus minor phosphoUb species.


A Structure of phosphoUb in space group P1 with 8 molecules in asymmetric unit ( Table ) . Ub molecules are colored differently , atoms for pSer65 are shown as spheres with red oxygen purple phosphorous atoms .

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A Structure of phosphoUb in space group P1 with 8 molecules in the asymmetric unit (Table ). Ub molecules are colored differently, and atoms for pSer65 are shown as spheres with red oxygen and purple phosphorous atoms.


B Six of eight phosphoUb molecules form similar dimer interactions , where phosphoUb molecules interact via their Ile44 patch . This resembles crystal structure of Lys48 ‐ linked diUb [ pdb ‐ id 1aar ( Cook et al , ) ] . Atoms for pSer65 are shown as in ( A ) , Ile44 side chain is shown as blue sticks .

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B Six of eight phosphoUb molecules form similar dimer interactions, where phosphoUb molecules interact via their Ile44 patch. This resembles the crystal structure of Lys48‐linked diUb [pdb‐id 1aar (Cook et al, )]. Atoms for pSer65 are shown as in (A), and the Ile44 side chain is shown as blue sticks.


C Representative 2 | Fo | ‐ | Fc | electron density , contoured at 1 σ , covering phosphorylation site . pSer65 is shown in ball ‐ ‐ stick representation , neighboring side chains are shown in stick representation with red oxygen blue nitrogen atoms .

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C Representative 2|Fo|‐|Fc| electron density, contoured at 1 σ, covering the phosphorylation site. pSer65 is shown in ball‐and‐stick representation, and neighboring side chains are shown in stick representation with red oxygen and blue nitrogen atoms.


D In wt Ub ( left ) , Ser65 forms a hydrogen bond with backbone amide of Gln62 . Upon phosphorylation ( right ) , an oxygen atom from phosphate forms same backbone hydrogen bond .

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D In wt Ub (left), Ser65 forms a hydrogen bond with the backbone amide of Gln62. Upon phosphorylation (right), an oxygen atom from the phosphate forms the same backbone hydrogen bond.


E Cartoon representation electrostatic surface potential of Ub ( left ) phosphoUb ( right ) in identical orientations . Electrostatics were calculated using CHARMM ( www . charmm - gui . org ) are colored from red ( negative potential ) to blue ( positive potential ) .

E Cartoon representation and electrostatic surface potential of Ub (left) and phosphoUb (right) in identical orientations. Electrostatics were calculated using CHARMM (www.charmm-gui.org) and are colored from red (negative potential) to blue (positive potential).


A Long ‐ range HNCO NMR experiments showing differences of hydrogen bonding patterns for major minor phosphoUb species . Through H bond correlations ( blue ) recorded for residue Ile44 in major minor species of phosphoUb with reference to a control HNCO spectrum ( red ) . In major form of phosphoUb ( left ) , HN of Ile44 is a H bond donor to His68 , as seen in wt Ub . In minor form of phosphoUb ( right ) , HN of Ile44 does not have a cross peak to His68 but instead has a correlation to Val70 .

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A Long‐range HNCO NMR experiments showing differences of hydrogen bonding patterns for major and minor phosphoUb species. Through H bond correlations (blue) recorded for residue Ile44 in the major and minor species of phosphoUb with reference to a control HNCO spectrum (red). In the major form of phosphoUb (left), the HN of Ile44 is a H bond donor to His68, as seen in wt Ub. In the minor form of phosphoUb (right), the HN of Ile44 does not have a cross peak to His68 but instead has a correlation to Val70.


C Structure of major form of phosphoUb , where phosphoUb residues 1 - 61 are shown under a surface , β5 strand is shown in stick representation with red oxygen , blue nitrogen purple phosphorus atoms . four Leu residues are shown in green . D Model of phosphoUb minor species as in ( C ) in which β5 strand is retracted into by two residues . This extends β4 - β5 loop significantly shortens C ‐ terminal tail of Ub .

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C Structure of the major form of phosphoUb, where phosphoUb residues 1-61 are shown under a surface, and the β5 strand is shown in stick representation with red oxygen, blue nitrogen and purple phosphorus atoms. The four core Leu residues are shown in green.D Model of the phosphoUb minor species as in (C) in which the β5 strand is retracted into the protein core by two residues. This extends the β4-β5 loop and significantly shortens the C‐terminal tail of Ub.


A E1 ‐ mediated charging of E2 enzymes by Ub phosphoUb in a time course . Non ‐ reducing Coomassie ‐ stained 4 - 12 % SDS - PAGE gradient gels are shown , bands are labeled . " ˜ Ub " refers to generation of a thioester , while " Ub ‐ " refers to additional covalent ubiquitination of E2 enzyme . * contaminant from E2 purification .

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A E1‐mediated charging of E2 enzymes by Ub and phosphoUb in a time course analysis. Non‐reducing Coomassie‐stained 4-12% SDS-PAGE gradient gels are shown, and bands are labeled. "˜Ub" refers to generation of a thioester, while "Ub‐"refers to additional covalent ubiquitination of the E2 enzyme. * contaminant from E2 purification.


B SAXS ‐ derived ensemble of UBE2D3 ( Pruneda et al , ) , where E2 is shown under a blue surface Ub is shown as a green ribbon . Ser65 is shown in sphere representation . Ser65 does not contact E2 enzyme in any orientation of Ub at E2 enzyme . PDB files were downloaded from Klevit ‐ lab website ( http : / / depts . washington . edu / klvtlab / ) .

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B SAXS‐derived ensemble of UBE2D3 (Pruneda et al, ), where the E2 is shown under a blue surface and the Ub is shown as a green ribbon. Ser65 is shown in sphere representation. Ser65 does not contact the E2 enzyme in any orientation of the Ub at the E2 enzyme. PDB files were downloaded from the Klevit‐lab website (http://depts.washington.edu/klvtlab/).


C Quantitative mass spectrometry for UBE2T , UBE2E1 UBE2R1 with Ub phosphoUb using UbAQUA peptides reveals impaired chain formation with phosphoUb ( see also Supplementary Fig S10 ) .

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C Quantitative mass spectrometry analysis for UBE2T, UBE2E1 and UBE2R1 with Ub and phosphoUb using UbAQUA peptides reveals impaired chain formation with phosphoUb (see also Supplementary Fig S10).


D Charging of UBE2N , UBE2N / UBE2V1 ‐ mediated Lys63 chain assembly with Ub phosphoUb . Charging of UBE2N as in ( A ) proceeds identically with both Ub phosphoUb ( left 6 lanes ) , while UBE2V1 ‐ mediated Lys63 chain assembly only proceeds with Ub but not phosphoUb ( right 6 lanes Western blot ) .

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D Charging of UBE2N, and UBE2N/UBE2V1‐mediated Lys63 chain assembly with Ub and phosphoUb. Charging of UBE2N as in (A) proceeds identically with both Ub and phosphoUb (left 6 lanes), while UBE2V1‐mediated Lys63 chain assembly only proceeds with Ub but not phosphoUb (right 6 lanes and Western blot).


E Structure of Saccharomyces cerevisiae Ubc13 ‐ Mms2 ( blue / red , Mms2 is a homolog of UBE2V1 ) with acceptor Ub ( cyan ) bound non ‐ covalently to Mms2 [ pdb ‐ id 2gmi ( Eddins et al , ) ] . Ser65 of acceptor Ub interacts with Mms2 , Ser65 phosphorylation would prevent binding due to Mms2 Glu18 .

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E Structure of Saccharomyces cerevisiae Ubc13‐Mms2 (blue/red, Mms2 is a homolog of UBE2V1) with the acceptor Ub (cyan) bound non‐covalently to Mms2 [pdb‐id 2gmi (Eddins et al, )]. Ser65 of the acceptor Ub interacts with Mms2, and Ser65 phosphorylation would prevent binding due to Mms2 Glu18.


F PhosphoUb competition assays for UBE2N / UBE2V1 ‐ mediated Lys63 diUb formation . Time course with K63R Ub ( donor ) Ub ΔGG ( acceptor ) generates Lys63 diUb is not inhibited upon increasing concentrations of phosphoUb , revealing phosphoUb does not compete with Ub for UBE2N / UBE2V1 ‐ mediated Lys63 diUb formation .

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F PhosphoUb competition assays for UBE2N/UBE2V1‐mediated Lys63 diUb formation. Time course with K63R Ub (donor) and Ub ΔGG (acceptor) generates Lys63 diUb and is not inhibited upon increasing concentrations of phosphoUb, revealing phosphoUb does not compete with Ub for UBE2N/UBE2V1‐mediated Lys63 diUb formation.


A Time course for a ligase reaction with GST ‐ tagged cIAP1 ( aa 363 - 614 ) , UBE2D1 Ub or phosphoUb . See Supplementary Fig S11D for a reaction with UBE2D3 .

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A Time course for a ligase reaction with GST‐tagged cIAP1 (aa 363-614), UBE2D1 and Ub or phosphoUb. See Supplementary Fig S11D for a reaction with UBE2D3.


B Anti ‐ Ub Western blot of an identical reaction run for 3 h . C Comparison of number of Ub chain linkages in reaction in ( B ) . D Ub chain linkage profile for reaction in ( B ) .

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B Anti‐Ub Western blot of an identical reaction run for 3 h.C Comparison of the number of Ub chain linkages in the reaction in (B).D Ub chain linkage profile for the reaction in (B).


E Reaction of GST ‐ tagged TRAF6 ( aa 1 - 285 ) with UBE2D3 as in ( A ) . See Supplementary Fig S11E F for additional TRAF6 reactions .

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E Reaction of GST‐tagged TRAF6 (aa 1-285) with UBE2D3 as in (A). See Supplementary Fig S11E and F for additional TRAF6 reactions.


F Anti ‐ Ub Western blot of GST ‐ TRAF6 / UBE2D1 run for 3 h . G Comparison of number of Ub chain linkages in reaction in ( F ) . H Linkage composition in GST ‐ TRAF6 ‐ UBE2D1 reactions reveals 58 % Lys63 chains with wt Ub UBE2D1 . Significantly smaller quantities of linkages are assembled with phosphoUb .

F Anti‐Ub Western blot of GST‐TRAF6/UBE2D1 run for 3 h.G Comparison of the number of Ub chain linkages in the reaction in (F).H Linkage composition in GST‐TRAF6‐UBE2D1 reactions reveals 58% Lys63 chains with wt Ub and UBE2D1. Significantly smaller quantities of linkages are assembled with phosphoUb.


I - K Chain assembly as in ( A ) with UBE2L3 ( I ) GST ‐ tagged NEDD4L ( aa 576 - 955 ) ( Mund Pelham , ) , ( J ) NleL ( aa 170 - 782 ) ( Hospenthal et al , ) ( K ) HOIP RBR ‐ LDD ( aa 699 - 1072 ) ( Smit et al , ) . While HECT E3 ligases NEDD4L NleL are active with Ub phosphoUb , HOIP is significantly less active with phosphoUb . See also Supplementary Fig S11G H .

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I-K Chain assembly as in (A) with UBE2L3 and (I) GST‐tagged NEDD4L (aa 576-955) (Mund Pelham, ), (J) NleL (aa 170-782) (Hospenthal et al, ) and (K) HOIP RBR‐LDD (aa 699-1072) (Smit et al, ). While the HECT E3 ligases NEDD4L and NleL are active with Ub and phosphoUb, HOIP is significantly less active with phosphoUb. See also Supplementary Fig S11G and H.


A Structure of TAB2 NZF ( red ) in with Lys63 diUb ( cyan ) [ pdb ‐ id 2wwz ( Kulathu et al , ) ] . Ser65 for both Ub moieties is shown as spheres highlighted with a blue circle .

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A Structure of TAB2 NZF (red) in complex with Lys63 diUb (cyan) [pdb‐id 2wwz (Kulathu et al, )]. Ser65 for both Ub moieties is shown as spheres and highlighted with a blue circle.


B Pull ‐ down assays with Lys63 ‐ specific TAB2 NZF against different tetraUb ( Ub4 ) species are shown . Phosphorylation of K63 Ub4 does not prevent binding to TAB2 NZF . Met1 ‐ linked tetraUb does not interact with TAB2 NZF , independent of Ser65 phosphorylation .

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B Pull‐down assays with the Lys63‐specific TAB2 NZF against different tetraUb (Ub4) species are shown. Phosphorylation of K63 Ub4 does not prevent binding to TAB2 NZF. Met1‐linked tetraUb does not interact with TAB2 NZF, independent of Ser65 phosphorylation.


A Structure of USP2 ( red ) bound to Ub ( cyan ) [ pdb ‐ id 2hd5 ( Renatus et al , ) ] , with a close ‐ up on Ub Ser65 in distal Ub . A negative charge at USP2 Asp345 position is conserved in most USP enzymes ( Ye et al , ) .

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A Structure of USP2 (red) bound to Ub (cyan) [pdb‐id 2hd5 (Renatus et al, )], with a close‐up on Ub Ser65 in the distal Ub. A negative charge at the USP2 Asp345 position is conserved in most USP enzymes (Ye et al, ).


B - H Ubiquitination reactions from Fig A were used as input for deubiquitinase ( DUB ) . Silver stained time course gels are shown . DUBs tested ( USP2 , USP8 , USP15 , USP30 , Ataxin ‐ 3 , USP21 ) hydrolyze Ub chains but have significantly lower activity against phosphoUb chains , with exception of vOTU USP21 .

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B-H Ubiquitination reactions from Fig A were used as input for deubiquitinase (DUB) analysis. Silver stained time course gels are shown. All DUBs tested (USP2, USP8, USP15, USP30, Ataxin‐3, USP21) hydrolyze Ub chains but have significantly lower activity against phosphoUb chains, with the exception of vOTU and USP21.


B - H Ubiquitination reactions from Fig A were used as input for deubiquitinase ( DUB ) . Silver ‐ stained time ‐ course gels are shown . DUBs tested ( USP2 , USP8 , USP15 , USP30 , Ataxin ‐ 3 , USP21 ) hydrolyze Ub chains but have significantly lower activity against phosphoUb chains , with exception of vOTU USP21 .

B-H Ubiquitination reactions from Fig A were used as input for deubiquitinase (DUB) analysis. Silver‐stained time‐course gels are shown. All DUBs tested (USP2, USP8, USP15, USP30, Ataxin‐3, USP21) hydrolyze Ub chains but have significantly lower activity against phosphoUb chains, with the exception of vOTU and USP21.


I USP30 activity was significantly lower for cIAP polyUb as compared to other USP DUBs . Cleavage assays against diUb panel reveals preference for Lys6 ‐ linked chains .

I USP30 activity was significantly lower for cIAP polyUb as compared to other USP DUBs. Cleavage assays against the diUb panel reveals preference for Lys6‐linked chains.


J USP30 activity is decreased against phosphorylated Lys6 diUb .

J USP30 activity is decreased against phosphorylated Lys6 diUb.


K - N Deubiquitinase assays against phosphorylated tetraUb . activity of AMSH ( K ) , TRABID ( L ) , OTUB1 ( M ) OTULIN ( N ) are inhibited with Lys63 , Lys33 , Lys48 Met1 phosphorylated tetraUb , respectively .

K-N Deubiquitinase assays against phosphorylated tetraUb. The activity of AMSH (K), TRABID (L), OTUB1 (M) and OTULIN (N) are inhibited with Lys63, Lys33, Lys48 and Met1 phosphorylated tetraUb, respectively.


qRT - PCR for INHBA relative to RPL27 using from SCC13 transduced with a lentiviral vector allowing expression of INHBA in a doxycycline ( DOX ) - inducible manner ( SCC13 Act clone 1 2 ) or empty vector ( EV ) ( N = 3 ) . SCC13 Act EV lines were generated from lentivirally transduced upon single clonal expansion .

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qRT-PCR for INHBA relative to RPL27 using RNA from SCC13 cells transduced with a lentiviral vector allowing expression of INHBA in a doxycycline (DOX)-inducible manner (SCC13 Act clone 1 and 2) or empty vector (EV) (N=3). SCC13 Act and EV cell lines were generated from lentivirally transduced cells upon single cell clonal expansion.


Western blot of lysate conditioned media ( CM ; bottom ) of transduced SCC13 for activin βA subunit GAPDH ( loading control for lysate ) under reducing conditions . higher molecular of recombinant activin βA results from HA - epitope tag . Ponceau S of was used as a loading control for CM .

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Western blot of cell lysate and conditioned media (CM; bottom) of transduced SCC13 cells for the activin βA subunit and GAPDH (loading control for cell lysate) under reducing conditions. The higher molecular weight of recombinant activin βA results from the HA-epitope tag. Ponceau S staining of the membrane was used as a loading control for the CM.


Representative pictures of 5 week - old tumors ( indicated by arrows ) formed in NOD / SCID mice upon intradermal injection of SCC13 Act ( clone 2 ) or SCC13 EV volume at different time points of development . N = 3 - 6 tumors .

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Representative pictures of 5 week-old tumors (indicated by arrows) formed in NOD/SCID mice upon intradermal injection of SCC13 Act (clone 2) or SCC13 EV cells and tumor volume at different time points of tumor development. N=3-6 tumors.


Representative of H & E stainings of tumors formed by SCC13 EV Act ( clone 2 ) at day 35 ( asterisk indicates cartilage , arrow indicates site of invasion ) . An inset panel indicates where invade into cartilage . sections were evaluated for invasion through basement . N = 6 ( EV ) , N = 6 Act ( 3 tumors from clone 1 3 tumors from clone 2 ) . Tumors from Act mice were pooled in graph ) .

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Representative images of H&E stainings of tumors formed by SCC13 EV and Act cells (clone 2) at day 35 (asterisk indicates cartilage, arrow indicates site of invasion). An inset panel indicates the area where tumor cells invade into the cartilage. Tumor sections were evaluated for invasion through the basement membrane. N=6 (EV), N=6 Act (3 tumors from clone 1 and 3 tumors from clone 2). Tumors from Act mice were pooled in the graph).


at endpoint ( 5 after injection ) . N = 3 - 6 tumors .

Tumor weight at endpoint (5 weeks after injection). N=3-6 tumors.


Herovici of of collagen - positive ( blue ) per field of view . N = 3 tumors , n = 3 - 4 histological sections .

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Herovici staining and quantification of percentage of collagen-positive (dark blue) area per field of view. N=3 tumors, n=3-4 histological sections.


Representative of sections from tumors formed by SCC13 EV SCC13 Act ( clone 2 ) stained for Ki67 ( red ) , PDGFR - α ( green ) counterstained with Hoechst ( blue ) . of Ki67 / PDGFR - α positive is shown in graph . N = 3 tumors , n = 3 - 4 histological sections .

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Representative images of sections from tumors formed by SCC13 EV and SCC13 Act (clone 2) stained for Ki67 (red), PDGFR-α (green) and counterstained with Hoechst (blue). Quantification of Ki67/PDGFR-α positive cells is shown in the graph. N=3 tumors, n=3-4 histological sections.


Representative of sections from 3D cultures of human skin fibroblasts with either SCC13 EV or SCC13 Act ( clones 1 2 ) stained with either H & E or Herovici .

Representative images of sections from 3D cultures of primary human skin fibroblasts with either SCC13 EV or SCC13 Act (clones 1 and 2) cells stained with either H&E or Herovici.


of Ki67 - positive in sections from SCC13 EV SCC13 Act ( clone 2 ) organotypic cultures ( co - stained with E - cadherin to indicate epithelial ) . N = 3 independent cultures , n = 3 histological sections .

Quantification of Ki67-positive cells in sections from SCC13 EV and SCC13 Act (clone 2) organotypic cultures (co-stained with E-cadherin to indicate epithelial cells). N=3 independent cultures, n=3 histological sections.


Representative of sections from SCC13 EV SCC13 Act ( clone 2 ) organotypic cultures stained for K14 ( red ) collagen IV ( green ) combined with Hoechst ( blue ) of K14 - positive below basement ( BM ) . N = 3 independent cultures , n = 4 histological sections . indicated with a rectangle in left picture is shown at higher magnification in right picture .

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Representative images of sections from SCC13 EV and SCC13 Act (clone 2) organotypic cultures stained for K14 (red) and collagen IV (green) combined with Hoechst staining (blue) and quantification of the K14-positive area below the basement membrane (BM). N=3 independent cultures, n=4 histological sections. The area indicated with a white rectangle in the left picture is shown at higher magnification in the right picture.


murine dermal fibroblasts , which had been treated with recombinant activin A at different concentrations , were analyzed for BrdU incorporation . N = 3 - 4 .

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Primary murine dermal fibroblasts, which had been treated with recombinant activin A at different concentrations, were analyzed for BrdU incorporation. N=3-4.


Representative of collagen gels of extent of gel contraction ( of initial gel ) in presence or absence of activin A ( 20 ng / ml ) / or TGF - β1 ( 1 ng / ml ) . N = 3 - 4 .

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Representative images of collagen gels and quantification of the extent of gel contraction (percentage of initial gel size) in the presence or absence of activin A (20 ng/ml) and/or TGF-β1 (1 ng/ml). N=3-4.


qRT - PCR Western blot analyses of ACTA2 / αSMA expression using total / lysates of fibroblasts treated with activin A or TGF - β1 for 6 h . N = 3 . was re - probed with GAPDH mDia2 antibodies .

qRT-PCR and Western blot analyses of ACTA2/αSMA expression using total RNA/lysates of fibroblasts treated with activin A or TGF-β1 for 6 h. N=3. The membrane was re-probed with GAPDH and mDia2 antibodies.


Representative of fibroblasts treated with activin A , TGF - β1 or vehicle ( control ) as in ( C ) , stained for αSMA ( green ) , counterstained with rhodamine - coupled phalloidin ( red ) Hoechst ( blue ) . Bar graph shows of fibroblasts with stress . N = 3 .

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Representative images of fibroblasts treated with activin A, TGF-β1 or vehicle (control) as in (C), stained for αSMA (green), counterstained with rhodamine-coupled phalloidin (red) and Hoechst (blue). Bar graph shows percentage of fibroblasts with stress fibers. N=3.


epresentative of control activin A - treated fibroblasts stained with rhodamine - coupled phalloidin ; scatter plot shows of filopodium in control , activin A - TGF - β1 - treated fibroblasts . N = 27 - 70 filopodia .

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epresentative images of control and activin A-treated fibroblasts stained with rhodamine-coupled phalloidin; scatter plot shows quantification of filopodium length in control, activin A- and TGF-β1-treated fibroblasts. N=27-70 filopodia.


Scratch of fibroblasts treated with activin A or vehicle for 24 h . N = 3 . covered by in original scratch was quantified . Migration in one control culture was set to 1 .

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Scratch assay of fibroblasts treated with activin A or vehicle for 24 h. N=3. The area covered by cells in the original scratch was quantified. Migration in one control culture was set to 1.


Chemotactic migration of mouse fibroblasts from wt mice for 24 h in a transwell towards increasing concentrations of activin A or vehicle . N = 3 .

Chemotactic migration of primary mouse fibroblasts from wt mice for 24 h in a transwell assay towards increasing concentrations of activin A or vehicle. N=3.


qRT - PCR of CAF marker genes relative to Rps29 using from fibroblasts treated with activin A for 6 h . N = 3 .

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qRT-PCR analysis of CAF marker genes relative to Rps29 using RNA from fibroblasts treated with activin A for 6 h. N=3.


qRT - PCR for INHBA relative to RPL27 using from human fibroblasts transduced with a lentiviral vector allowing expression of INHBA in an inducible manner after treatment with DOX for 24 h ( Fb Act , clones 1 2 ) or with empty vector ( Fb EV ) ( N = 3 ) . Fb Act Fb EV cultures were generated from lentivirally - transduced upon clonal expansion of resistant single .

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qRT-PCR for INHBA relative to RPL27 using RNA from primary human fibroblasts transduced with a lentiviral vector allowing expression of INHBA in an inducible manner after treatment with DOX for 24 h (Fb Act, clones 1 and 2) or with empty vector (Fb EV) (N=3). Fb Act and Fb EV cultures were generated from lentivirally-transduced cells upon clonal expansion of resistant single cells.


Western blot of conditioned media ( CM ) of transduced fibroblasts showing mature activin βA . Ponceau S of was used as a loading control .

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Western blot of conditioned media (CM) of transduced fibroblasts showing mature activin βA. Ponceau S staining of the membrane was used as a loading control.


Chemotactic migration of SCC13 for 24 h in a transwell containing CM from Fb Act ( clone 2 ) or Fb EV . N = 4 . Migration in one control culture was set to 1 .

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Chemotactic migration of SCC13 cells for 24 h in a transwell assay containing CM from Fb Act (clone 2) or Fb EV. N=4. Migration in one control culture was set to 1.


Relative colony of SCC13 upon culture in CM from Fb Act ( clone 2 ) or EV . N = 3 .

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Relative colony area of SCC13 cells upon culture in CM from Fb Act (clone 2) or EV. N=3.


Representative immunofluorescence of de - cellularized matrix from Fb Act ( clone 2 ) Fb EV stained for fibronectin or collagen I .

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Representative immunofluorescence images of de-cellularized matrix from Fb Act (clone 2) and Fb EV stained for fibronectin or collagen I.


Relative colony of SCC13 plated on either de - cellularized matrix from Fb Act ( clone 2 ) or Fb EV . N = 3 .

Relative colony area of SCC13 cells plated on either de-cellularized matrix from Fb Act (clone 2) or Fb EV. N=3.


Migration of SCC13 plated on either de - cellularized matrix from Fb Act ( clone 2 ) or Fb EV . N = 6 . experimental setup is shown schematically above graph .

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Migration of SCC13 cells plated on either de-cellularized matrix from Fb Act (clone 2) or Fb EV. N=6. The experimental setup is shown schematically above the graph.


Fb Act ( clone 2 ) or Fb EV were cultured together in 2D with SCC13 . After 7 day of culture SCC13 were identified by K14 number of K14 - positive colony was quantified . N = 6 .

Fb Act (clone 2) or Fb EV cells were cultured together in 2D with SCC13 cells. After 7 day of culture SCC13 cells were identified by K14 and the number of K14-positive colony area was quantified. N=6.


of SCC13 spheroid in single hanging drop including CM from Fb EV or Fb Act ( clone 2 ) ( including 1 % FBS ) , DMEM / 1 % FBS , or DMEM / 1 % FBS plus 20 ng / mL recombinant activin A . of spheroid formed in one Fb EV sample was set to 1 . N = 9 - 10 hanging drops .

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Quantification of SCC13 tumor spheroid area in single hanging drop including CM from Fb EV or Fb Act (clone 2) (including 1% FBS), DMEM/1% FBS, or DMEM/1% FBS plus 20 ng/mL recombinant activin A. The area of the spheroid formed in one Fb EV sample was set to 1. N=9-10 hanging drops.


Representative of K14 - stained areas from 3D cultures of SCC13 seeded on top of either Fb Act ( clone 2 ) or Fb EV . N = 3 independent cultures , n = 3 histological sections .

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Representative images and quantification of K14-stained areas from 3D cultures of SCC13 cells seeded on top of either Fb Act (clone 2) or Fb EV. N=3 independent cultures, n=3 histological sections.


Representative pictures of tumors formed in NOD / SCID mice 6 - 7 after intradermal injection of SCC13 with Fb Act ( clone 2 ) or Fb EV . volume at different time points of development ( 6 - 7 after injection ) . N = 5 tumors per group .

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Representative pictures of tumors formed in NOD/SCID mice 6-7 weeks after intradermal injection of SCC13 cells with Fb Act (clone 2) or Fb EV. Tumor volume at different time points of tumor development (6-7 weeks after injection). N=5 tumors per group.


mDia2 ( Diap3 ) expression quantified by sequencing of fibroblasts isolated from ear skin . N = 3 pools of 3 - 6 mice .

mDia2 (Diap3) expression quantified by RNA sequencing of fibroblasts isolated from ear skin. N=3 pools of 3-6 mice.


mouse fibroblasts were treated with activin A or TGF - β1 or vehicle for 6h analyzed by qRT - PCR ( N = 3 ) Western blot for mDia2 ( Diap3 ) mDia2 expression , respectively . GAPDH loading control is identical to one in Fig 2C , since same was probed for mDia2 , αSMA GAPDH .

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Primary mouse fibroblasts were treated with activin A or TGF-β1 or vehicle for 6h and analyzed by qRT-PCR (N=3) and Western blot for mDia2 (Diap3) and mDia2 expression, respectively. The GAPDH loading control is identical to the one in Fig 2C, since the same membrane was probed for mDia2, αSMA and GAPDH.


Immortalized mouse fibroblasts were transduced with a lentivirus expressing a DOX - inducible dominant - negative ActRIB mutant ( dnActRIB ) or an empty lentivirus ( EV ) , treated with activin A for 2 . 5 h analyzed by Western blot for mDia2 GAPDH .

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Immortalized mouse fibroblasts were transduced with a lentivirus expressing a DOX-inducible dominant-negative ActRIB mutant (dnActRIB) or an empty lentivirus (EV), treated with activin A for 2.5 h and analyzed by Western blot for mDia2 and GAPDH.


Representative sections of ear skin tumors formed by SCC13 EV or SCC13 Act ( clone 2 ) stained for mDia2 or K14 ( green ) ( top ) , mDia2 ( green ) or MECA32 / LYVE1 ( red , bottom ) counterstained with Hoechst ( blue ) .

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Representative sections of ear skin tumors formed by SCC13 EV or SCC13 Act cells (clone 2) stained for mDia2 or K14 (green) (top), mDia2 (green) or MECA32/LYVE1 (red, bottom) and counterstained with Hoechst (blue).