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 .

B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O B-GENEPROD O O O O O O O O O O O O O O O O O O B-GENEPROD 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 sem . One - way ANOVA , Bonferroni ' s post hoc test , * * * p < 0 . 001 , N = 3 .

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

B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O O O O O O O B-EXP_ASSAY O B-EXP_ASSAY O O O O O B-GENEPROD 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 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 .

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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 viruses in combination . The lysates were processed for Western blot with antibody that recognizes all forms of CLCs .

O O O O O O O B-CELL_TYPE I-CELL_TYPE O O B-ORGANISM O O O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O B-GENEPROD 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 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 .

B-CELL_TYPE I-CELL_TYPE O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O B-GENEPROD O O O O O O O O O O B-GENEPROD O O B-ORGANISM O O O B-GENEPROD O O O O O O O O O O O O O O O O O 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 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 .

O O O O O O O O O B-ORGANISM B-TISSUE O O O O O O O O O O O O O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O O O B-TISSUE O O O O O O O O O O O B-TISSUE O O O O O O O O B-GENEPROD O O O O B-GENEPROD O O O B-GENEPROD O O O O O O O O O O O O O O B-GENEPROD O O O O O B-TISSUE O O B-TISSUE O O O O O O O B-GENEPROD O O O O B-GENEPROD O O O B-GENEPROD O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O B-TISSUE O O B-TISSUE O O O O B-GENEPROD O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O B-TISSUE O O O O O O O O B-GENEPROD O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O B-TISSUE 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 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 / + ) .

O O O O O O O O O B-ORGANISM B-TISSUE O O O O O O O O O B-GENEPROD O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O B-GENEPROD O O O O O B-TISSUE O O B-TISSUE O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O B-TISSUE O O O O O O O O B-GENEPROD 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 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 .

O O O O O O O O O B-ORGANISM B-TISSUE O O O O O O O O O O O O O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O O O B-TISSUE O O O O O O O O B-TISSUE O O O O O O O O O B-GENEPROD O O O O B-GENEPROD O O O O O B-GENEPROD O O O O O O O O O O O B-GENEPROD O O O O O O B-TISSUE O O O O B-GENEPROD O O O O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O O O O O O O O B-GENEPROD O O O O O O B-TISSUE O O O O B-GENEPROD O O O O B-GENEPROD O O O O O O O B-GENEPROD O O O O O O O O O O O B-GENEPROD O O O O O O B-TISSUE O O O O O O O O B-TISSUE O O O O O O O O O O O O O O O O B-GENEPROD O O O B-GENEPROD O O O O B-GENEPROD O O O O O O O O O O O O B-TISSUE 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 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 .

O O O B-GENEPROD O O B-SMALL_MOLECULE O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-TISSUE O B-SMALL_MOLECULE O O O O B-ORGANISM O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O B-GENEPROD O B-GENEPROD O O O O B-TISSUE O O O B-EXP_ASSAY O B-SMALL_MOLECULE O O O B-TISSUE O O O O O O O B-SMALL_MOLECULE O O O B-TISSUE O O O O O O O O O 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-TISSUE O O O O O O O O O O O O O O O O O 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 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 .

O O O B-GENEPROD O B-GENEPROD O O O O O B-GENEPROD B-EXP_ASSAY I-EXP_ASSAY O O B-SMALL_MOLECULE O O O O O B-ORGANISM O O O O O O O O O O O O B-GENEPROD O O O O B-TISSUE O O O O O B-EXP_ASSAY O B-GENEPROD O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 ) 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 .

O O O O O O O O O B-GENEPROD O B-GENEPROD O O B-GENEPROD O B-GENEPROD O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O B-TISSUE O O O O O O B-GENEPROD O O O O O O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD 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 ) 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 .

O O O O O O O B-EXP_ASSAY O O B-GENEPROD O O O O O O O O B-GENEPROD B-ORGANISM O O O O O O O O O B-TISSUE O O O O O B-EXP_ASSAY O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 . 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 .

O O O B-GENEPROD O B-EXP_ASSAY O O O O O O O B-EXP_ASSAY O B-EXP_ASSAY O O B-GENEPROD O O B-GENEPROD O O O B-EXP_ASSAY O O O B-GENEPROD O B-EXP_ASSAY O O O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 .

O O O B-GENEPROD O O B-GENEPROD O B-GENEPROD O O O B-SMALL_MOLECULE O O O O O O O O B-GENEPROD O B-ORGANISM O O O O O O O O O O O O O O B-TISSUE O B-TISSUE O O O O O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD O O O O O O O B-EXP_ASSAY B-GENEPROD O B-EXP_ASSAY B-GENEPROD O O O O O B-GENEPROD O O O B-GENEPROD O O O O 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 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 .

O O O O O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O O B-GENEPROD O O O O O O O O O O O O B-GENEPROD O O O B-GENEPROD O O O B-GENEPROD O O B-GENEPROD O O O O O O O O O B-EXP_ASSAY B-GENEPROD O B-EXP_ASSAY B-GENEPROD O O O O O B-GENEPROD O O O B-GENEPROD O 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 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 .

O O O B-GENEPROD O O O O B-EXP_ASSAY O B-ORGANISM I-ORGANISM O B-GENEPROD O O O O O O B-GENEPROD O B-ORGANISM O O O O O B-TISSUE O O O 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-TISSUE O 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-TISSUE O O O O O O O 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 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 .

O O O B-GENEPROD O O B-GENEPROD O B-GENEPROD O O O B-SMALL_MOLECULE O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O B-ORGANISM O O O O O O O O O O O O O O B-GENEPROD O O O O O O O B-EXP_ASSAY B-GENEPROD O B-EXP_ASSAY B-GENEPROD O O O O O B-GENEPROD O O O B-GENEPROD O O O O O 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 ) . 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 ) .

O O O O O O B-ORGANISM O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O B-ORGANISM I-ORGANISM I-ORGANISM O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O 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-ORGANISM O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O B-ORGANISM I-ORGANISM I-ORGANISM O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-ORGANISM O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O B-ORGANISM I-ORGANISM I-ORGANISM O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-ORGANISM O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O B-ORGANISM I-ORGANISM I-ORGANISM O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O 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).


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 .

O O O O B-EXP_ASSAY O O O O O B-ORGANISM B-TISSUE O O O O 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-ORGANISM O O O O B-SMALL_MOLECULE O O O B-ORGANISM I-ORGANISM O B-ORGANISM I-ORGANISM I-ORGANISM O O 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 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 .

O O O B-ORGANISM I-ORGANISM O B-ORGANISM I-ORGANISM I-ORGANISM O B-SMALL_MOLECULE O B-EXP_ASSAY I-EXP_ASSAY O O O O O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O B-TISSUE O O O O O O O B-ORGANISM I-ORGANISM O B-ORGANISM I-ORGANISM I-ORGANISM O B-SMALL_MOLECULE O O O B-SMALL_MOLECULE O O O O O O O B-EXP_ASSAY O B-SMALL_MOLECULE O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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.


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 .

O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-EXP_ASSAY O O O O O B-ORGANISM B-TISSUE O O O O 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-ORGANISM O O O O B-SMALL_MOLECULE O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O 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 ) 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 .

O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-SMALL_MOLECULE O B-EXP_ASSAY I-EXP_ASSAY O O B-TISSUE O O O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O B-EXP_ASSAY O B-SMALL_MOLECULE O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 .

O O O O B-TISSUE I-TISSUE O O O O O O O O O O O O O B-DISEASE O O O O B-SUBCELLULAR O O O O O O O B-TISSUE I-TISSUE O O O O O O O B-SUBCELLULAR O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O B-EXP_ASSAY O O O O B-SMALL_MOLECULE B-EXP_ASSAY O O O O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE O B-EXP_ASSAY O O O O B-TISSUE 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 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 ) .

O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O B-TISSUE O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-CELL_TYPE O O O O O O O O O O O O O O O O O O O O O B-CELL_TYPE O O B-ORGANISM O O O B-DISEASE I-DISEASE I-DISEASE O B-DISEASE I-DISEASE I-DISEASE 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 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 .

O O O B-TISSUE B-EXP_ASSAY O O O O O O O O O O O O O O B-TISSUE O B-TISSUE O B-TISSUE O O O O O O O O O O O O O O O O O O O B-TISSUE I-TISSUE O O O O B-TISSUE B-EXP_ASSAY O O O O O O O O O O O O O O O O O O B-TISSUE I-TISSUE 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 RNA sequencing reads in P1 ( red ) and two control muscle samples ( blue and green ) at the exons 3 - 5 of BET1 .

O O O O O O B-TISSUE B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD 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 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 ) .

O O O B-EXP_ASSAY O B-ORGANISM B-CELL_TYPE O O O O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O B-GENEPROD B-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O 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 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 .

O O O O B-EXP_ASSAY O B-GENEPROD O O O B-ORGANISM B-CELL_TYPE O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-EXP_ASSAY O O B-GENEPROD O O O O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O B-GENEPROD O O O O O O O O O B-EXP_ASSAY O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-EXP_ASSAY O O B-GENEPROD O O O O B-SUBCELLULAR O B-GENEPROD O O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 - 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 .

O O O B-CELL_LINE O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O 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 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 .

O O O B-EXP_ASSAY O B-SMALL_MOLECULE I-SMALL_MOLECULE O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE 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 B-EXP_ASSAY O B-EXP_ASSAY O O B-GENEPROD O B-GENEPROD O B-SMALL_MOLECULE O

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


( 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 .

O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O O O O B-CELL_LINE O O O O B-GENEPROD O O O O B-EXP_ASSAY O O O B-GENEPROD O O B-EXP_ASSAY O O O O O O O B-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-GENEPROD O O O B-GENEPROD O O O B-GENEPROD O O O B-GENEPROD 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 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 .

O O O O B-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD O B-CELL_TYPE O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-EXP_ASSAY O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-GENEPROD O O O O B-CELL_TYPE O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-EXP_ASSAY 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 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 .

O O O B-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O O O O O O B-CELL_TYPE O O B-EXP_ASSAY O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-GENEPROD O O O O B-CELL_TYPE O O O O O O O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 ) 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 .

O O O O B-EXP_ASSAY O O O O B-SUBCELLULAR O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 the TNF - α - up - regulated genes . Genes were classified based on the TNF - α - treated time points , using the algorithm " HOPACH " .

O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O 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 . 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 .

O O O O O O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O 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-EXP_ASSAY I-EXP_ASSAY O O O O O O O O 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-ORGANISM B-CELL_TYPE I-CELL_TYPE O B-CELL_TYPE 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 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 .

O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O O O O O O O O B-ORGANISM B-CELL_TYPE O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O 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 , and β - actin ( ACTB ) in lysates from ECs treated with or without TNF - α ( 4 hrs ) ± miR - 3679 - 5p .

O O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O B-GENEPROD O B-GENEPROD O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O O O O B-CELL_TYPE O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD 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 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 .

O O O O B-EXP_ASSAY O O O O O O O O O O O O B-EXP_ASSAY O B-SMALL_MOLECULE O O B-CELL_LINE B-CELL_TYPE O B-CELL_TYPE O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O 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-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O 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 ) 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 .

O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY O O O O B-EXP_ASSAY O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O 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 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 .

O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O O O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O B-CELL_TYPE O O O O O O B-GENEPROD O O O O O O O O O O O O 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 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 .

O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O O O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O B-CELL_TYPE O O O O O O B-GENEPROD O O O O O O O O O O O O O O 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 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 " .

O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O 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 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 .

O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O B-GENEPROD O O B-GENEPROD O O O O O O B-CELL_TYPE O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O 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-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O 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 , and ACTB in lysates from ECs treated with or without TNF - α ( 4 hrs ) ± ( siKDM7A + siUTX ) .

O O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O B-GENEPROD O B-GENEPROD O O B-GENEPROD O O O B-CELL_TYPE O O O O B-GENEPROD I-GENEPROD I-GENEPROD 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 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 .

O O O O B-EXP_ASSAY O O O O O O O O O O O O B-EXP_ASSAY O B-SMALL_MOLECULE O O B-CELL_LINE B-CELL_TYPE O B-CELL_TYPE O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O 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-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O 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 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 .

O O O O O O O O O O O B-GENEPROD O B-GENEPROD B-EXP_ASSAY O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD O O B-CELL_TYPE O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O B-GENEPROD O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O B-GENEPROD O O O O B-GENEPROD O O 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 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 .

O O O O O O O O B-GENEPROD O O O O B-GENEPROD O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD 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 ) , 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 .

O O O O O O B-GENEPROD O O O O B-GENEPROD O O O O B-GENEPROD O O O O O B-GENEPROD O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O B-CELL_TYPE O O O O O O O O O O B-GENEPROD O O O O B-GENEPROD O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY 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 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 / ) .

O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O B-GENEPROD O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O B-GENEPROD O O O O B-GENEPROD O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-CELL_TYPE O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY 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 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 .

O O O O O B-EXP_ASSAY O B-EXP_ASSAY O B-GENEPROD O O O O B-GENEPROD O O O O O B-GENEPROD O O O O O O 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-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O 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 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 .

O O O O O B-EXP_ASSAY O B-EXP_ASSAY O B-GENEPROD O O O O B-GENEPROD O O O O O B-GENEPROD O O O O O O 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-GENEPROD I-GENEPROD I-GENEPROD O O 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 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 .

O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD O B-GENEPROD O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-GENEPROD I-GENEPROD I-GENEPROD 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 and H3K27me3 around the VCAM1 locus in TNF - α ( - ) and TNF - α ( + ) ECs .

O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD O B-GENEPROD O O B-GENEPROD O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-CELL_TYPE O

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


( 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 .

O O O O O B-EXP_ASSAY O B-EXP_ASSAY O O O O O O O B-GENEPROD O O O O B-GENEPROD O O O O O B-GENEPROD O O O O O O O O O O O O O O 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-GENEPROD I-GENEPROD I-GENEPROD O O 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 and H3K27me3 around the SELE locus in TNF - α ( - ) and 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 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 .

(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 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 .

O O O O O B-EXP_ASSAY O B-EXP_ASSAY O O O O O O O B-GENEPROD O B-GENEPROD O O B-GENEPROD O O O O B-GENEPROD O O O O O 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-GENEPROD I-GENEPROD I-GENEPROD O O 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 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 .

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(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 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 .

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


( B 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 ) .

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


( D ) Pirate plots showing ChIA - PET interaction changes at control - specific , common , 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 .

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


( E 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 .

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


( G 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 .

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(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 and H3K27me3 around VCAM1 ( I ) , IL8 ( J ) , and SELE ( K ) in TNF - α ( - ) and TNF - α ( + ) ECs . Red bars show TNF - α - specific SEs .

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


( B ) Representative snapshot from 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 .

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


( C ) Quantitative analyses of leukocyte adhesive interactions in 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 .

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


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

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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 the loading control .

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


F Immunoblot 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 .

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


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

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 .

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


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 .

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


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

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


B Immunoblots showing 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 .

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


F Immunoblots showing 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 .

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


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

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


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

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


E - G Following 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 ) .

O O O O O O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE I-CELL_LINE I-CELL_LINE O B-GENEPROD O B-GENEPROD O O O O O O O O O O O B-DISEASE B-ORGANISM O O O O O O B-GENEPROD O B-GENEPROD O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O B-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY O O O O O O O O O O O O O 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-ORGANISM O 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-CELL_LINE I-CELL_LINE I-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE I-CELL_LINE I-CELL_LINE O O O B-GENEPROD O O O O O O O O O O O O B-ORGANISM O O O O B-GENEPROD O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O B-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY O O O O O O O O O O O O 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-ORGANISM O O 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 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 .

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


B Immunoblot 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 .

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


C 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 .

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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 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 .

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

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(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 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 .

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


( E ) Immunoblotting against 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 .

O O O B-EXP_ASSAY O O B-GENEPROD O O B-GENEPROD O O O O B-CELL_LINE O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O B-GENEPROD 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 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 .

O O O B-EXP_ASSAY O O B-GENEPROD O O O B-GENEPROD O O O O B-CELL_LINE O O O O O O O O O O O O B-ORGANISM O B-GENEPROD O O O O O O O O O B-GENEPROD 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 the N - terminal part of the HA - immunoprecipitated and LysC digested polyPG protein 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 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 .

(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 ) 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 .

O O O B-EXP_ASSAY I-EXP_ASSAY O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE B-EXP_ASSAY O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE B-CELL_TYPE I-CELL_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 B-GENEPROD O O O B-GENEPROD 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 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 .

O O O B-EXP_ASSAY O O B-GENEPROD O O B-GENEPROD O O B-GENEPROD O O O O B-CELL_LINE O O O O O O O O O O O O B-ORGANISM O B-GENEPROD O O O O B-GENEPROD O O O O O O O O O O O O O O B-GENEPROD 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 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 .

O O O B-EXP_ASSAY O O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O O O B-CELL_LINE O O O O O O O O O O O O B-ORGANISM O B-GENEPROD O O O O B-GENEPROD O O O O O O O O O O O O O O O B-GENEPROD O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE 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 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 .

(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 ) 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 .

O O O B-EXP_ASSAY I-EXP_ASSAY O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE B-EXP_ASSAY O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE B-CELL_TYPE I-CELL_TYPE O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O B-GENEPROD 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 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 .

O O O O O O B-EXP_ASSAY O O B-GENEPROD O O O B-GENEPROD O O O O B-CELL_LINE O O O O O O O O O O O O O O O B-ORGANISM O B-GENEPROD O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O B-GENEPROD 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.