Annexin - V & Dead ( 7 - AAD ) flow cytometry of MDA - MB - 231 / ACC - DKO Wt cultured in DFBS medium . right panel shows of annexin V - positive in left panels .

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

Annexin-V & Dead cell (7-AAD) flow cytometry analysis of MDA-MB-231/ACC-DKO and Wt cultured in DFBS medium. The right panel shows the quantification of annexin V-positive cells in the left panels.


relative survival of MDA - MB - 231 / ACC - DKO or Wt with control vector or Bcl - 2 or Bcl - xL overexpression cultured in FBS medium or DFBS medium for 5 days . left panels show survival , right panels verify overexpression of Bcl - 2 Bcl - xL .

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

The relative cell survival of MDA-MB-231/ACC-DKO or Wt with control vector or Bcl-2 or Bcl-xL overexpression cultured in FBS medium or DFBS medium for 5 days. The left panels show the cell survival, and the right panels verify the overexpression of Bcl-2 and Bcl-xL.


transmembrane potential in mitochondria of MDA - MB - 231 / ACC - DKO Wt cultured in FBS medium or DFBS medium was detected by FACS . bar graphs show of JC - 1 monomer .

O O O O O O O O O O O O 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 transmembrane potential in mitochondria of MDA-MB-231/ACC-DKO and Wt cells cultured in FBS medium or DFBS medium was detected by FACS analysis. The bar graphs show the percentage of JC-1 monomer.


MDA - MB - 231 / ACC - DKO Wt were stained for Mito Tracker to visualize mitochondria . Scale bar , 10 μm . Mitochondrial was scored in types indicated . Data represent three independent experiments , at least 100 were counted per type .

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

MDA-MB-231/ACC-DKO and Wt cells were stained for Mito Tracker to visualize mitochondria. Scale bar, 10 μm. Mitochondrial morphology was scored in the cell types indicated. Data represent three independent experiments, and at least 100 cells were counted per cell type.


MDA - MB - 231 / ACC - DKO were in FBS medium with or without exogenous LCFA or VLCFA supplementation , were stained for MitoTracker to visualize mitochondria . Scale bar , 10 μm . Mitochondrial in ( D ) was scored in types indicated . Data represent three independent experiments , at least 100 were counted per type . Error bars represent mean ± SD , data are from triplicate experiments .

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

MDA-MB-231/ACC-DKO cells were in FBS medium with or without exogenous LCFA or VLCFA supplementation, and were stained for MitoTracker to visualize mitochondria. Scale bar, 10 μm.   Mitochondrial morphology in (D) was scored in the cell types indicated. Data represent three independent experiments, and at least 100 cells were counted per cell type. Error bars represent mean ± SD, and data are from triplicate experiments.


A . Equal aliquots ( 2 mg ) of a Triton X - 100 solubilized lysate from rat brain were incubated with GST or GST - ROC . Specifically bound were detected by Western blot with antibodies recognizing indicated .

O O O O O O O O O O O O O O O O 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. Equal aliquots (2 mg) of a Triton X-100 solubilized lysate from rat brain were incubated with GST or GST-ROC. Specifically bound proteins were detected by Western blot with antibodies recognizing the indicated proteins.


B . Brain lysate as in ( A ) was incubated with GST or GST - nCLCb . Specifically bound were detected by Western blot with antibodies recognizing indicated .

O O O O O O 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. Brain lysate as in (A) was incubated with GST or GST-nCLCb. Specifically bound proteins were detected by Western blot with antibodies recognizing the indicated proteins.


C . Full - GST - nCLCb ( 1 - 228 ) various truncation mutants were purified from . Equal aliquots of purified were incubated with GST or GST - ROC . Specifically bound were detected by Western blot with antibody against CLCs .

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

C. Full-length GST-nCLCb (1-228) and various truncation mutants were purified from bacteria. Equal aliquots of purified protein were incubated with GST or GST-ROC. Specifically bound proteins were detected by Western blot with antibody against CLCs.


D . Brain lysate as in ( A ) was incubated with GST or GST - nCLCb with boundaries as indicated . Specifically bound were detected by Western blot with antibody against CLCs .

O O O O O O O O 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

D. Brain lysate as in (A) was incubated with GST or GST-nCLCb proteins with boundaries as indicated. Specifically bound proteins were detected by Western blot with antibody against CLCs.


F . Highly enriched triskelia were stripped from purified CCVs . triskelia were incubated with GST or GST - ROC . Specifically bound were detected by Western blot with antibodies recognizing indicated . For A - D / F , aliquots of starting material ( SM ) equal to 10 % of that added to beads , was analyzed in parallel .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Highly enriched triskelia were stripped from purified CCVs. The triskelia were incubated with GST or GST-ROC. Specifically bound proteins were detected by Western blot with antibodies recognizing the indicated proteins. For A-D/F, aliquots of the starting material (SM) equal to 10% of that added to the beads, was analyzed in parallel.


PCR results of LRRK2 - WT from clone E1 ( 1 ) using primers that detect endogenous LRRK2 . Clone E1 is positive for 3xHA - LRRK2 ( 2 ) using a primer pair with antisense in 3xHA insert sense primer in endogenous LRRK2 . ( 3 ) Control unedited COS - 7 using same primer combination as in ( 2 ) . ( 4 ) 1Kb marker .

O O O B-MEASURED_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 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

PCR results of LRRK2-WT from clone E1 (1) using primers that detect endogenous LRRK2. Clone E1 is positive for 3xHA-LRRK2 (2) using a primer pair with the antisense in the 3xHA insert and the sense primer in endogenous LRRK2. (3) Control unedited COS-7 cells using the same primer combination as in (2). (4) 1Kb marker.


COS - 7 clone E1 were serum starved followed by 20 min incubation with Alexa488 - EGF , after which were fixed processed for immunofluorescence using HA antibody . Scale bar = 10 m for bottom 6 panels 25 m for top 3 panels .

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

COS-7 clone E1 cells were serum starved followed by 20 min incubation with Alexa488-EGF, after which the cells were fixed and processed for immunofluorescence using HA antibody. Scale bar = 10 m for bottom 6 panels and 25 m for top 3 panels.


COS - 7 clone E1 were fixed processed for immunofluorescence using HA CLC antibodies . Scale bar = 10 m .

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

COS-7 clone E1 cells were fixed and processed for immunofluorescence using HA and CLC antibodies. Scale bar = 10 m.


COS - 7 clone E1 were fixed an processed for immunofluorescence using HA EEA1 antibodies . Scale bar = 10 m for bottom 6 panels 25 m for top 3 panels .

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

COS-7 clone E1 cells were fixed an processed for immunofluorescence using HA and EEA1 antibodies. Scale bar = 10 m for bottom 6 panels and 25 m for top 3 panels.


Lysates from COS - 7 transfected with siRNA as indicated were processed for Western blot with antibodies recognizing indicated . Band intensities of blots as in A are presented as % control siRNA . Two - Student t - , Mann - Whiteney post hoc , * * * p < 0 . 001 , N = 3 .

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

Lysates from COS-7 cells transfected with siRNA as indicated were processed for Western blot with antibodies recognizing the indicated proteins.Band intensities of blots as in A are presented as % control siRNA. Two-tailed Student t-test, Mann-Whiteney post hoc test, ***p < 0.001, N = 3.


NP - 40 soluble lysates were prepared from COS - 7 transfected with siRNA as indicated equal aliquots ( 1 . 5 mg ) were incubated with GST - PAK - CRIB domain . Specifically bound were detected by Western blot with antibody recognizing Rac1 . An aliquot of starting material ( SM ) equal to 5 % of that added to GST - PAK - CRIB was analyzed in parallel .

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

NP-40 soluble lysates were prepared from COS-7 cells transfected with siRNA as indicated and equal protein aliquots (1.5 mg) were incubated with GST-PAK-CRIB domain. Specifically bound proteins were detected by Western blot with antibody recognizing Rac1. An aliquot of the starting material (SM) equal to 5% of that added to GST-PAK-CRIB was analyzed in parallel.


Band intensities of blots as in A were presented as % control siRNA . Bars represent mean . One - way ANOVA , Bonferroni ' s post hoc , * * * p < 0 . 001 , N = 3 .

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

Band intensities of blots as in A were presented as % control siRNA. Bars represent mean sem. One-way ANOVA, Bonferroni's post hoc test, ***p < 0.001, N=3.


COS - 7 transfected with siRNA as indicated were fixed processed for immunofluorescence using Phalloidin - 647 antibody recognizing CLCs . Scale bar = 10 m .

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

COS-7 cells transfected with siRNA as indicated were fixed and processed for immunofluorescence using Phalloidin-647 and antibody recognizing CLCs. Scale bar = 10 m.


Perimeter of as in A were measured a perimeter : was plotted as a % of control siRNA treated . One - way ANOVA , Bonferroni ' s post hoc , * * * p < 0 . 001 , N = 3 .

Perimeter and area of cells as in A were measured and a perimeter:area ratio was plotted as a % of the control siRNA treated cells. One-way ANOVA, Bonferroni's post hoc test, ***p < 0.001, N=3.


Lysates from 21 day in vitro cultured hippocampal neurons transduced with lentivirus ( at 7 days in vitro ) driving expression of control shRNAmiR or shRNAmiRs specific for CLCa , CLCb , or both in combination . lysates were processed for Western blot with antibody that recognizes forms of CLCs .

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

Lysates from 21 day in vitro cultured hippocampal neurons transduced with lentivirus (at 7 days in vitro) driving expression of control shRNAmiR or shRNAmiRs specific for CLCa, CLCb, or both viruses in combination. The lysates were processed for Western blot with antibody that recognizes all forms of CLCs.


Hippocampal neurons transduced as in G were fixed at 21 days in vitro processed for immunofluorescence with polyclonal antibody specific for pre - synaptic synapsin monoclonal antibody specific for post - synaptic PSD95 . lentivirus drives expression of GFP . areas indicated by boxes in low power ( squares ) are shown enlarged immediately below . scale bars = 10 µm for low mag 2 . 5 µm for high mag .

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 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

Hippocampal neurons transduced as in G were fixed at 21 days in vitro and processed for immunofluorescence with polyclonal antibody specific for the pre-synaptic protein synapsin and monoclonal antibody specific for the post-synaptic protein PSD95. The lentivirus drives expression of GFP. The areas indicated by white boxes in the low power images (squares) are shown enlarged immediately below. The scale bars = 10 µm for the low mag and 2.5 µm for the high mag.


Ai - x . Lateral view of adult fly head . Scale bar = 100 μm . i . longGMR , UAS - Rac1W / TM6B , Hu , Tb causes a mild rough eye phenotype with fused ( arrows ) disorganized but still separated ommatidia ( arrowheads ) . ii / iii . Rac1 overexpression with KD of dlrrk ( UAS - dlrrk - RNAi ( v22139 or v22140 ) / longGMR - GAL4 , UAS - Rac1W ) , results in smaller eyes where most ommatidia are fused . iv / v . Rac1 overexpression with KD of Clc ( UAS - Clc - RNAi ( v22318 or v106632 ) / + ; longGMR - GAL4 , UAS - Rac1W / + ) , results in smaller eyes with some ommatidia fused . vi . Rac1 overexpression with KD of inwardly rectifying potassium channel 3 ( irk3 ) , a non - relevant RNAi control ( UAS - irk3 - RNAi ( v101174 ) / + ; longGMR - GAL4 , UAS - Rac1W / + ) , has no effect . vii / viii . KD of dlrrk with GMR - GAL4 / + ; UAS - dlrrk - RNAi ( v22139 or 22140 ) / + , respectively as control have no effect on eye . ix / x . KD of Clc with GMR - GAL4 / UAS - Clc - RNAi ( v22318 or v106632 ) , respectively as control have no effect on eye .

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

Ai-x. Lateral view of the adult fly head. Scale bar = 100 μm.i. longGMR,UAS-Rac1W/TM6B, Hu, Tb causes a mild rough eye phenotype with fused (arrows) and disorganized but still separated ommatidia (arrowheads).ii/iii. Rac1 overexpression with KD of dlrrk (UAS-dlrrk-RNAi (v22139 or v22140)/longGMR-GAL4, UAS-Rac1W), results in smaller eyes where most ommatidia are fused.iv/v. Rac1 overexpression with KD of Clc (UAS-Clc-RNAi (v22318 or v106632)/+;longGMR-GAL4,UAS-Rac1W/+), results in smaller eyes with some ommatidia fused.vi. Rac1 overexpression with KD of the inwardly rectifying potassium channel 3 (irk3), a non-relevant RNAi control (UAS-irk3-RNAi(v101174)/+; longGMR-GAL4,UAS-Rac1W/+), has no effect.vii/viii. KD of dlrrk with GMR-GAL4/+; UAS-dlrrk-RNAi(v22139 or 22140)/+, respectively as control have no effect on eye morphology.ix/x. KD of Clc with GMR-GAL4/UAS-Clc-RNAi (v22318 or v106632), respectively as control have no effect on eye morphology.


Bi - ii . Lateral view of adult fly head . Scale bar : 100 μm . i . Rac1 overexpression with removing one copy of dlrrk ( dlrrke03680 / longGMR - GAL4 , UAS - Rac1W ) , results in smaller eyes where most ommatidia are fused together . ii . Removing one copy of dlrrke03680 in GMR - GAL4 background , as control has no effect on eye ( GMR - GAL4 / + ; dlrrke03680 / + ) .

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

Bi-ii. Lateral view of the adult fly head. Scale bar: 100 μm.i. Rac1 overexpression with removing one copy of dlrrk (dlrrke03680/longGMR-GAL4,UAS-Rac1W), results in smaller eyes where most ommatidia are fused together.ii. Removing one copy of dlrrke03680 in GMR-GAL4 background, as control has no effect on eye morphology (GMR-GAL4/+;dlrrke03680/+).


Ci - viii . Lateral view of adult fly head . Scale bar : 100 μm . i . longGMR , UAS - Rac1W / TM6B , Hu , Tb causes a mild rough eye phenotype with fused disorganized but still separated ommatidia ( as explained in Ai ) . ii . Rac1 co - expression with dlrrk - WT using UAS - dlrrk - WT / + ; longGMR - GAL4 , UAS - Rac1W / + , results in rescued eye . iii . Rac1 co - expression with Clc using UAS - GFP - Clc / + ; longGMR - GAL4 , UAS - Rac1W / + , results in rescued eye . iv . Rac1 co - expression with dlrrk - WT - 3KD using UAS - dlrrk - 3KD / + ; longGMR - GAL4 , UAS - Rac1W / + , results in rescued eye . v . A wild - type eye of longGMR - GAL4 promoter as a control . vi - viii . Overexpression of dlrrk - WT , dlrrk - 3KD , or Clc alone with GMR - GAL4 as control , have no effect on eye .

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

Ci-viii. Lateral view of the adult fly head. Scale bar: 100 μm.i. longGMR,UAS-Rac1W/TM6B, Hu, Tb causes a mild rough eye phenotype with fused and disorganized but still separated ommatidia (as explained in Ai).ii. Rac1 co-expression with dlrrk-WT using UAS-dlrrk-WT/+;longGMR-GAL4,UAS-Rac1W/+, results in rescued eye morphology.iii. Rac1 co-expression with Clc using UAS-GFP-Clc/+;longGMR-GAL4,UAS-Rac1W/+, results in rescued eye morphology.iv. Rac1 co-expression with dlrrk-WT-3KD using UAS-dlrrk-3KD/+;longGMR-GAL4,UAS-Rac1W/+, results in rescued eye morphology.v. A wild-type eye of the longGMR-GAL4 promoter as a control.vi-viii. Overexpression of dlrrk-WT, dlrrk-3KD, or Clc alone with GMR-GAL4 as control, have no effect on eye morphology.


( B ) flg22 - induced ROS production in Col - 0 fer - 4 mutant roots under Pi - starvation conditions . Seedlings grown in either HP or LP media were treated with 1 μM flg22 , RALF23 or both flg22 RALF23 for 15 mins . Roots were harvested stained with H2DCFDA . One representative root was shown . ( C ) Average H2DCFDA signal in roots shown in ( B ) . was quantified with ImageJ . data shown indicate means ± SDs ( n = 30 , n refers to number of roots per group ) ; n . s . , not significant ; * * , p < 0 . 01 ( Student ' s t ) . Data information : experiments were repeated three times with similar results .

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

(B) flg22-induced ROS production in Col-0 and fer-4 mutant roots under Pi-starvation conditions. Seedlings grown in either HP or LP media were treated with 1 μM flg22, RALF23 or both flg22 and RALF23 for 15 mins. Roots were harvested and stained with H2DCFDA. One representative root was shown. (C) Average H2DCFDA signal intensity in roots shown in (B). The fluorescence intensity was quantified with ImageJ. The data shown indicate the means ± SDs (n = 30, n refers to the number of roots per group); n.s., not significant; **, p < 0.01 (Student's t test). Data information: All experiments were repeated three times with similar results.


( F ) RALF23 inhibits flg22 - induced MAMP - responsive FRK1 gene expression in a Pi content - dependent manner . Seedlings grown in either HP or LP media were treated with water or flg22 for 15 mins roots were harvested . relative expression of FRK1 was quantified via RT - qPCR . data shown indicate means ± SDs ( n = 3 , n refers to technical replicates ) ; n . s . , not significant ; * * , p < 0 . 01 ( Student ' s t ) . Data information : experiments were repeated three times with similar results .

O O O B-CONTROLLED_VAR O B-CONTROLLED_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 B-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 O O O O O O O O O O O O O O O O O O O O O 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) RALF23 inhibits flg22-induced MAMP-responsive FRK1 gene expression in a Pi content-dependent manner. Seedlings grown in either HP or LP media were treated with water or flg22 for 15 mins and roots were harvested. The relative expression of FRK1 was quantified via RT-qPCR. The data shown indicate the means ± SDs (n = 3, n refers to technical replicates); n.s., not significant; **, p < 0.01 (Student's t test). Data information: All experiments were repeated three times with similar results.


( A ) PSR induces cleavage of PRORALF23 - GFP into mature RALF23 - GFP . 35S promoter - driven RALF23 - GFP was grown in either HP or LP media for 5 days roots were harvested . Premature mature RALF23 were determined with anti - GFP . β - actin was used as a loading control . Data information : experiments were repeated three times with similar results .

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 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

(A) The PSR induces the cleavage of PRORALF23-GFP into mature RALF23-GFP. The 35S promoter-driven RALF23-GFP line was grown in either HP or LP media for 5 days and roots were harvested. Premature and mature RALF23 levels were determined with anti-GFP. β-actin was used as a loading control. Data information: All experiments were repeated three times with similar results.


( E ) PSR activates RALF expression in a PHR1 - dependent manner . Col - 0 phr1 seedlings were grown in LP media for 5 days roots were harvested . relative expression of RALFs was quantified via RT - qPCR . data shown indicate means ± SDs ( n = 3 , n refers to technical replicates ) ; n . s . , not significant ; * * , p < 0 . 01 ( Student ' s t ) . Data information : experiments were repeated three times with similar results .

O O O O O O O O O B-CONTROLLED_VAR O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) The PSR activates RALF expression in a PHR1-dependent manner. Col-0 and phr1 seedlings were grown in LP media for 5 days and roots were harvested. The relative expression of RALFs was quantified via RT-qPCR. The data shown indicate the means ± SDs (n = 3, n refers to technical replicates); n.s., not significant; **, p < 0.01 (Student's t test). Data information: All experiments were repeated three times with similar results.


( G ) PHR1 directly binds to RALF promoters , as shown via ChIP - PCR . Myc - labelled PHR1 overexpression was immunoprecipitated with anti - Myc . ChIP was performed on Col - 0 , CRY1 - Myc PHR1 - Myc transformed , with Col - 0 CRY1 - Myc transformed as negative controls . data shown indicate means ± SDs ( n = 3 , n refers to technical replicates ) ; n . s . , not significant ; * * , p < 0 . 01 ( Student ' s t ) . Data information : experiments were repeated three times with similar results .

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

(G) PHR1 directly binds to RALF promoters, as shown via ChIP-PCR. The Myc-labelled PHR1 overexpression line was immunoprecipitated with anti-Myc. ChIP analysis was performed on Col-0, CRY1-Myc and PHR1-Myc transformed plants, with Col-0 and CRY1-Myc transformed plants as negative controls. The data shown indicate the means ± SDs (n = 3, n refers to technical replicates); n.s., not significant; **, p < 0.01 (Student's t test). Data information: All experiments were repeated three times with similar results.


( B ) PHR1 negatively regulates FLS2 - BAK1 formation under Pi - starvation conditions . Col - 0 , phr1 mutant seedlings were grown in either HP or LP media for 5 days , then lysing roots into protoplasts , transformed with water or PHR1 - Myc plasmid treated with flg22 for 6 hours . amount of immunoprecipitated FLS2 coimmunoprecipitated BAK1 were determined using anti - FLS2 anti - BAK1 antibodies , respectively . Preimmune serum ( IgG ) was used as negative control . Data information : experiments were repeated three times with similar results .

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

(B) PHR1 negatively regulates FLS2-BAK1 complex formation under Pi-starvation conditions. Col-0, phr1 mutant seedlings were grown in either HP or LP media for 5 days, then lysing roots into protoplasts, transformed with water or PHR1-Myc plasmid and treated with flg22 for 6 hours. The amount of immunoprecipitated FLS2 and coimmunoprecipitated BAK1 were determined using anti-FLS2 and anti-BAK1 antibodies, respectively. Preimmune serum (IgG) was used as negative control. Data information: All experiments were repeated three times with similar results.


( D ) Application of RALF23 / 34 disrupted FLS2 - BAK1 formation in phr1 mutants . phr1 mutants grown in LP media were treated with water , 1 μM flg22 , 1 μM RALF23 , 1 μM RALF34 or both flg22 RALF for 6 hours . amount of immunoprecipitated FLS2 coimmunoprecipitated BAK1 were determined using anti - FLS2 anti - BAK1 antibodies , respectively . IgG was used as a negative control . Data information : experiments were repeated three times with similar results .

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

(D) Application of RALF23/34 disrupted FLS2-BAK1 complex formation in phr1 mutants. phr1 mutants grown in LP media were treated with water, 1 μM flg22, 1 μM RALF23, 1 μM RALF34 or both flg22 and RALF for 6 hours. The amount of immunoprecipitated FLS2 and coimmunoprecipitated BAK1 were determined using anti-FLS2 and anti-BAK1 antibodies, respectively. IgG was used as a negative control. Data information: All experiments were repeated three times with similar results.


( E ) RALF23 / 34 promoted growth of Pto DC3000 in phr1 mutants . Col - 0 phr1 mutant seedlings grown in LP media were root inoculated ( by soaking ) with a suspension ( OD600 = 0 . 2 ) for 2 min , number of from root was quantified at 5 dpi . data shown indicate means ± SDs ( n = 18 , n refers to number of roots per group ) . different lowercase letters indicate statistical significance ( one - way ANOVA ) . Data information : experiments were repeated three times with similar results .

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

(E) RALF23/34 promoted the growth of Pto DC3000 in phr1 mutants. Col-0 and phr1 mutant seedlings grown in LP media were root inoculated (by soaking) with a bacterial suspension (OD600 = 0.2) for 2 min, and the number of bacteria from individual root was quantified at 5 dpi. The data shown indicate the means ± SDs (n = 18, n refers to the number of roots per group). The different lowercase letters indicate statistical significance (one-way ANOVA). Data information: All experiments were repeated three times with similar results.


( F ) FER negatively regulates FLS2 - BAK1 formation under Pi - starvation conditions . Col - 0 , fer - 4 mutant FER / fer - 4 complementation lines seedlings were grown in either HP or LP media for 5 days treated with flg22 for 6 hours . amount of immunoprecipitated FLS2 coimmunoprecipitated BAK1 was determined using anti - FLS2 anti - BAK1 antibodies , respectively . Preimmune serum ( IgG ) was used as a negative control . Data information : experiments were repeated three times with similar results .

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

(F) FER negatively regulates FLS2-BAK1 complex formation under Pi-starvation conditions. Col-0, fer-4 mutant and FER/fer-4 complementation lines seedlings were grown in either HP or LP media for 5 days and treated with flg22 for 6 hours. The amount of immunoprecipitated FLS2 and coimmunoprecipitated BAK1 was determined using anti-FLS2 and anti-BAK1 antibodies, respectively. Preimmune serum (IgG) was used as a negative control. Data information: All experiments were repeated three times with similar results.


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

(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 .

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

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 .

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

(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 .

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 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

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 .

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

(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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) 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 ) .

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

(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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) 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 .

O O O O 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

(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 ) .

O O O O 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 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

(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 ± .

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

(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 .

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

(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 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 O O O O O O 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) 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 .

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

(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 .

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

(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 .

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 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 B-MEASURED_VAR O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) 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 .

O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O 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 B-MEASURED_VAR O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) 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 " .

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 O O O O O O O

(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 .

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

(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 .

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

(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 .

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

(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 .

O O 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 B-CONTROLLED_VAR I-CONTROLLED_VAR I-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 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

(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 .

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

(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 .

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

(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 .

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

(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 " .

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

(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 .

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

(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 ) .

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

(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 .

O O 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 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 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

(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 .

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

(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 .

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

(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 .

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

(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 / ) .

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

(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 .

O O O O O O O O B-MEASURED_VAR 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 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 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

(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 .

O O O O O O O O B-MEASURED_VAR 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 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

(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 .

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

(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 .

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

(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 .

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

(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 .

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

(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 .

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

(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 .

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 I-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 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

(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 ) .

O O O O O O O B-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 O O

(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 .

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

(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 .

O O O O O O O O O O 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

(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 .

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

(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 .

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

(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 .

O O O O O O O O O O O O O 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) 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 .

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

(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 .

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

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 .

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

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 .

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

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 .

O O 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 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 .

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

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 .

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

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 .

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

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 .

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 O O O O O O O

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 .

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

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 .

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

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 .

O O O B-MEASURED_VAR 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

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 ) .

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

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 .

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

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 .

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-MEASURED_VAR O O O O O O O O O O

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 .

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

C 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 .

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

(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 ) .

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

(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 .

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

(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 .

O O O O O O O O O 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

(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 .

O 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

(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 .

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

(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 .

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

(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 .

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

(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 .

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

(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 .

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

(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 .

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

(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.