( H ) Western Blot of CREBH K6 , K11 , K27 , K29 K33 only ubiquitination level after HRD1 co - expression .

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(H) Western Blot analysis of the CREBH K6, K11, K27, K29 and K33 only ubiquitination level after HRD1 co-expression.


( I ) Western Blot of CREBH WT , K27R ubiquitination level after HRD1 full - co - expression .

(I) Western Blot analysis of the CREBH WT, K27R ubiquitination level after HRD1 full-length co-expression.


( J ) Western Blot of CREBH WT , K27 K48 only ubiquitination level after HRD1 C - terminal co - expression .

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(J) Western Blot analysis of the CREBH WT, K27 and K48 only ubiquitination level after HRD1 C-terminal co-expression.


( B ) Hepatic CREBH 5 after AAV - shCrebh injection . ( n = 5 for each group ) .

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(B) Hepatic CREBH protein levels 5 weeks after AAV-shCrebh injection. (n=5 for each group).


( C ) Hepatic Fgf21 mRNA in WT L - HRD1 KO mice 5 after AAV - shCrebh injection . ( n = 5 for each group ) .

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(C) Hepatic Fgf21 mRNA in the WT and L-HRD1 KO mice 5 weeks after AAV-shCrebh injection. (n=5 for each group).


( D ) Serum FGF21 in WT L - HRD1 KO mice 5 after AAV - shCrebh injection . ( n = 5 for each group ) .

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(D) Serum FGF21 protein levels in the WT and L-HRD1 KO mice 5 weeks after AAV-shCrebh injection. (n=5 for each group).


( E ) height tibia of WT L - HRD1 KO mice 5 after AAV - shCrebh injection ( n = 5 for each group ) .

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(E) Body height and tibia length of the WT and L-HRD1 KO mice 5 weeks after AAV-shCrebh injection (n=5 for each group).


( F ) Hepatic Igf1 , Cyp2d9 Cyp4a12 mRNA in WT L - HRD1 KO mice 5 after AAV - shCrebh injection . ( n = 5 for each group ) .

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(F) Hepatic Igf1, Cyp2d9 and Cyp4a12 mRNA in the WT and L-HRD1 KO mice 5 weeks after AAV-shCrebh injection. (n=5 for each group).


( G , H ) of mated estrus cycle of WT L - HRD KO 5 after AAV - shCrebh injection . Data information : data are representative of three independent experiments ( mean ± s . d . ) . * : P < 0 . 05 . * * : P < 0 . 01 by unpaired student ' s t .

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(G, H) Percentage of the mated and estrus cycle of the WT and L-HRD KO 5 weeks after AAV-shCrebh injection. Data information: The data are representative of three independent experiments (mean ± s.d.). *: P<0.05. **: P<0.01 by unpaired student's t test.


( A ) Examples of sizes of WT , L - HRD KO HRD1 / FGF21 DKO mice . ( B , C ) height of WT , L - HRD KO HRD1 / FGF21 DKO mice . ( n = 5 for each group ) . Data information : data are representative of three independent experiments ( mean ± s . d . ) . * : P < 0 . 05 . * * : P < 0 . 01 by unpaired student ' s t .

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

(A) Examples of the body sizes of the WT, L-HRD KO and HRD1/FGF21 DKO mice. (B, C) Body weight and body height of the WT, L-HRD KO and HRD1/FGF21 DKO mice. (n=5 for each group). Data information: The data are representative of three independent experiments (mean ± s.d.). *: P<0.05. **: P<0.01 by unpaired student's t test.


( D ) Tibia lengths of WT , L - HRD KO HRD1 / FGF21 DKO mice fed with normal chow diet . ( n = 5 for each group ) . Data information : data are representative of three independent experiments ( mean ± s . d . ) . * : P < 0 . 05 . * * : P < 0 . 01 by unpaired student ' s t .

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

(D) Tibia lengths of the WT, L-HRD KO and HRD1/FGF21 DKO mice fed with normal chow diet. (n=5 for each group). Data information: The data are representative of three independent experiments (mean ± s.d.). *: P<0.05. **: P<0.01 by unpaired student's t test.


( E ) Hepatic Hsd3b5 , Cyp2d9 Cyp4a12 mRNA in WT , L - HRD1 KO HRD1 / FGF21 DKO mice . ( n = 5 for each group ) . Data information : data are representative of three independent experiments ( mean ± s . d . ) . * : P < 0 . 05 . * * : P < 0 . 01 by unpaired student ' s t .

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

(E) Hepatic Hsd3b5, Cyp2d9 and Cyp4a12 mRNA in the WT, L-HRD1 KO and HRD1/FGF21 DKO mice. (n=5 for each group). Data information: The data are representative of three independent experiments (mean ± s.d.). *: P<0.05. **: P<0.01 by unpaired student's t test.


( F ) Estrus cycle of L - HRD KO HRD1 / FGF21 DKO mice at of 4 months . Data information : data are representative of three independent experiments ( mean ± s . d . ) . * : P < 0 . 05 . * * : P < 0 . 01 by unpaired student ' s t .

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(F) Estrus cycle of L-HRD KO and HRD1/FGF21 DKO mice at the age of 4 months. Data information: The data are representative of three independent experiments (mean ± s.d.). *: P<0.05. **: P<0.01 by unpaired student's t test.


( G ) of Activity , feeding water intake of WT , L - HRD KO HRD1 / FGF21 DKO mice . ( n = 5 for each group ) . Data information : data are representative of three independent experiments ( mean ± s . d . ) . * : P < 0 . 05 . * * : P < 0 . 01 by unpaired student ' s t .

(G) Percentage of the Activity, feeding and water intake of the WT, L-HRD KO and HRD1/FGF21 DKO mice. (n=5 for each group). Data information: The data are representative of three independent experiments (mean ± s.d.). *: P<0.05. **: P<0.01 by unpaired student's t test.


Immunoblot of p62 LC3 I / II in KRIT1 wt KRIT1 - KO endothelial . Actin was used as a loading control . of total LC3 on actin is reported ( * P = 0 . 02712 ) . results are representative of three independent experiments .

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Immunoblot analysis of p62 and LC3 I/II in KRIT1 wt and KRIT1-KO endothelial cells. Actin was used as a loading control. Quantification of total LC3 on actin is reported (*P = 0.02712). The results are representative of three independent experiments.


Representative of p62 dots in KRIT1 wt KRIT1 - KO endothelial . Scale bar , 20 μm . Magnifications in insets . Right , quantitative of p62 distribution of dots is reported ( four independent experiments ; n = 35 per group ) . * P = 0 . 00542 ( dotted ) ; * P = 0 . 00014 ( ) .

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Representative images of p62 dots in KRIT1 wt and KRIT1-KO endothelial cells. Scale bar, 20 μm. Magnifications in insets. Right, quantitative analysis of p62 distribution of dots is reported (four independent experiments; n = 35 cells per group). *P = 0.00542 (dotted); *P = 0.00014 (nuclear).


Immunoblot of p62 LC3 I / II in KRIT1 - KO KRIT1 - KO re - expressing KRIT1 ( KO + KRIT1 ) MEFs . Left , immunoblot showing KRIT1 in KRIT1 - KO KO + KRIT1 . Right , immunoblots for p62 LC3 I / II . Actin was used as a loading marker . of total LC3 on actin is reported ( * P = 0 . 01248 ) . results are representative of three independent experiments .

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

Immunoblot analysis of p62 and LC3 I/II in KRIT1-KO and KRIT1-KO re-expressing KRIT1 (KO+KRIT1) MEFs. Left, immunoblot showing KRIT1 levels in KRIT1-KO and KO+KRIT1 cells. Right, immunoblots for p62 and LC3 I/II. Actin was used as a loading marker. Quantification of total LC3 on actin is reported (*P = 0.01248). The results are representative of three independent experiments.


Representative of p62 dots in KO + KRIT1 ( top ) KRIT1 - KO ( bottom ) . Scale bar , 50 μm . Magnifications in insets . Right , quantitative of number of p62 dots per is shown ( four independent experiments ; n = 50 per group ) . * P = 7 . 18e − 14 .

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Representative images of p62 dots in KO+KRIT1 (top) and KRIT1-KO cells (bottom). Scale bar, 50 μm. Magnifications in insets. Right, quantitative analysis of the number of p62 dots per cell is shown (four independent experiments; n = 50 cells per group). *P = 7.18e−14.


Immunoblot of hBMECs transiently transfected with control siRNA or KRIT1 siRNA . Left , evaluation of siRNA efficiency with antibody directed against KRIT1 . Right , immunoblots for p62 LC3 I / II . Actin was used as a loading marker . of total LC3 on actin is reported ( * P = 0 . 03071 ) . results are representative of three independent experiments .

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

Immunoblot analysis of hBMECs transiently transfected with control siRNA or KRIT1 siRNA. Left, evaluation of siRNA efficiency with antibody directed against KRIT1. Right, immunoblots for p62 and LC3 I/II. Actin was used as a loading marker. Quantification of total LC3 on actin is reported (*P = 0.03071). The results are representative of three independent experiments.


Immunoblot of EA . hy926 transiently transfected with control siRNA or KRIT1 siRNA . Left , evaluation of siRNA efficiency with antibody directed against KRIT1 . Right , immunoblot for p62 LC3 I / II . Actin was used as a loading marker . of total LC3 on actin is reported ( * P = 0 . 02527 ) . results are representative of three independent experiments .

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

Immunoblot analysis of EA.hy926 cells transiently transfected with control siRNA or KRIT1 siRNA. Left, evaluation of siRNA efficiency with antibody directed against KRIT1. Right, immunoblot for p62 and LC3 I/II. Actin was used as a loading marker. Quantification of total LC3 on actin is reported (*P = 0.02527). The results are representative of three independent experiments.


Immunofluorescence of p62 ( green ) ProteoStat Aggresome detection reagent ( red ) in KRIT1 wt KRIT1 - KO lung endothelial . yellow signal in merged represents an overlapping spatial relationship between green red . Magnification in insets . Scale bar , 50 μm . are representative of four independent experiments .

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Immunofluorescence analysis of p62 (green) and ProteoStat Aggresome staining detection reagent (red) in KRIT1 wt and KRIT1-KO lung endothelial cells. The yellow signal in the merged images represents an overlapping spatial relationship between green and red fluorescence. Magnification in insets. Scale bar, 50 μm. The images are representative of four independent experiments.


Immunofluorescence of p62 ( green ) ProteoStat Aggresome detection reagent ( red ) in KRIT1 - KO re - expressing KRIT1 ( KO + KRIT1 ) KRIT1 - KO MEFs . yellow signal in merged represents an overlapping spatial relationship between green red . Magnification in insets . Scale bar , 50 μm . are representative of four independent experiments .

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Immunofluorescence analysis of p62 (green) and ProteoStat Aggresome staining detection reagent (red) in KRIT1-KO re-expressing KRIT1 (KO+KRIT1) and KRIT1-KO MEFs. The yellow signal in the merged images represents an overlapping spatial relationship between green and red fluorescence. Magnification in insets. Scale bar, 50 μm. The images are representative of four independent experiments.


Immunoblot with antibodies directed against phosphorylated mTOR ( Ser 2448 ) , total mTOR , phosphorylated p70 S6 Kinase ( Ser 371 ) , total p70 S6 Kinase , phosphorylated 4E - BP1 ( Thr 37 / 46 ) , total 4E - BP1 ; actin was used as a loading marker . Where indicated , KRIT1 wt KRIT1 - KO endothelial were treated with 100 nM Torin1 for 4 h . results are representative of three independent experiments .

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

Immunoblot analysis with antibodies directed against phosphorylated mTOR (Ser 2448), total mTOR, phosphorylated p70 S6 Kinase (Ser 371), total p70 S6 Kinase, phosphorylated 4E-BP1 (Thr 37/46), and total 4E-BP1; actin was used as a loading marker. Where indicated, KRIT1 wt and KRIT1-KO endothelial cells were treated with 100 nM Torin1 for 4 h. The results are representative of three independent experiments.


Immunoblot of total ULK1 actin in KRIT1 wt KRIT1 - KO endothelial . Where indicated , were treated with 100 nM Torin1 for 4 h . results are representative of three independent experiments .

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Immunoblot analysis of total ULK1 and actin in KRIT1 wt and KRIT1-KO endothelial cells. Where indicated, cells were treated with 100 nM Torin1 for 4 h. The results are representative of three independent experiments.


Immunoblot of p62 , LC3 I / II , actin in KRIT1 wt KRIT1 - KO endothelial treated with 100 nM Torin1 or 500 nM rapamycin for 4 h . results are representative of three independent experiments .

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Immunoblot analysis of p62, LC3 I/II, and actin in KRIT1 wt and KRIT1-KO endothelial cells treated with 100 nM Torin1 or 500 nM rapamycin for 4 h. The results are representative of three independent experiments.


Immunoblot with antibodies directed against phosphorylated mTOR ( Ser 2448 ) , total mTOR , phosphorylated p70 S6 Kinase ( Ser 371 ) , total p70 S6 Kinase , phosphorylated 4E - BP1 ( Thr 37 / 46 ) , total 4E - BP1 ; actin was used as a loading marker . Where indicated , KRIT1 - KO re - expressing KRIT1 ( KO + KRIT1 ) KRIT1 - KO MEFs were treated with 100 nM Torin1 for 4 h . results are representative of three independent experiments .

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

Immunoblot analysis with antibodies directed against phosphorylated mTOR (Ser 2448), total mTOR, phosphorylated p70 S6 Kinase (Ser 371), total p70 S6 Kinase, phosphorylated 4E-BP1 (Thr 37/46), and total 4E-BP1; actin was used as a loading marker. Where indicated, KRIT1-KO re-expressing KRIT1 (KO+KRIT1) and KRIT1-KO MEFs were treated with 100 nM Torin1 for 4 h. The results are representative of three independent experiments.


Immunoblot of phosphorylated ULK1 ( Ser 757 ) , total ULK1 , actin in KRIT1 KO + KRIT1 , KRIT1 KO MEFs . Where indicated , were treated with 100 nM Torin1 for 4 h . results are representative of three independent experiments .

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Immunoblot analysis of phosphorylated ULK1 (Ser 757), total ULK1, and actin in KRIT1 KO+KRIT1, and KRIT1 KO MEFs. Where indicated, cells were treated with 100 nM Torin1 for 4 h. The results are representative of three independent experiments.


Immunoblot of p62 , actin , LC3 I / II in KO + KRIT1 KRIT1 - KO . Where indicated , were treated with 100 nM Torin1 for 4 h or 500 nM rapamycin for 4 h . results are representative of three independent experiments .

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Immunoblot analysis of p62, actin, LC3 I/II in KO+KRIT1 and KRIT1-KO cells. Where indicated, cells were treated with 100 nM Torin1 for 4 h or 500 nM rapamycin for 4 h. The results are representative of three independent experiments.


KRIT1 wt KRIT1 - KO endothelial were transiently transfected with mRFP - GFP - LC3 . Where indicated , were treated with 100 nM Torin1 for 4 h or 2 μM xestospongin B for 4 h . differences in autophagic flux were evaluated by counting yellow LC3 I / II dots / ( RFP + GFP + ) red LC3 dots / ( RFP + GFP − ) for each condition . Yellow dots : autophagosomes ; red dots : autophagolysosomes . * P = 5 . 74e − 5 ( red dots , WT ctrl vs . WT Tor1 ) ; * P = 9 . 62e − 5 ( red dots , WT ctrl vs . WT xesto ) ; * P = 0 . 00727 ( red dots , WT ctrl vs . KO ctrl ) ; # P = 0 . 00046 ( red dots , KO ctrl vs . KO Tor1 ) . data are expressed as mean ± s . e . m .

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

KRIT1 wt and KRIT1-KO endothelial cells were transiently transfected with mRFP-GFP-LC3. Where indicated, the cells were treated with 100 nM Torin1 for 4 h or 2 μM xestospongin B for 4 h. The differences in the autophagic flux were evaluated by counting the yellow LC3 I/II dots/cell (RFP+GFP+) and red LC3 dots/cell (RFP+GFP−) for each condition. Yellow dots: autophagosomes; red dots: autophagolysosomes. *P = 5.74e−5 (red dots, WT ctrl vs. WT Tor1); *P = 9.62e−5 (red dots, WT ctrl vs. WT xesto); *P = 0.00727 (red dots, WT ctrl vs. KO ctrl); #P = 0.00046 (red dots, KO ctrl vs. KO Tor1). The data are expressed as the mean ± s.e.m.


KO + KRIT1 KRIT1 - KO MEFs were transiently transfected with mRFP - GFP - LC3 tandem construct . Where indicated , were treated with 100 nM Torin1 for 4 h or 2 μM xestospongin B for 4 h . differences in autophagic flux were evaluated by counting yellow LC3 I / II dots / ( RFP + GFP + ) red LC3 dots / ( RFP + GFP − ) for each condition . Yellow dots : autophagosomes ; red dots : autophagolysosomes . * P = 0 . 00023 ( red dots , KO + KRIT1 ctrl vs . KO + KRIT1 Tor1 ) ; * P = 0 . 00045 ( red dots , KO + KRIT1 ctrl vs . KO + KRIT1 xesto ) ; # P = 3 . 08e − 6 ( red dots , KO ctrl vs . KO Tor1 ) ; # # P = 6 . 73e − 5 ( yellow dots , KO + KRIT1 ctrl vs . KO ctrl ) . data are expressed as mean ± s . e . m . of four independent experiments .

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

KO+KRIT1 and KRIT1-KO MEFs were transiently transfected with the mRFP-GFP-LC3 tandem construct. Where indicated, the cells were treated with 100 nM Torin1 for 4 h or 2 μM xestospongin B for 4 h. The differences in the autophagic flux were evaluated by counting the yellow LC3 I/II dots/cell (RFP+GFP+) and red LC3 dots/cell (RFP+GFP−) for each condition. Yellow dots: autophagosomes; red dots: autophagolysosomes. *P = 0.00023 (red dots, KO+KRIT1 ctrl vs. KO+KRIT1 Tor1); *P = 0.00045 (red dots, KO+KRIT1 ctrl vs. KO+KRIT1 xesto); #P = 3.08e−6 (red dots, KO ctrl vs. KO Tor1); ##P = 6.73e−5 (yellow dots, KO+KRIT1 ctrl vs. KO ctrl). The data are expressed as the mean ± s.e.m. of four independent experiments.


Cd44 , PAI1 ( also known as Serpine1 ) , Id1 mRNA expression in KRIT1 wt KRIT1 - KO endothelial were assessed by quantitative real - time PCR . Where indicated , KRIT1 wt KRIT1 - KO endothelial were treated with 100 nM Torin1 or 500 nM rapamycin for 16h . data are expressed as mean ± s . e . m . Cd44 : * P = 0 . 02848 ( KO ctrl vs . KO Rapa ) ; * P = 0 . 02605 ( KO ctrl vs . KO Tor1 ) . PAI1 : * P = 0 . 04446 ( KO ctrl vs . KO Rapa ) ; * P = 0 . 03996 ( KO ctrl vs . KO Tor1 ) . Id1 : * P = 0 . 00266 ( KO ctrl vs . KO Rapa ) ; * P = 0 . 01554 ( KO ctrl vs . KO Tor1 ) . n = 3 independent experiments .

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

Cd44, PAI1 (also known as Serpine1), and Id1 mRNA expression levels in KRIT1 wt and KRIT1-KO endothelial cells were assessed by quantitative real-time PCR. Where indicated, KRIT1 wt and KRIT1-KO endothelial cells were treated with 100 nM Torin1 or 500 nM rapamycin for 16h. The data are expressed as the mean ± s.e.m. Cd44: *P = 0.02848 (KO ctrl vs. KO Rapa); *P = 0.02605 (KO ctrl vs. KO Tor1). PAI1: *P = 0.04446 (KO ctrl vs. KO Rapa); *P = 0.03996 (KO ctrl vs. KO Tor1). Id1: *P = 0.00266 (KO ctrl vs. KO Rapa); *P = 0.01554 (KO ctrl vs. KO Tor1). n = 3 independent experiments.


Immunoblot of CD31 / Pecam - 1 , vascular endothelial cadherin ( VE - cadherin ) , actin in KRIT1 - KO endothelial that were treated with 100 nM Torin1 or 500 nM rapamycin for 24 h . results are representative of three independent experiments .

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Immunoblot analysis of CD31/Pecam-1, vascular endothelial cadherin (VE-cadherin), and actin in KRIT1-KO endothelial cells that were treated with 100 nM Torin1 or 500 nM rapamycin for 24 h. The results are representative of three independent experiments.


Immunoblot of CD31 / Pecam - 1 , vascular endothelial cadherin ( VE - cadherin ) , N - cadherin , alpha - smooth muscle actin ( alpha - SMA ) , actin in HUVECs transfected with control siRNA or ATG7 siRNA .

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

Immunoblot analysis of CD31/Pecam-1, vascular endothelial cadherin (VE-cadherin), N-cadherin, alpha-smooth muscle actin (alpha-SMA), and actin in HUVECs transfected with control siRNA or ATG7 siRNA.


Formation of capillary - like structures measured by tube formation assays . HUVECs were transfected with control siRNA or ATG7 siRNA for 72 h . Representative phase - contrast microscopy ( Scale bar , 100 μm ) calcein - ( Scale bar , 50 μm ) were reported . data are presented as ± s . e . m from three different experiments performed in duplicate . * P = 1 . 29e − 11 .

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

Formation of capillary-like structures measured by tube formation assays. HUVECs were transfected with control siRNA or ATG7 siRNA for 72 h. Representative phase-contrast microscopy (Scale bar, 100 μm) and calcein-fluorescent (Scale bar, 50 μm) images were reported. All data are presented as percentage ± s.e.m from three different experiments performed in duplicate. *P = 1.29e−11.


Immunoblot of p62 , LC3I / II , rapamycin in CCM3 wt CCM3 - KO rapamycin treated with 100 nM Torin1 or 500 nM rapamycin for 4 h . results are representative of three independent experiments .

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Immunoblot analysis of p62, LC3I/II, and rapamycin in CCM3 wt and CCM3-KO rapamycin treated with 100 nM Torin1 or 500 nM rapamycin for 4 h. The results are representative of three independent experiments.


Representative immunostaining of retina sections from wt a model of inducible endothelial - specific CCM3 - KO ( CCM3 - ECKO ) mice at postnatal day 14 . Endothelium was stained with isolectin B4 ( ISOB4 ) ( blue ) . A , artery ; V , vein . p62 can be observed in endothelial forming retinal lesions in CCM3 - ECKO ( scale bar : 200 μm ) . Scale bar of magnifications : 100 μm .

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Representative immunostaining of retina sections from wt and a model of inducible and endothelial-specific CCM3-KO (CCM3-ECKO) mice at postnatal day 14. Endothelium was stained with isolectin B4 (ISOB4) (blue). A, artery; V, vein. p62 aggregates can be observed in endothelial cells forming retinal lesions in CCM3-ECKO animals (scale bar: 200 μm). Scale bar of magnifications: 100 μm.


Representative immunostaining of brain sections from wt a model of inducible endothelial - specific CCM3 - knockout mice ( CCM3 - ECKO ) at postnatal day 9 . p62 can be observed in proximity of CCM lesions ( arrows ) . nuclei ( DAPI ) are in blue . Scale bar , 30 μm . Smaller panel shows magnifications of blood vessels ( green ) . Scale bar , 10 μm .

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Representative immunostaining of brain sections from wt and a model of inducible and endothelial-specific CCM3-knockout mice (CCM3-ECKO) at postnatal day 9. p62 aggregates can be observed in the proximity of CCM lesions (arrows). Cell nuclei (DAPI) are in blue. Scale bar, 30 μm. Smaller panel shows the magnifications of blood vessels (green). Scale bar, 10 μm.


p62 immunohistochemistry ( ) in humanbrain tissue . A , B Normal vascular endothelium of autoptic brain parenchyma samples is lacking typical autophagic p62 granules as shown by negative for p62 . Scale bars : ( A ) 200 μm ; ( B ) 100 μm .

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p62 immunohistochemistry (IHC) staining in humanbrain tissue.A, B Normal vascular endothelium of autoptic brain parenchyma samples is lacking the typical autophagic p62 granules as shown by the negative staining for p62. Scale bars: (A) 200 μm; (B) 100 μm.


p62immunohistochemistry ( ) in humanbrain tissue . C - F Two different representative samples of CCM lesions with a thin , single layer brain endothelium displaying either moderate ( C , D ) or marked ( E , F ) positive perinuclear " pearl necklace - like " immunostaining for p62 granules . ( C , D ) , case n ° 4 ( p62 + + ) , ( E , F ) , case n ° 8 ( p62 + + + ) are representative of CCM cases listed in Appendix Table S1 . Scale bars : ( C , E ) 200 μm ; ( D , F ) 100 μm . Arrows indicate endothelial p62 positive .

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p62immunohistochemistry (IHC) staining in humanbrain tissue.C-F Two different representative samples of CCM lesions with a thin, single layer brain endothelium displaying either moderate (C, D) or marked (E, F) positive perinuclear "pearl necklace-like" immunostaining for p62 granules. (C, D), case n° 4 (p62++), and (E, F), case n° 8 (p62+++) are representative of CCM cases listed in Appendix Table S1. Scale bars: (C, E) 200 μm; (D, F) 100 μm. Arrows indicate endothelial p62 positive staining.


p62immunohistochemistry ( ) in humanbrain tissue . G - I Hematoxylin eosin ( H & amp ; amp ; E ) ( G ) p62immunohistochemistry ( H , I ) of a CCM surgical sample ( case n ° 6 in Appendix Table S1 ) containing normal vessels in peri - lesional , which served as an internal control . Notice marked p62 - positive in endothelial lining a CCM lesion ( H , arrows ) p62 - negative in endothelial lining a normal peri - lesional vessel ( I , arrows ) . Scale bars : ( G ) 300 μm ; ( H , I ) 100 μm . Background in brainparenchyma surrounding CCM lesions may be attributed to either debris or p62 immunoreactivity in neuronal glial .

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p62immunohistochemistry (IHC) staining in humanbrain tissue.G-I Hematoxylin and eosin (H&amp;E) staining (G) and p62immunohistochemistry analysis (H, I) of a CCM surgical sample (case n° 6 in Appendix Table S1) containing normal vessels in the peri-lesional area, which served as an internal control. Notice marked p62-positive staining in endothelial cells lining a CCM lesion (H, arrows) and p62-negative staining in endothelial cells lining a normal peri-lesional vessel (I, arrows). Scale bars: (G) 300 μm; (H, I) 100 μm. Background staining in brainparenchyma surrounding CCM lesions may be attributed to either cell debris or p62 immunoreactivity in neuronal cells and glial cells.


( A ) Representative micro - CT H & E of trabecular bone from femoral metaphysis of 6 - week - old Sp7 - Cre ; Cdc20f / f littermate control mice . Scale bar , 500 μm . ( B ) Histomorphometric analyses of 6 - week - old femurs . ( n = 6 )

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(A) Representative micro-CT images and H&E staining of trabecular bone from the femoral metaphysis of 6-week-old male Sp7-Cre;Cdc20f/f and littermate control mice. Scale bar, 500 μm. (B) Histomorphometric analyses of 6-week-old male femurs. (n=6)


( C ) Representative micro - CT H & E of trabecular bone from femoral metaphysis of 6 - week - old Sp7 - Cre ; Cdc20f / f littermate control mice . Scale bar , 500 μm . ( D ) Histomorphometric analyses of 6 - week - old femurs . ( n = 6 )

(C) Representative micro-CT images and H&E staining of trabecular bone from the femoral metaphysis of 6-week-old female Sp7-Cre;Cdc20f/f and littermate control mice. Scale bar, 500 μm. (D) Histomorphometric analyses of 6-week-old female femurs. (n=6)


( E ) Representative micro - CT of tibial cortical bone defects in Sp7 - Cre ; Cdc20f / f littermate control mice . green lines show position of original defect position . green circle represents regenerated bone . Scale bar , 500 μm . ( F ) Histomorphometric of regenerated bone in tibial cortical gaps . ( n = 5 )

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(E) Representative micro-CT images of tibial cortical bone defects in Sp7-Cre;Cdc20f/f and littermate control mice. The green lines show the position of the original defect position. The green circle represents the regenerated bone. Scale bar, 500 μm. (F) Histomorphometric analysis of the regenerated bone in tibial cortical gaps. (n=5)


( B , C ) ALP ( B ) ALP ( C ) of Cdc20f / f Sp7 - Cre ; Cdc20f / f BMSCs after 7 days osteogenic differentiation . ( n = 6 )

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(B, C) The ALP staining (B) and ALP quantification (C) of Cdc20f/f and Sp7-Cre;Cdc20f/f BMSCs after 7 days osteogenic differentiation. (n=6)


( E , F ) ALP ( E ) ALP ( F ) of NC CDC20sh hBMSCs after 7 days osteogenic differentiation . ( n = 5 )

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(E, F) The ALP staining (E) and ALP quantification (F) of NC and CDC20sh hBMSCs after 7 days osteogenic differentiation. (n=5)


( H , I ) ALP ( H ) ALP ( I ) of Vector truncated fragments overexpression of CDC20 in CDC20sh hBMSCs after 7 days osteogenic differentiation . ( n = 5 )

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(H, I) The ALP staining (H) and ALP quantification (I) of Vector and truncated fragments overexpression of CDC20 in CDC20sh hBMSCs after 7 days osteogenic differentiation. (n=5)


( E ) Western blot analyses of degradation of p65 after overexpression of CDC20 . of overexpressed Myc - CDC20 Vector HEK293T were extracted after TNF - α treatment for 30 minutes .

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(E) Western blot analyses of the degradation of nuclear p65 protein after the overexpression of CDC20. The cytoplasmic and nuclear protein of overexpressed Myc-CDC20 and Vector HEK293T cells were extracted after TNF-α treatment for 30 minutes.


( G ) Western blot analyses of degradation of p65 after overexpression of Myc - CDC20 . hBMSCs were transfected with Vector Myc - CDC20 plasmids for 36 h , were treated with or without 10 μM MG132 for 6 h before collected .

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(G) Western blot analyses of the degradation of p65 protein after the overexpression of Myc-CDC20. hBMSCs were transfected with Vector and Myc-CDC20 plasmids for 36 h, cells were treated with or without 10 μM MG132 for 6 h before collected.


( E , F ) Co - immunoprecipitation of endogenous CDC20 with endogenous p65 in HEK293T .

(E, F) Co-immunoprecipitation of endogenous CDC20 with endogenous p65 in HEK293T cells.


( G ) Co - immunoprecipitation of purified GST - CDC20 with endogenous p65 in HEK293T construct of GST - CDC20 . Whole lysates ( WCL ) were subjected to perform GST pull - down analyses with purified GST - CDC20 GST as a negative control .

(G) Co-immunoprecipitation of purified GST-CDC20 protein with endogenous p65 in HEK293T cells and the construct of GST-CDC20. Whole cell lysates (WCL) were subjected to perform GST pull-down analyses with purified GST-CDC20 and GST as a negative control.


( G , H ) Co - immunoprecipitation of ectopically expressed Myc - CDC20 171 - 499 with ectopically expressed Flag - p65 1 - 310 fragment in HEK293T .

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(G, H) Co-immunoprecipitation of ectopically expressed Myc-CDC20 171-499 with ectopically expressed Flag-p65 1-310 fragment in HEK293T cells.


( C ) Western blot analyses of degradation of p65 in NC APC11si HEK293T .

(C) Western blot analyses of the degradation of p65 protein in NC and APC11si HEK293T cells.


( F ) Western blot analyses of p65 expression in NC or CDC20sh HEK293T after overexpression of Vector or HA - APC11 plasmids .

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(F) Western blot analyses of p65 expression in NC or CDC20sh HEK293T cells after the overexpression of Vector or HA-APC11 plasmids.


( H , I ) ALP ( H ) ALP ( I ) of NC APC11si hBMSCs after 7 days osteogenic differentiation . ( n = 6 )

(H, I) The ALP staining (H) and ALP quantification (I) of NC and APC11si hBMSCs after 7 days osteogenic differentiation. (n=6)


( A ) Immunoblot of Flag - p65 - linked ubiquitination promoted by Myc - CDC20 . HEK293T were co - transfected with Flag - p65 , HA - Ubiquitin , with or without Myc - CDC20 or Vector plasmids for 36 h . Transfected were then treated with 10 μM MG132 for 6 h before collection . ( B ) Immunoblot of Flag - p65 - linked ubiquitination promoted by HA - APC11 . HEK293T were transfected with Flag - p65 , His - Ubiquitin , with or without HA - APC11 or Vector plasmids for 36 h . Transfected were then treated with 10 μM MG132 for 6 h before collection .

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(A) Immunoblot of Flag-p65-linked ubiquitination promoted by Myc-CDC20. HEK293T cells were co-transfected with Flag-p65, HA-Ubiquitin, with or without Myc-CDC20 or Vector plasmids for 36 h. Transfected cells were then treated with 10 μM MG132 for 6 h before collection. (B) Immunoblot of Flag-p65-linked ubiquitination promoted by HA-APC11. HEK293T cells were transfected with Flag-p65, His-Ubiquitin, with or without HA-APC11 or Vector plasmids for 36 h. Transfected cells were then treated with 10 μM MG132 for 6 h before collection.


( F ) Immunoblot of Flag - p65 - linked ubiquitination promoted by HA - APC11 in NC CDC20sh . NC or CDC20sh HEK293T were co - transfected with Flag - p65 , His - Ubiquitin , with or without HA - APC11 Vector plasmids . Transfected were treated with 10 μM MG132 for 6 h before collection .

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(F) Immunoblot of Flag-p65-linked ubiquitination promoted by HA-APC11 in NC and CDC20sh cells. NC or CDC20sh HEK293T cells were co-transfected with Flag-p65, His-Ubiquitin, with or without HA-APC11 and Vector plasmids. Transfected cells were treated with 10 μM MG132 for 6 h before collection.


( A ) Representative micro - CT of trabecular bone from femoral metaphysis of Cdc20f / f with negative control lentivirus , Sp7 - Cre ; Cdc20f / f with negative control lentivirus , Sp7 - Cre ; Cdc20f / f with p65sh lentivirus . Scale bar , 500 μm . ( B ) Histomorphometric analyses of Cdc20f / f with negative control lentivirus , Sp7 - Cre ; Cdc20f / f with negative control lentivirus , Sp7 - Cre ; Cdc20f / f with p65sh lentivirus . ( n = 6 )

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(A) Representative micro-CT images of trabecular bone from the femoral metaphysis of Cdc20f/f with negative control lentivirus, Sp7-Cre;Cdc20f/f with negative control lentivirus, and Sp7-Cre;Cdc20f/f with p65sh lentivirus. Scale bar, 500 μm. (B) Histomorphometric analyses of Cdc20f/f with negative control lentivirus, Sp7-Cre;Cdc20f/f with negative control lentivirus, and Sp7-Cre;Cdc20f/f with p65sh lentivirus. (n=6)


( C , D ) ALP ( C ) ALP ( D ) of BMSCs from Cdc20f / f mice Sp7 - Cre ; Cdc20f / f mice after 7 days osteogenic differentiation treated with negative control or p65si . ( n = 5 )

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(C, D) The ALP staining (C) and ALP quantification (D) of BMSCs from Cdc20f/f mice and Sp7-Cre;Cdc20f/f mice after 7 days osteogenic differentiation treated with negative control or p65si. (n=5)


( G ) expression of RUNX2 in BMSCs from Cdc20f / f mice Sp7 - Cre ; Cdc20f / f mice after 7 days osteogenic differentiation treated with negative control or p65si by western blot analyses .

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(G) The expression of RUNX2 in BMSCs from Cdc20f/f mice and Sp7-Cre;Cdc20f/f mice after 7 days osteogenic differentiation treated with negative control or p65si by western blot analyses.


F : IMAF for CSF , plasma urine of with samples collected at 6 months follow - up ( n = 3 ) . Matched MRI scans are added for annotation .

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F: IMAF for the CSF, plasma and urine of the patients with samples collected at 6 months follow-up (n=3). Matched MRI scans are added for annotation.


A : Mutant allele fraction ( MAF ) measured in , CSF , plasma urine samples ( on y axis ) for detected mutations , in 52 genes that are most frequently mutated in gliomas on TCGA databases ( on x axis ) , for 8 analysed by targeted sequencing panels . MAF from are derived from exome sequencing . MAF from CSF , plasma urine samples are derived from capture panels sequencing .

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A: Mutant allele fraction (MAF) measured in tumor, CSF, plasma and urine samples (on the y axis) for the detected mutations, in the 52 genes that are most frequently mutated in gliomas based on the TCGA databases (on the x axis), for the 8 patients analysed by targeted sequencing panels. MAF from tumor are derived from exome sequencing. MAF from CSF, plasma and urine samples are derived from the capture panels sequencing.


A - C : Fragment distributions for mutant ( blue ) non - mutant ( red ) cfDNA reads , determined from capture sequencing data for CSF samples ( A ) , plasma samples ( B ) urine samples ( C ) .

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A-C: Fragment size distributions for mutant (blue) and non-mutant (red) cfDNA reads, determined from the capture sequencing data for CSF samples (A), plasma samples (B) and urine samples (C).


( B ) SEC - MALLS traces of different TRIM25

(B) SEC-MALLS traces of different TRIM25 and


( C ) TRIM32 constructs . are colour coded domain architecture is reported next to respective MALLS curve . constructs were analysed over different ranges data are reported for : T25R , 4 mg / mL ; T25RB1 , 3 . 2 mg / mL ; T25RB1B2 , 3 . 6 mg / mL ; T25RBCC , 0 . 5 mg / mL ; T32R , 3 mg / mL ; T32RB , 4 mg / mL ; T32RBCC , 4 mg / mL .

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(C) TRIM32 constructs. The proteins are colour coded and the domain architecture is reported next to the respective MALLS curve. The constructs were analysed over different concentration ranges and the data are reported for: T25R, 4 mg/mL; T25RB1, 3.2 mg/mL; T25RB1B2, 3.6 mg/mL; T25RBCC, 0.5 mg/mL; T32R, 3 mg/mL; T32RB, 4 mg/mL; T32RBCC, 4 mg/mL.


( A , C ) UBE2D3 ~ Ub discharge assays with different TRIM25 TRIM32 constructs , respectively . Assays were carried out with TRIM constructs as indicated reaction was monitored over 30 min . asterisk indicates band for TRIM construct .

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(A, C) UBE2D3~Ub discharge assays with different TRIM25 and TRIM32 constructs, respectively. Assays were carried out with TRIM constructs as indicated and the reaction was monitored over 30 min. The asterisk indicates the band for the TRIM construct.


( B , D ) of discharge assays using UBE2D1 ~ UbAtto TRIM25 or TRIM32 , respectively . loss of UBE2D1 ~ UbAtto is plotted as average of experimental triplicates ( ± s . d . ) .

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(B, D) Quantification of the discharge assays using UBE2D1~UbAtto and TRIM25 or TRIM32, respectively. The loss of UBE2D1~UbAtto is plotted as the average of experimental triplicates (± s.d.).


( E , G ) K63 poly - ubiquitination assays using UBE2N / UBE2V1 TRIM25 TRIM32 , respectively . Reactions were incubated for 30 minutes samples taken at indicated times . asterisk indicates band for TRIM construct .

(E, G) K63 poly-ubiquitination assays using UBE2N/UBE2V1 and TRIM25 and TRIM32, respectively. Reactions were incubated for 30 minutes and samples taken at indicated times. The asterisk indicates the band for the TRIM construct.


( F , H ) Assays were quantified ( see Materials Methods ) by supplementing reaction with 1 uM UbAtto integrating loss of free UbAtto for TRIM25 TRIM32 , respectively . Assays were carried out in triplicate .

(F, H) Assays were quantified (see Materials and Methods) by supplementing the reaction with 1 uM UbAtto and integrating the loss of free UbAtto for TRIM25 and TRIM32, respectively. Assays were carried out in triplicate.


( A ) Structure of TRIM32 RING dimer in ribbon representation with each RING monomer coloured in cyan blue Zn2 + ions as grey spheres .

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(A) Structure of the TRIM32 RING dimer in ribbon representation with each RING monomer coloured in cyan and blue and the Zn2+ ions as grey spheres.


( B ) Overlap of RING dimers of TRIM32 ( cyan , 5FEY . pdb ) , TRIM25 ( yellow , 5FER . pdb ) TRIM5 ( orange , 4TKP . pdb ) . structures were overlapped on circled RING domain . This overlap shows that structures of RINGs are very similar but that there are differences in relative orientations of two RINGs .

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(B) Overlap of the RING dimers of TRIM32 (cyan, 5FEY.pdb), TRIM25 (yellow, 5FER.pdb) and TRIM5 (orange, 4TKP.pdb). The structures were overlapped on the circled RING domain. This overlap shows that the structures of the RINGs are very similar but that there are differences in the relative orientations of the two RINGs.


( C ) Structure of TRIM25 RING / E2 ~ Ub with RING domains in same colour scheme as in ( A ) , UBE2D1 in grey ubiquitin molecules in salmon red .

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(C) Structure of the TRIM25 RING/E2~Ub complex with the RING domains in the same colour scheme as in (A), UBE2D1 in grey and the ubiquitin molecules in salmon and red.


( A ) Close - up of TRIM25 RING dimer interface highlighting hydrophobic interactions made between four helices ( left ) . Close - up of interface between each RING monomer proximal ubiquitin ( right ) .

(A) Close-up of the TRIM25 RING dimer interface highlighting the hydrophobic interactions made between the four helices (left). Close-up of the interface between each RING monomer and the proximal ubiquitin (right).


( B ) UBE2D1 ~ UbAtto discharge assays with TRIM25 wild - type RING , fused RING constructs different mutants important for dimerization or interaction with ubiquitin . Time point zero for T25 - R Linker T25 - R Linker V72R samples was taken before addition of E3 as discharge is very fast . loss of UBE2D1 ~ UbAtto is plotted as average of experimental duplicates ( ± s . d . ) .

(B) UBE2D1~UbAtto discharge assays with TRIM25 wild-type RING, the fused RING constructs and different mutants important for dimerization or the interaction with ubiquitin. Time point zero for the T25-R Linker and T25-R Linker V72R samples was taken before the addition of E3 as discharge is very fast. The loss of UBE2D1~UbAtto is plotted as the average of experimental duplicates (± s.d.).


( C ) UBE2D3 ~ Ub discharge assays with same mutants as in ( B ) , stained with InstantBlue . ( D ) K63 poly - ubiquitination assays using UBE2N / UBE2V1 . asterisk indicates band for TRIM construct .

(C) UBE2D3~Ub discharge assays with the same mutants as in (B), stained with InstantBlue. (D) K63 poly-ubiquitination assays using UBE2N/UBE2V1. The asterisk indicates the band for the TRIM construct.


( A ) Role of E10 in RING of TRIM25 ( blue ) in stabilizing closed E2 ~ Ub conformation by contacting K11 from proximal ubiquitin ( salmon ) N71 of opposite RING ( cyan ) which in turn contacts K33 of same ubiquitin .

(A) Role of E10 in the RING of TRIM25 (blue) in stabilizing the closed E2~Ub conformation by contacting K11 from the proximal ubiquitin (salmon) and N71 of the opposite RING (cyan) which in turn contacts K33 of the same ubiquitin.


( B ) E2 ~ Ub discharge K63 poly - ubiquitination assays to role of E9 E10 in TRIM25 activity . Substitution of Glu9 with Arg has no significant effect on activity , whereas E10R mutation almost completely abolishes catalytic activity .

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(B) E2~Ub discharge and K63 poly-ubiquitination assays to test the role of E9 and E10 in TRIM25 activity. Substitution of Glu9 with Arg has no significant effect on activity, whereas the E10R mutation almost completely abolishes catalytic activity.


( C ) Discharge K63 poly - ubiquitination assays to role of equivalent residue E16 in TRIM32 role of I85R . Mutation of E16R abolishes catalytic activity indicating that role of glutamate is conserved .

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(C) Discharge and K63 poly-ubiquitination assays to test the role of the equivalent residue E16 in TRIM32 and the role of I85R. Mutation of E16R abolishes catalytic activity indicating that the role of the glutamate is conserved.


( D E ) of UBE2D1 ~ UbAtto discharge assays . loss of E2 ~ Ub is plotted as average of experimental duplicates ( ± s . d . ) .

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(D and E) Quantification of UBE2D1~UbAtto discharge assays. The loss of E2~Ub is plotted as the average of experimental duplicates (± s.d.).


A subplantar injection of 5 % of formalin ( 20 µl ) was administered in right hind paw of Balb / c mice an equal volume of PBS was injected in left hind paw . After 1 hour of formalin injection , Balb / c mice were treated with a single dose of either PBS ( vehicle control ) or Diclofenac ( 10 mg / kg ) or different concentrations of rPPE2 ( 2 mg / kg 3 mg / kg ) via intraperitoneal route after three hours of treatment , mice were sacrificed paw sections were prepared stained with Toluidine blue to quantify mast population . ( A ) of representative sections were visualized at 20X magnification ( scale bar = 50 µm ) . ( B ) Counting of mast was performed in Toluidine blue stained paw sections using ImageJ software were normalized per unit ( mm2 ) . Data information : Data shown are Mean ± of 8 mice . For ( B , unpaired t - was applied to calculate p values .

O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 subplantar injection of 5% of formalin (20 µl) was administered in the right hind paw of Balb/c mice and an equal volume of PBS was injected in left hind paw. After 1 hour of formalin injection, Balb/c mice were treated with a single dose of either PBS (vehicle control) or Diclofenac (10 mg/kg) or different concentrations of rPPE2 (2 mg/kg and 3 mg/kg) via intraperitoneal route and after three hours of treatment, mice were sacrificed and the paw sections were prepared and stained with Toluidine blue to quantify mast cell population. (A) Photographs of representative sections were visualized at 20X magnification (scale bar = 50 µm). (B) Counting of mast cells was performed in Toluidine blue stained paw sections using ImageJ software and were normalized per unit area (mm2). Data information: Data shown are Mean ± SEM of 8 mice. For (B, unpaired t-test was applied to calculate p values.


( B - E ) In another experiment , NIH - 3T3 ( B , C ) or MEFs ( D , E ) were incubated with either PBS or rPPE2 ( 3 µg / ml ) . After 45 minutes of rPPE2 treatment , were fixed , permeabilized , stained with anti - PPE2 Ab . DAPI was used to stain nucleus . localization of rPPE2 in NIH - 3T3 ( C ) MEFs ( E ) was measured by calculating colocalization coefficient . Data information : Data represent Mean ± of more than 50 of three independent experiments . For Figure C , E unpaired t - was applied to calculate p values .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-E) In another experiment, NIH-3T3 cells (B, C) or MEFs (D, E) were incubated with either PBS or rPPE2 (3 µg/ml). After 45 minutes of rPPE2 treatment, cells were fixed, permeabilized, and stained with anti-PPE2 Ab. DAPI was used to stain the nucleus. Nuclear localization of rPPE2 in NIH-3T3 (C) and MEFs (E) was measured by calculating the colocalization coefficient. Data information: Data represent Mean ± SEM of more than 50 cells of three independent experiments. For Figure C, E unpaired t-test was applied to calculate p values.


( F - G ) ( F ) NIH - 3T3 were transiently transfected with either pEGFP or pEGFP - PPE2 . After 24 hours of transfection , were fixed observed under a confocal microscope . DAPI was used to stain nucleus . ( G ) localization of rPPE2 was measured by calculating colocalization coefficient . Data information : Data represent Mean ± of more than 50 of three independent experiments . For Figure G , unpaired t - was applied to calculate p values .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-G) (F) NIH-3T3 cells were transiently transfected with either pEGFP or pEGFP-PPE2. After 24 hours of transfection, cells were fixed and observed under a confocal microscope. DAPI was used to stain the nucleus. (G) Nuclear localization of rPPE2 was measured by calculating colocalization coefficient. Data information: Data represent Mean ± SEM of more than 50 cells of three independent experiments. For Figure G, unpaired t-test was applied to calculate p values.


( D ) Lysates prepared from same samples used in C were resolved on SDS - PAGE checked for GFP expression by Western blotting using anti - GFP Ab . GAPDH was used as a loading control .

(D) Lysates prepared from the same samples used in C were resolved on SDS-PAGE and checked for GFP expression by Western blotting using anti-GFP Ab. GAPDH was used as a loading control.


( E ) Varying concentrations of rPPE2 were incubated with [ γ - 32P ] - ATP oligonucleotides of 60 bp spanning SCF promoter . - complexes were resolved on 7 % native polyacrylamide gel . In cold competition reactions , equimolar cold / unlabeled 60 bp oligonucleotides were used ( arrow represents PPE2 - oligo ) . data shown are representative of 3 independent experiments .

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

(E) Varying concentrations of rPPE2 protein were incubated with [γ-32P]-ATP oligonucleotides of 60 bp spanning SCF promoter. DNA-protein complexes were resolved on 7% native polyacrylamide gel. In cold competition reactions, equimolar cold/unlabeled 60 bp oligonucleotides were used (arrow represents PPE2-oligo complex). The data shown are representative of 3 independent experiments.


A subplantar injection of 5 % of formalin ( 20 µl ) was administered in right hind paw of Balb / c mice , an equal volume of PBS was injected in left hind paw . After induction of inflammation , Balb / c mice were treated with PBS or peptide ( 8 mg / kg ) via intraperitoneal route . After 3 hours , paw tissues were harvested used for cDNA synthesis . B ) ( B ) Next , Toluidine blue was done to check mast population . of representative sections were visualized at 20X magnification ( scale bar = 100 µm ) .

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

A subplantar injection of 5% of formalin (20 µl) was administered in the right hind paw of Balb/c mice, and an equal volume of PBS was injected in left hind paw. After induction of inflammation, Balb/c mice were treated with PBS or peptide (8 mg/kg) via the intraperitoneal route. After 3 hours, paw tissues were harvested and used for cDNA synthesis. B) (B) Next, Toluidine blue staining was done to check the mast cell population. Photographs of representative sections were visualized at 20X magnification (scale bar = 100 µm).


A subplantar injection of 5 % of formalin ( 20 µl ) was administered in right hind paw of Balb / c mice , an equal volume of PBS was injected in left hind paw . After induction of inflammation , Balb / c mice were treated with PBS or peptide ( 8 mg / kg ) via intraperitoneal route . After 3 hours , paw tissues were harvested used for cDNA synthesis . ( C ) Counting of mast was performed in Toluidine blue - stained paw sections using ImageJ software was normalized per unit ( mm2 ) . Data information : Data shown are Mean ± of 8 mice . unpaired t - was applied to calculate p values .

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

A subplantar injection of 5% of formalin (20 µl) was administered in the right hind paw of Balb/c mice, and an equal volume of PBS was injected in left hind paw. After induction of inflammation, Balb/c mice were treated with PBS or peptide (8 mg/kg) via the intraperitoneal route. After 3 hours, paw tissues were harvested and used for cDNA synthesis. (C) Counting of mast cells was performed in Toluidine blue-stained paw sections using ImageJ software and was normalized per unit area (mm2). Data information: Data shown are Mean ± SEM of 8 mice. unpaired t-test was applied to calculate p values.


( B ) confirmative RT - PCRs were resolved in a 2 % agarose gel . Untreated reverse transcriptase RNAs ( - RT ) genomic served as negative positive controls , respectively .

(B) The confirmative RT-PCRs were resolved in a 2% agarose gel. Untreated reverse transcriptase RNAs (-RT) and genomic DNA served as negative and positive controls, respectively.


( D ) Left panel : Absorption spectra of - adapted XccBphP after illumination with red , far - red , lamp , sunlight or sunlight filtered through green leaves ( LF ) . Pr / Pfr equilibriums scheme derived from absorption spectra is depicted in bottom panel . Right panel : XccBphP - C13S mutant was purified in presence of BV its absorption spectra recorded upon illumination with red , far - red or adaptation .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Left panel: Absorption spectra of dark-adapted XccBphP after illumination with red, far-red, fluorescent white lamp light, sunlight or sunlight filtered through green leaves (LF). Pr/Pfr equilibriums scheme derived from absorption spectra is depicted in the bottom panel. Right panel: The XccBphP-C13S mutant was purified in the presence of BV and its absorption spectra recorded upon illumination with red, far-red and white light or dark adaptation.


( A - B ) A . thaliana were inoculated with wild - type , XccbphP , pXccBphP or pC13S strains cultured prior to inoculation under ( A ) normal laboratory illumination or ( B ) under or conditions . After 1 , 2 or 3 d . p . i . , CFU per mg were determined ( n = 3 replicates ) . Data presented here derive from more than 4 independent experiments .

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

(A-B) A. thaliana plants were inoculated with wild-type, XccbphP, pXccBphP or pC13S bacterial strains cultured prior to inoculation under (A) normal laboratory illumination or (B) under light or dark conditions. After 1, 2 or 3 d.p.i., CFU per plant mg were determined (n = 3 replicates). Data presented here derive from more than 4 independent experiments.


( C ) Stomatal closure in presence of strains or not ( untreated ) after 1 h under conditions . ( D ) Stomatal opening after 3 h of incubation with strains or not ( untreated ) in . A control treatment without illuminated was included ( ) . ( C - D ) Stomatal apertures were recorded ( n = 80 replicates ) . Data are representative of two independent experiments .

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

(C) Stomatal closure in the presence of bacterial strains or not (untreated) after 1 h under light conditions. (D) Stomatal opening after 3 h of incubation with bacterial strains or not (untreated) in the dark. A control treatment without bacteria and illuminated was included (Light). (C-D) Stomatal apertures were recorded (n = 80 replicates). Data are representative of two independent experiments.


( E ) A . thalianaleaves were inoculated with wild - type , XccbphP , pXccBphP pC13S strains , stained for callose deposits observed by microscopy . MgCl2 buffer ( untreated ) flg22 peptide were used as negative positive controls , respectively . Top panel : representative pictures of three independent experiments . Scale bar represents 200 µm . Bottom panel : number of callose deposits per field of view ( 0 . 45 mm2 ) were determined ( n = 8 replicates ) . Data are representative of two independent experiments . ( A - E ) Values are expressed as mean ± s . e . m . Statistical was performed by a two - Mann - Whitney ( * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 )

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

(E) A. thalianaleaves were inoculated with wild-type, XccbphP, pXccBphP and pC13S strains, stained for callose deposits and observed by fluorescence microscopy. MgCl2 buffer (untreated) and flg22 peptide were used as negative and positive controls, respectively. Top panel: representative pictures of three independent experiments. Scale bar represents 200 µm. Bottom panel: the number of callose deposits per field of view (0.45 mm2) were determined (n = 8 replicates). Data are representative of two independent experiments. (A-E) Values are expressed as mean ± s.e.m. Statistical analysis was performed by a two-tailed Mann-Whitney test (*P < 0.05, **P < 0.01, ***P < 0.001)


( A ) Five µL supernatants from wild - type , XccbphP , pXccBphP pC13 strains cultures ( OD600 = 1 ) grown under or conditions were plated onto PYM - carboxymethyl cellulose ( CMC ) agar plates revealed with Congo redstaining ( n = 2 replicates ) . extracellular β - 1 , 4 - endoglucanase production correlated with CMC degradation halo radiuses ( top panel ) . Halo measurements are presented in bottom panel . Data derive from 10 independent experiments . ( A - B ) Values are expressed as mean ± s . e . m . Statistical was performed by a Kruskal - Wallis Dunn ´ s multiple comparison ( * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 ) .

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

(A) Five µL supernatants from wild-type, XccbphP, pXccBphP and pC13 strains bacterial cultures (OD600 = 1) grown under light or dark conditions were plated onto PYM-carboxymethyl cellulose (CMC) agar plates and revealed with Congo redstaining (n = 2 replicates). The extracellular β-1,4-endoglucanase production levels correlated with CMC degradation halo radiuses (top panel). Halo measurements are presented in the bottom panel. Data derive from 10 independent experiments. (A-B) Values are expressed as mean ± s.e.m. Statistical analysis was performed by a Kruskal-Wallis test and Dunn´s multiple comparison test (*P < 0.05, **P < 0.01, ***P < 0.001).


( B ) extracellular β - 1 , 4 - endoglucanase activity from cultures supernatants ( OD600 = 1 ) was determined by a colorimetric in solution ( n = 2 replicates ) . Wild - type , XccbphP , pXccBphP pC13 strains were assayed under normal laboratory conditions . Data derive from 3 independent experiments . ( A - B ) Values are expressed as mean ± s . e . m . Statistical was performed by a Kruskal - Wallis Dunn ´ s multiple comparison ( * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) The extracellular β-1,4-endoglucanase activity from bacterial cultures supernatants (OD600 = 1) was determined by a colorimetric assay in solution (n = 2 replicates). Wild-type, XccbphP, pXccBphP and pC13 strains were assayed under normal laboratory conditions. Data derive from 3 independent experiments. (A-B) Values are expressed as mean ± s.e.m. Statistical analysis was performed by a Kruskal-Wallis test and Dunn´s multiple comparison test (*P < 0.05, **P < 0.01, ***P < 0.001).


( A ) Five µL from wild - type , XccbphP , pXccBphP pC13S cultures ( OD600 = 1 ) were plated onto PYM - glucose agar plates under or conditions . Xanthan production was determined measuring colony diameters ( n = 4 replicates ) for 20 independent experiments . ( A - B ) Values are expressed as mean ± s . e . m . Statistical was performed by a Kruskal - Wallis Dunn ´ s multiple comparison ( * P < 0 . 05 ) .

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

(A) Five µL from wild-type, XccbphP, pXccBphP and pC13S bacterial cultures (OD600 = 1) were plated onto PYM-glucose agar plates under light or dark conditions. Xanthan production was determined measuring colony diameters (n = 4 replicates) for 20 independent experiments. (A-B) Values are expressed as mean ± s.e.m. Statistical analysis was performed by a Kruskal-Wallis test and Dunn´s multiple comparison test (*P < 0.05).


( B ) Wild - type , XccbphP , pXccBphP pC13S were grown in 20 ml PYM liquid medium under or conditions . Extracellular xanthan was purified by KCl addition ethanol precipitation , then dried weighted ( n = 2 replicates ) for 4 independent experiments . ( A - B ) Values are expressed as mean ± s . e . m . Statistical was performed by a Kruskal - Wallis Dunn ´ s multiple comparison ( * P < 0 . 05 ) .

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

(B) Wild-type, XccbphP, pXccBphP and pC13S were grown in 20 ml PYM liquid medium under light or dark conditions. Extracellular xanthan was purified by KCl addition and ethanol precipitation, then dried and weighted (n = 2 replicates) for 4 independent experiments. (A-B) Values are expressed as mean ± s.e.m. Statistical analysis was performed by a Kruskal-Wallis test and Dunn´s multiple comparison test (*P < 0.05).


( C ) Representative confocal - scanning microscopy pictures of biofilm development of cultured for 4 d in minimal medium in chambered cover slides under or conditions . scale bar represents 30 µm .

O O O O O O O O O O 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) Representative confocal laser-scanning microscopy pictures of biofilm development of bacteria cultured for 4 d in minimal medium in chambered cover slides under light or dark conditions. The scale bar represents 30 µm.


A - E Microvascular leak extravasation were modeled in vitro using modified Boyden chamber experiments where lung HMVECs ( A - C ) dermal HMVECs ( D - E ) were grown to 100 % confluence over 8 - μm - pore insert membranes . ECs were first treated with Vasculotide ( VT ) , PBS ( vehicle / negative control ) , Ang1 ( positive control ) , PEG - Cys ( polyethylene glycol backbone ) , T7c ( non - PEGylated CHHHRHSF peptides ) , or VT in presence of 100 - fold molar excess of T7c . concentrations used , 10 - 20 ng / mL VT 200 - 400 ng / mL Ang1 , are estimated molar equivalents ( 0 . 71 - 1 . 43 nM ) . Thirty minutes later , ECs were stimulated with thrombin , 0 . 1 % BSA ( vehicle / negative control ) , or EDTA ( positive control ) . Another 30mins later , amount of FITC - dextran diffusion into lower chambers ( A , D ) provides a measure of endothelial permeability . CMTPX - labeled ( TCs ) were then dispensed into inserts , amount of TC emitting from underside of insert membranes after 20 h ( B - C ) or 28 h ( E ) reflects efficiency of trans - endothelial TC migration . Representative ( 10 × ) of undersides are shown in ( C ) , where DAPI - stained nuclei of ECs / TCs are shown in blue , ' extravasated ' CMTPX - labeled TCs are shown in red , # of CMTPX + pixels are shown numerically in red . Means ± are shown ( A , B , D , E ) . Three experiments ( twice with HMVEC - LBl once with HMVEC - DBl ) were run with n = 3 - 5 inserts ( independent biological replicates ) per group analyzed by two - sampled unpaired t - .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O B-CONTROLLED_VAR O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-E Microvascular leak and tumor cell extravasation were modeled in vitro using modified Boyden chamber experiments where lung HMVECs (A-C) and dermal HMVECs (D-E) were grown to 100% confluence over 8-μm-pore insert membranes. ECs were first treated with Vasculotide (VT), PBS (vehicle/negative control), Ang1 (positive control), PEG-Cys (polyethylene glycol backbone), T7c (non-PEGylated CHHHRHSF peptides), or VT in the presence of 100-fold molar excess of T7c. The concentrations used, 10-20 ng/mL VT and 200-400 ng/mL Ang1, are estimated molar equivalents (0.71-1.43 nM). Thirty minutes later, ECs were stimulated with thrombin, 0.1% BSA (vehicle/negative control), or EDTA (positive control). Another 30mins later, the amount of FITC-dextran diffusion into the lower chambers (A, D) provides a measure of endothelial permeability. CMTPX-labeled tumor cells (TCs) were then dispensed into inserts, and the amount of TC fluorescence emitting from the underside of insert membranes after 20 h (B-C) or 28 h (E) reflects the efficiency of trans-endothelial TC migration. Representative fluorescent images (10×) of membrane undersides are shown in (C), where DAPI-stained nuclei of ECs/TCs are shown in blue, 'extravasated' CMTPX-labeled TCs are shown in red, and the # of CMTPX+ pixels are shown numerically in red. Means ± SEM are shown (A,B,D,E). Three experiments (twice with HMVEC-LBl and once with HMVEC-DBl) were run with n = 3-5 inserts (independent biological replicates) per group and analyzed by two-sampled unpaired t-tests.


A - H LM2 - 4luctumor were implanted intravenously into SCIDmice , generating predominantly lung - specific metastases in one experiment ( n = 5 - 6 ; A - D ) predominantly lymphatic - specific metastases in another experiment ( n = 12 ; E - H ) . ( C G ) of typical metastases in lungs caudal lymphatics , respectively . Kaplan - Meier survival showed that Vasculotide ( VT ) significantly delayed mortality due to lungmetastases ( A ) but not lymphaticmetastases ( E ) . Quantitative of in vivo bioluminescent taken at 24DPI ( B ) showed that concurrent VT treatment effectively reversed sunitinib ( SU ) - induced acceleration of lungmetastases ( D ) . With regard to lymphaticmetastases ( F , H ) , SU treatment did not accelerate their progression . Geometric means ± 95 % CI are depicted in ( D H ) ; P values were derived by one - way ANOVA ( D ) Kruskal - Wallis ( H ) . same trends were reproduced in confirmatory experiments summarized in Supplementary Fig S5 .

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

A-H LM2-4luctumor cells were implanted intravenously into SCIDmice, generating predominantly lung-specific metastases in one experiment (n = 5-6; A-D) and predominantly lymphatic-specific metastases in another experiment (n = 12; E-H). (C and G) Photographs of typical metastases in the lungs and caudal lymphatics, respectively. Kaplan-Meier survival analysis showed that Vasculotide (VT) significantly delayed mortality due to lungmetastases (A) but not lymphaticmetastases (E). Quantitative analysis of in vivo bioluminescent images taken at 24DPI (B) showed that concurrent VT treatment effectively reversed the sunitinib (SU)-induced acceleration of lungmetastases (D). With regard to lymphaticmetastases (F, H), SU treatment did not accelerate their progression. Geometric means ± 95%CI are depicted in (D and H); P values were derived by one-way ANOVA (D) and Kruskal-Wallis test (H). The same trends were reproduced in confirmatory experiments summarized in Supplementary Fig S5.


A - D An experiment where HT29luc were implanted intravenously into YFP - SCID mice . ( A ) Kaplan - Meier survival . ( B ) of total metastatic burden in liver lymphatics , at 35 days post - implantation ( DPI ) , from whole - bioluminescent imaging ( C ) . ( D ) Photograph of typical liver metastases .

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A-D An experiment where HT29luc tumor cells were implanted intravenously into YFP-SCID mice. (A) Kaplan-Meier survival analysis. (B) Quantification of total metastatic burden in the liver and lymphatics, at 35 days post-implantation (DPI), from whole-body bioluminescent imaging (C). (D) Photograph of typical liver metastases.


E - G An experiment where SN12luc were implanted intravenously into YFP - SCID mice . ( E ) Kaplan - Meier survival . ( F ) of total metastatic burden at 50DPI , mostly within lungs , from whole - bioluminescent imaging ( G ) .

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E-G An experiment where SN12luc tumor cells were implanted intravenously into YFP-SCID mice. (E) Kaplan-Meier survival analysis. (F) Quantification of total metastatic burden at 50DPI, mostly within the lungs, from whole-body bioluminescent imaging (G).


A , B In modified Boyden chamber experiments , confluent dermal HMVECs were stimulated with - conditioned media ( TC - CM ) - that is , media conditioned by either LM2 - 4luc , SN12luc , or HT29luc - treated with 20 ng / mL Vasculotide vs . 400 ng / mL Ang1 ( positive control ) vs . PBS ( negative control ) . Meta - analyses of treatment effects on dextran permeability ( A ) migration ( B ) , where each datapoint represents mean of an independent experiment , averaged over 2 - 3 biological replicates ( inserts ) per treatment group normalized internally within that same experiment . Statistical significance of overall Ang1 or VT treatment effects was determined by paired t - , n = 11 ( A ) or 6 ( B ) experimental replicates . C Representative ( 10 × ) of ' extravasated ' CMTPX - labeled TCs ( red ) fixed on undersides .

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

A, B In modified Boyden chamber experiments, confluent dermal HMVECs were stimulated with tumor cell-conditioned media (TC-CM)-that is, media conditioned by either LM2-4luc, SN12luc, or HT29luc tumor cells-and treated with 20 ng/mL Vasculotide vs. 400 ng/mL Ang1 (positive control) vs. PBS (negative control). Meta-analyses of treatment effects on dextran permeability (A) and tumor cell migration (B), where each datapoint represents the mean of an independent experiment, averaged over 2-3 biological replicates (inserts) per treatment group and normalized internally within that same experiment. Statistical significance of overall Ang1 or VT treatment effects was determined by paired t-tests, n = 11 (A) or 6 (B) experimental replicates.C Representative fluorescent images (10×) of 'extravasated' CMTPX-labeled TCs (red) fixed on membrane undersides.


D LM2 - 4luc produced highest of VEGF IL - 8 , but lower of IL - 6 , compared to other TC types ; * * * P < 0 . 001 * P < 0 . 05 ( unpaired t - ) .

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D LM2-4luc produced the highest levels of VEGF and IL-8, but lower levels of IL-6, compared to other TC types; ***P < 0.001 and *P < 0.05 (unpaired t-tests).


A - C Vasculotide ( VT ) did not alter growth kinetics of orthotopic LM2 - 4luc tumors ; n = 8 mice per group . Sunitinib ( SU ) treatment significantly lowered burden as measured by volume ( B : one - way ANOVA , P < 0 . 01 ) or bioluminescent activity ( C : one - way ANOVA , P < 0 . 001 ) .

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A-C Vasculotide (VT) did not alter the growth kinetics of orthotopic LM2-4luc tumors; n = 8 mice per group. Sunitinib (SU) treatment significantly lowered primary tumor burden as measured by volume (B: one-way ANOVA, P < 0.01) or bioluminescent activity (C: one-way ANOVA, P < 0.001).


D VT treatment did not alter mean thickness of viable peripheral regions of tumors , as quantified by H & amp ; amp ; E histological .

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D VT treatment did not alter the mean thickness of the viable peripheral regions of primary tumors, as quantified by H&amp;E histological staining.


E VT treatment did not alter mean thickness of CAIX + hypoxic edge between viable periphery necrotic of a ; control sample taken at 5 × are shown .

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E VT treatment did not alter the mean thickness of the CAIX+ hypoxic edge between the viable periphery and necrotic core of a primary tumor; IHC control and sample images taken at 5× are shown.


F Ex vivo bioluminescent imaging suggests that VT treatment lowered proportion of mice with metastatic involvement in lungs , without corresponding effect on lymphatic metastases . Representative bioluminescent vs . photographic of dissected lungs in a 24 - well plate are shown .

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F Ex vivo bioluminescent imaging suggests that VT treatment lowered the proportion of mice with metastatic involvement in the lungs, without the corresponding effect on lymphatic metastases. Representative bioluminescent vs. photographic images of dissected lungs in a 24-well plate are shown.


G Human LM2 - 4luc can be identified on histological sections of mouse lung tissue by of human vimentin , a cytoskeletal . distribution show VT monotherapy reducing occurence of bigger metastatic nodules .

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G Human LM2-4luc cells can be identified on histological sections of mouse lung tissue by IHC staining of human vimentin, a cytoskeletal protein. Size distribution analysis show VT monotherapy reducing the occurence of bigger metastatic nodules.


A Kaplan - Meier survival curves . Number of days gained in median survival due to treatments is listed in brackets . Yellow arrows mark timepoints at which bioluminescent were taken .

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A Kaplan-Meier survival curves. Number of days gained in median survival due to treatments is listed in brackets. Yellow arrows mark the timepoints at which the bioluminescent images were taken.


B Bioluminescent of burden . C - D Total bioluminescent fluxes , at 35 days post - implantation ( while ' SU _ 3 wk ' group was still receiving 60 mg / kg / day sunitinib treatment ) , were separately quantified from thoracic ( C ) abdominal ( D ) regions . Geometric means P values from Mann - Whitney are depicted in ( C D ) . Percentages listed below x - axes in panels ( C D ) refer to treatment - associated changes in geometric mean bioluminescence , relative to vehicle group .

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B Bioluminescent images of tumor burden.C-D Total bioluminescent fluxes, at 35 days post-implantation (while the 'SU_3 wk' group was still receiving 60 mg/kg/day sunitinib treatment), were separately quantified from the thoracic (C) and abdominal (D) regions. Geometric means and P values from Mann-Whitney tests are depicted in (C and D). Percentages listed below the x-axes in panels (C and D) refer to treatment-associated changes in the geometric mean bioluminescence, relative to the vehicle group.


A - F human umbilical vein endothelial ( HUVECs ) or human dermal microvascular endothelial ( HMVEC - DBls ) were stimulated at 80 % confluency with Vasculotide ( treatment ) , Ang1 ( positive control ; ligand of Tie2 ) , PBS ( vehicle control ) , or VEGF ( negative control ; non - ligand of Tie2 ) . Tie2 was immunoprecipitated from lysates immunoblotted for total Tie2 or phosphotyrosine . Vasculotide treatment did not lead to increases in pTie2 / Tie2 in HUVECs after 45 - min ( A - D ) or 15 - min ( E ) treatments , with ( E ) or without ( A - D ) serum / supplement starvation . Vasculotide also did not activate Tie2 receptors in HMVEC - DBls , whether in presence or absence of serum , growth factor , cytokine supplements ( F ) .

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A-F Primary human umbilical vein endothelial cells (HUVECs) or primary human dermal microvascular endothelial cells (HMVEC-DBls) were stimulated at 80% confluency with Vasculotide (treatment), Ang1 (positive control; ligand of Tie2), PBS (vehicle control), or VEGF (negative control; non-ligand of Tie2). Tie2 was immunoprecipitated from cell lysates and immunoblotted for total Tie2 or phosphotyrosine. Vasculotide treatment did not lead to increases in the pTie2/Tie2 ratio in HUVECs after 45-min (A-D) or 15-min (E) treatments, with (E) or without (A-D) serum/supplement starvation. Vasculotide also did not activate Tie2 receptors in HMVEC-DBls, whether in the presence or absence of serum, growth factor, and cytokine supplements (F).


Healthy non - - bearing mice ( n = 8 - 9 per group ) were treated for 1 week with vehicle ( Veh ) ; Vasculotide 250 ng / 2 days IP ( VT ) ; sunitinib 60 mg / kg / day PO ( SU ) ; or VT + SU . Tie2 immunoprecipitates from lung , kidney , or liver homogenates were pooled from two mice per treatment group immunoblotted with an anti - Tie2 antibody ( ' Tie2 ' ) or an anti - phosphotyrosine antibody ( ' p - Tie2 ' ) . Densitometry of immunoblots from ( A ) . pTie2 / Tie2 ratios were internally normalized to vehicle group within each blot . Means ± P values ( unpaired t - ) are depicted .

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Healthy non-tumor-bearing mice (n = 8-9 per group) were treated for 1 week with vehicle (Veh); Vasculotide 250 ng/2 days IP (VT); sunitinib 60 mg/kg/day PO (SU); or VT + SU. Tie2 immunoprecipitates from lung, kidney, or liver homogenates were pooled from two mice per treatment group and immunoblotted with an anti-Tie2 antibody ('Tie2') or an anti-phosphotyrosine antibody ('p-Tie2').Densitometry analysis of immunoblots from (A). pTie2/Tie2 ratios were internally normalized to the vehicle group within each blot. Means ± SEM and P values (unpaired t-tests) are depicted.


A - B Pull - down . Input ligands - rhAng1 ( A ) VT ( B ) - were incubated with either A sepharose beads only , bead - conjugated mouse Tie2 - Fc ( mTie2 ) , or bead - conjugated human Tie2 - Fc ( hTie2 ) . ' bound ' fraction shows total amount pulled down by beads . ' unbound ' fraction is 7 % of residual / free ligands remaining in supernatant .

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A-B Pull-down assay. Input ligands-rhAng1 (A) and VT (B)-were incubated with either protein A sepharose beads only, bead-conjugated mouse Tie2-Fc (mTie2), or bead-conjugated human Tie2-Fc (hTie2). The 'bound' fraction shows the total amount pulled down by beads. The 'unbound' fraction is 7% of residual/free ligands remaining in the supernatant.


C Quantitative kinetics by SPR . analytes were run at increasing concentrations of 0 . 625 , 1 . 25 , 2 . 5 , 5 , 10 nM , at 37 ° C pH 7 . 4 . positive controls - rhAng1 , BowAng1 , COMP - Ang1 - bound strongly to immobilized hTie2 - Fc . There was no detectable binding of VT , or negative control , rhVEGF , to immobilized hTie2 - Fc .

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C Quantitative kinetics analysis by SPR. All analytes were run at increasing concentrations of 0.625, 1.25, 2.5, 5, and 10 nM, at 37°C and pH 7.4. The positive controls-rhAng1, BowAng1, COMP-Ang1-all bound strongly to immobilized hTie2-Fc. There was no detectable binding of VT, or the negative control, rhVEGF, to immobilized hTie2-Fc.


F . A physical 650 - kb window of genomic distribution of genes surrounding SNP rs4971059 , such as EFNA4 , KRTCAP2 , MUC1 , MIR92B , THBS3 , DAP3 ( top ) . Total RNAs were extracted from [ GG ] , [ GA ] , or [ AA ] MCF - 10A analyzed for expression of EFNA4 , KRTCAP2 , TRIM46 , MUC1 , THBS3 , MIR92B , or DAP3 by RT - qPCR . TRIM28 was examined as a negative control ( bottom ) .

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F. A physical 650-kb window of the genomic distribution of genes surrounding SNP rs4971059, such as EFNA4, KRTCAP2, MUC1, MIR92B, THBS3,and DAP3 (top). Total RNAs were extracted from [GG], [GA], or [AA] MCF-10A cells and analyzed for the expression of EFNA4, KRTCAP2, TRIM46, MUC1, THBS3, MIR92B, or DAP3 by RT-qPCR. TRIM28 was examined as a negative control (bottom).


B . MCF - 7 were transfected with a control vector or FLAG - TRIM46 for 40 h treated with DMSO or MG132 ( 2μM ) for 10h . lysates were then collected for western blotting with antibodies against indicated .

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B. MCF-7 cells were transfected with a control vector or FLAG-TRIM46 for 40 h and treated with DMSO or MG132 (2μM) for 10h. Cellular lysates were then collected for western blotting with antibodies against the indicated proteins.


F . Schematic diagrams of HDAC1 deletion mutants . GST pull - down experiments were performed with bacterially expressed GST - fused as indicated in vitro transcribed / translated TRIM46 .

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F. Schematic diagrams of HDAC1 deletion mutants. GST pull-down experiments were performed with bacterially expressed GST-fused proteins as indicated and in vitro transcribed/translated TRIM46.


F . MCF - 7 were transfected with empty vector or FLAG - TRIM46 for forty - eight hours then treated with 50 μg / mL cycloheximide ( CHX ) for indicated hours . DMSO was added to 10 h before were collected for western blotting ( left ) . On right panel , quantitation was done by densitometry of immunoblots expressed as signals of HDAC1 . G . MCF - 7 were treated as described in F , except that MG132 ( 10 μM ) was added to 10 h before were collected for western blotting ( left ) . Quantitation was performed on right panel as in F . H . MCF - 7 were treated with control siRNA or TRIM46 siRNA . Forty - eight hours after transfection , were treated with 50 μg / mL CHX for indicated hours before were extracted for western blotting ( left ) . Quantitation was performed on right panel as in F .

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F. MCF-7 cells were transfected with empty vector or FLAG-TRIM46 for forty-eight hours and then treated with 50 μg/mL cycloheximide (CHX) for the indicated hours. DMSO was added to cells 10 h before cellular proteins were collected for western blotting (left). On the right panel, quantitation was done by densitometry analysis of the immunoblots and expressed as signals of HDAC1. G. MCF-7 cells were treated as described in F, except that MG132 (10 μM) was added to cells 10 h before cellular proteins were collected for western blotting (left). Quantitation was performed on the right panel as in F. H. MCF-7 cells were treated with control siRNA or TRIM46 siRNA. Forty-eight hours after transfection, cells were treated with 50 μg/mL CHX for the indicated hours before cellular proteins were extracted for western blotting analysis (left). Quantitation was performed on the right panel as in F.


F . MCF - 7 were transfected with empty vector or FLAG - TRIM46 for forty - eight hours then treated with 50 μg / mL cycloheximide ( CHX ) for indicated hours . DMSO was added to 10 h before were collected for western blotting ( left ) . On right panel , quantitation was done by densitometry of immunoblots expressed as signals of HDAC1 .

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F. MCF-7 cells were transfected with empty vector or FLAG-TRIM46 for forty-eight hours and then treated with 50 μg/mL cycloheximide (CHX) for the indicated hours. DMSO was added to cells 10 h before cellular proteins were collected for western blotting (left). On the right panel, quantitation was done by densitometry analysis of the immunoblots and expressed as signals of HDAC1.


In vivo ubiquitination assays performed in MCF - 7 . ( B ) Stable shCTR - or shTRIM46 - expressing MCF7 were transfected with His - Ub for 40 h . were then treated with MG132 ( 10 μM ) for 12 h before Ni - NTA bead precipitation followed by IB with anti - HDAC1 .

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In vivo ubiquitination assays performed in MCF-7 cells. (B) Stable shCTR- or shTRIM46-expressing MCF7 cells were transfected with His-Ub for 40 h. Cells were then treated with MG132 (10 μM) for 12 h before Ni-NTA bead precipitation followed by IB with anti-HDAC1.


( C ) ( D ) MCF - 7 were co - transfected with indicated plasmids . Forty - eight hours after transfection , were treated with MG132 for 10 h before extracts were harvested for Ni - NTA bead precipitation followed by IB with anti - HDAC1 .

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(C) and (D) MCF-7 cells were co-transfected with the indicated plasmids. Forty-eight hours after transfection, cells were treated with MG132 for 10 h before cellular extracts were harvested for Ni-NTA bead precipitation followed by IB with anti-HDAC1.


E - G . Verification of representative genes targeted by TRIM46 - HDAC1 axis . ( E ) MCF - 7 were transfected with empty vector , FLAG - TRIM46 , or FLAG - TRIM46 together with FLAG - HDAC1 for 48 h . ( F ) MCF - 7 were transfected with control siRNA , TRIM46 siRNA , or TRIM46 siRNA together with HDAC1 siRNA for 72 h . ( G ) MCF - 7 were transfected with control siRNA , TRIM46 siRNA , or a siRNA - resistant TRIM46 construct together with TRIM46 siRNA for 72 h . Total RNAs were extracted from RT - qPCR was performed with primers specific for indicted genes .

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E-G. Verification of representative genes targeted by the TRIM46-HDAC1 axis. (E) MCF-7 cells were transfected with empty vector, FLAG-TRIM46, or FLAG-TRIM46 together with FLAG-HDAC1 for 48 h. (F) MCF-7 cells were transfected with control siRNA, TRIM46 siRNA, or TRIM46 siRNA together with HDAC1 siRNA for 72 h. (G) MCF-7 cells were transfected with control siRNA, TRIM46 siRNA, or a siRNA-resistant TRIM46 construct together with TRIM46 siRNA for 72 h. Total RNAs were extracted from cells and RT-qPCR was performed with primers specific for indicted genes.


B . EdU assays were performed in MCF - 7 treated with control siRNA , TRIM46 siRNA or TRIM46 siRNA HDAC1 siRNA for 72 h , or MCF - 10A treated with vector , FLAG - TRIM46 or FLAG - TRIM46 FLAG - HDAC1 for 48 h . Representative from FACS are shown on left panel , numbers of in S phase was quantified by FlowJo software on right panel .

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B. EdU assays were performed in MCF-7 cells treated with control siRNA, TRIM46 siRNA or TRIM46 siRNA and HDAC1 siRNA for 72 h, or MCF-10A cells treated with vector, FLAG-TRIM46 or FLAG-TRIM46 and FLAG-HDAC1 for 48 h. Representative images from FACS analysis are shown on the left panel, the percentage numbers of cells in S phase was quantified by FlowJo software on the right panel.


G . Accumulation of γH2AX in depleted of TRIM46 or HDAC1 after cisplatin stimulation . MCF - 7 were treated with 4 μM cisplatin for 8 h before harvesting . lysates were analyzed by western blotting .

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G. Accumulation of γH2AX in cells depleted of TRIM46 or HDAC1 after cisplatin stimulation. MCF-7 cells were treated with 4 μM cisplatin for 8 h before harvesting. Cell lysates were analyzed by western blotting.


B . MCF - 7 stably transfected with indicated shRNAs or expression plasmids or MCF - 10A stably transfected with expression plasmids were tested for colony formation .

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B. MCF-7 cells stably transfected with indicated shRNAs or expression plasmids or MCF-10A cells stably transfected with expression plasmids were tested for colony formation assay.


D . Control MCF - 7 or MCF - 7 depleted with TRIM46 were treated with different concentrations of cisplatin ( cis ) , VP16 , or adriamycin ( ADM ) for 72 h , proportions of viable were examined . E . Control MCF - 10A or MCF - 10A overexpressed TRIM46 were treated with different concentrations of cisplatin , VP16 , or adriamycin for 72 h , proportions of viable were examined .

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D. Control MCF-7 cells or MCF-7 cells depleted with TRIM46 were treated with different concentrations of cisplatin (cis), VP16, or adriamycin (ADM) for 72 h, and the proportions of viable cells were examined. E. Control MCF-10A cells or MCF-10A cells overexpressed TRIM46 were treated with different concentrations of cisplatin, VP16, or adriamycin for 72 h, and the proportions of viable cells were examined.


D . Control MCF - 7 or MCF - 7 depleted with TRIM46 were treated with different concentrations of cisplatin ( cis ) , VP16 , or adriamycin ( ADM ) for 72 h , proportions of viable were examined .

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D. Control MCF-7 cells or MCF-7 cells depleted with TRIM46 were treated with different concentrations of cisplatin (cis), VP16, or adriamycin (ADM) for 72 h, and the proportions of viable cells were examined.


A . Immunohistochemical of tissue arrays containing 142 breast carcinoma samples ( grade I : n = 29 , grade II : n = 69 , grade III : n = 44 ) 20 normal mammary tissues for TRIM46 HDAC1 expression . Representative are shown on left panel . positively stained were analyzed , mean was scored by Image - Pro Plus software on right panel . In boxplot , central lines , box limits , whiskers represented medians , 25th / 75th percentile , min / max , respectively . ( * P < 0 . 05 , * * P < 0 . 01 , * * * * P < 0 . 0001 , one - way ANOVA ) .

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A. Immunohistochemical staining of tissue arrays containing 142 breast carcinoma samples (grade I: n=29, grade II: n=69, grade III: n=44) and 20 normal mammary tissues for TRIM46 and HDAC1 expression. Representative images are shown on the left panel. The positively stained cells were analyzed, and the mean intensity was scored by Image-Pro Plus software on the right panel. In the boxplot, the central lines, the box limits, and the whiskers represented medians, 25th/75th percentile, and min/max, respectively. (*P<0.05, **P<0.01, ****P < 0.0001, one-way ANOVA).