J . Analysis of cell death after overnight recovery of HeLa cells treated or untreated with CDKi ( SNS - 032 ; Flavopiridol or Dinaciclib , 1 µM , 150 min ) and then treated or not with SA ( 500 µM , 3 h ) . Images were acquired automatically by OPERETTA microscope . Values indicate the percentage of the total number of nuclei ( stained with DAPI ) positive for BoBo - 3 staining . Mean ± s . d . of one representative experiment out of three independent replicates . ns : not significant , * * * * p < 0 . 0001 , One - way ANOVA with Dunnett ' s multiple comparison test .

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J. Analysis of cell death after overnight recovery of HeLa cells treated or untreated with CDKi (SNS-032; Flavopiridol or Dinaciclib, 1 µM, 150 min) and then treated or not with SA (500 µM, 3 h). Images were acquired automatically by OPERETTA microscope. Values indicate the percentage of the total number of nuclei (stained with DAPI) positive for BoBo-3 staining. Mean ± s.d. of one representative experiment out of three independent replicates. ns: not significant, ****p<0.0001, One-way ANOVA with Dunnett's multiple comparison test.


K . Analysis of cell death in HeLa cells that were left untreated , treated with non - targeting siRNAs or siRNAs against TRAPPC2 , and in two cell clones knocked out for TRAPPC2 , with and without SA treatment . Mean ± s . d . of three independent replicates .

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K. Analysis of cell death in HeLa cells that were left untreated, treated with non-targeting siRNAs or siRNAs against TRAPPC2, and in two cell clones knocked out for TRAPPC2, with and without SA treatment. Mean ± s.d. of three independent replicates.


A . Left , control or SA - treated ( 500 µM , 120 min ) human fibroblasts ( HFs ) were incubated at 40 ° C for 180 min and stained for eIF3 , PCI , and Sec24C . Right , the cells were imaged 10 min after shifting the temperature from 40 ° C to 32 ° C and stained for eIF3 , PCI , and Giantin ( to label the Golgi ) . Data information Scale bars , 10 µm .

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A. Left, control or SA-treated (500 µM, 120 min) human fibroblasts (HFs) were incubated at 40°C for 180 min and stained for eIF3, PCI, and Sec24C. Right, the cells were imaged 10 min after shifting the temperature from 40°C to 32°C and stained for eIF3, PCI, and Giantin (to label the Golgi). Data information Scale bars, 10 µm.


B . HeLa cells were exposed to SA ( 200 µM ) for the indicated time , alone or in combination with ISRIB and SGs were analyzed . Quantification of SG area ( µm ) of three independent experiments . Mean ± s . e . m . n = 60 - 80 cells per experiment , N = 3 .

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B. HeLa cells were exposed to SA (200 µM) for the indicated time, alone or in combination with ISRIB and SGs were analyzed. Quantification of SG area (µm) of three independent experiments. Mean ± s.e.m. n = 60-80 cells per experiment, N = 3.


C . Quantification of Sec24C localization at SGs over time ( the ratio between Sec24C ( mean fluorescence intensity ) in SG puncta and cytosolic Sec24C ) . Mean ± SD . n = 60 - 80 cells per experiment , N = 3 .

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

C. Quantification of Sec24C localization at SGs over time (the ratio between Sec24C (mean fluorescence intensity) in SG puncta and cytosolic Sec24C). Mean ± SD. n = 60-80 cells per experiment, N = 3.


D . HeLa cells expressing VSV - G - UVR8 mEOS were left untreated or treated with SA alone or in combination with ISRIB ( 1 µM ) ( 120 min ISRIB pre - treatment and 30 min SA + ISRIB ) and then pulsed with blue light . Images were taken 10 min after the UV pulse and processed for staining with the indicated markers . E . Cells were treated with SA for 30 min , the SA was washed out ( WO ) , and cells were left to recover for 120 min in the presence of cycloheximide , followed by a blue light pulse and processing for staining as described in ( D ) . F . Quantification of VSV - G ( mean intensity ) in the Golgi area to the total VSV - G per cell under the conditions described in ( D , E ) . Data are expressed as percentage of the control . Mean ± s . e . m . of three independent experiment . * * * * p < 0 . 0001 ; ns : not significant , One - way ANOVA with Dunnett ' s multiple comparison test . Data information : Scale bars , 10 µm .

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D. HeLa cells expressing VSV-G-UVR8 mEOS were left untreated or treated with SA alone or in combination with ISRIB (1 µM) (120 min ISRIB pre-treatment and 30 min SA+ISRIB) and then pulsed with blue light. Images were taken 10 min after the UV pulse and processed for staining with the indicated markers. E. Cells were treated with SA for 30 min, the SA was washed out (WO), and cells were left to recover for 120 min in the presence of cycloheximide, followed by a blue light pulse and processing for staining as described in (D). F. Quantification of VSV-G (mean intensity) in the Golgi area to the total VSV-G per cell under the conditions described in (D,E). Data are expressed as percentage of the control. Mean ± s.e.m. of three independent experiment. ****p<0.0001; ns: not significant, One-way ANOVA with Dunnett's multiple comparison test. Data information: Scale bars, 10 µm.


A . AiryScan images of HeLa cells treated with SA for the indicated times and stained with GM130 ( gray ) . Scale bar , 10 µm . The graph shows quantification of Golgi objects in SA - treated cells . Mean ± s . e . m . of three independent experiments . n = 90 - 100 . * * * * p < 0 . 0001 ; ns : not significant , Student ' s unpaired two - tailed t - test .

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A. AiryScan images of HeLa cells treated with SA for the indicated times and stained with GM130 (gray). Scale bar, 10 µm. The graph shows quantification of Golgi objects in SA-treated cells. Mean ± s.e.m. of three independent experiments. n=90-100. ****p<0.0001; ns: not significant, Student's unpaired two-tailed t-test.


B . Control cells and cells treated with Dinaciclib ( CDKi ) were treated with SA and stained for GM130 ( in gray ) . Insets , eIF3 . Blue , nuclear DAPI staining . The graph shows quantification of Golgi objects in HeLa cells untreated or treated with SNS - 032 , Flavopiridol , or Dinaciclib for 150 min and then treated with SA ( 300 µM , 30 min ) . Mean ± s . e . m . of three independent experiments . * * * * p < 0 . 0001 , One - way ANOVA with Dunnett ' s multiple comparison test .

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B. Control cells and cells treated with Dinaciclib (CDKi) were treated with SA and stained for GM130 (in gray). Insets, eIF3. Blue, nuclear DAPI staining. The graph shows quantification of Golgi objects in HeLa cells untreated or treated with SNS-032, Flavopiridol, or Dinaciclib for 150 min and then treated with SA (300 µM, 30 min). Mean ± s.e.m. of three independent experiments. ****p<0.0001, One-way ANOVA with Dunnett's multiple comparison test.


C . Quantification of Golgi objects in cells plated at different percentage of confluency and treated or untreated with SA . Mean ± s . e . m . of three independent experiments . * * p < 0 . 001 ; * * * p < 0 . 0002 ; ns : not significant , One - way ANOVA with Dunnett ' s multiple comparison test .

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C. Quantification of Golgi objects in cells plated at different percentage of confluency and treated or untreated with SA. Mean ± s.e.m. of three independent experiments. **p<0.001; ***p<0.0002; ns: not significant, One-way ANOVA with Dunnett's multiple comparison test.


D . Quantification of Golgi objects in cells pre - treated with ISRIB ( 1 µM ) for 3 h and then exposed to SA ( 30 min ) in the presence of ISRIB . * * * p < 0 . 002 , Student ' s unpaired two - tailed t - test .

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D. Quantification of Golgi objects in cells pre-treated with ISRIB (1 µM) for 3 h and then exposed to SA (30 min) in the presence of ISRIB. ***p<0.002, Student's unpaired two-tailed t-test.


E . Electron microscopy images of cells treated ( 300 µM , 30 min ) or untreated with SA . The arrow indicates the Golgi complex . Scale bar , 200 nm .

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E. Electron microscopy images of cells treated (300 µM, 30 min) or untreated with SA. The arrow indicates the Golgi complex. Scale bar, 200 nm.


F . AiryScan images of HeLa cells at steady state or exposed to SA for the indicated times . A Rab1 - GTP specific antibody was used to monitor the pool of active Rab1 , and GM130 to stain the Golgi complex . Insets , eIF3 . Scale bar , 10 µm . The graph shows quantification of Rab1 - GTP at the Golgi complex expressed as a percentage of the control . Mean ± s . e . m . of three independent experiments . * p < 0 . 011 , * * p < 0 . 002 , Student ' s unpaired two - tailed t - test .

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F. AiryScan images of HeLa cells at steady state or exposed to SA for the indicated times. A Rab1-GTP specific antibody was used to monitor the pool of active Rab1, and GM130 to stain the Golgi complex. Insets, eIF3. Scale bar, 10 µm. The graph shows quantification of Rab1-GTP at the Golgi complex expressed as a percentage of the control. Mean ± s.e.m. of three independent experiments. *p<0.011, **p<0.002, Student's unpaired two-tailed t-test.


G . HeLa cells transfected or not with WT GFP - Rab1B were left untreated or treated with SA and stained for GM130 and G3BP to monitor SGs . Dashed white line , WT GFP - Rab1B transfected cells . Scale bar , 10 µm . The graph shows quantification of Golgi objects in the different conditions . NT : non - transfected . Mean ± s . e . m . of three independent experiments . * * * * p < 0 . 0001 , Student ' s unpaired two - tailed t - test .

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G. HeLa cells transfected or not with WT GFP-Rab1B were left untreated or treated with SA and stained for GM130 and G3BP to monitor SGs. Dashed white line, WT GFP-Rab1B transfected cells. Scale bar, 10 µm. The graph shows quantification of Golgi objects in the different conditions. NT: non-transfected. Mean ± s.e.m. of three independent experiments. ****p<0.0001, Student's unpaired two-tailed t-test.


A Western blot analysis of the localization of endogenous PHGDH in PLC , HepG2 , and THLE3 cells . Lamin B1 , Tubulin , and Tom20 were used as markers for nuclear ( Nuc ) , cytosolic ( Cyto ) , and mitochondrial ( Mito ) proteins , respectively .

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A Western blot analysis of the localization of endogenous PHGDH in PLC, HepG2, and THLE3 cells. Lamin B1, Tubulin, and Tom20 were used as markers for nuclear (Nuc), cytosolic (Cyto), and mitochondrial (Mito) proteins, respectively.


B Western blot analysis of the localization of endogenous PHGDH in tumour tissues and adjacent normal tissues . Lamin B1 , GAPDH , and Tom20 were used as markers for Nuc , Cyto , and Mito proteins , respectively .

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

B Western blot analysis of the localization of endogenous PHGDH in tumour tissues and adjacent normal tissues. Lamin B1, GAPDH, and Tom20 were used as markers for Nuc, Cyto, and Mito proteins, respectively.


C Western blot analysis of the localization of endogenous and exogenous Flag - tagged PHGDH in PLC cells . Lamin B1 , Tubulin , and Tom20 were used as markers for Nuc , Cyto , and Mito proteins , respectively .

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

C Western blot analysis of the localization of endogenous and exogenous Flag-tagged PHGDH in PLC cells. Lamin B1, Tubulin, and Tom20 were used as markers for Nuc, Cyto, and Mito proteins, respectively.


D Immunofluorescence images showing colocalization of Flag - PHGDH ( green ) and mitochondria ( red ) acquired with a laser confocal microscope system in PLC cells . Nuclei were stained with DAPI ( blue ) , and mitochondria were stained with MitoTracker Red . Scale bar , 10 μm ( left panel ) , 2 μm ( right panel ) .

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D Immunofluorescence images showing colocalization of Flag-PHGDH (green) and mitochondria (red) acquired with a laser confocal microscope system in PLC cells. Nuclei were stained with DAPI (blue), and mitochondria were stained with MitoTracker Red. Scale bar, 10 μm (left panel), 2 μm (right panel).


F Subcellular localization of PHGDH was determined by western blot in PLC cells overexpressing of Flag - tagged wild - type PHGDH , PHGDH ∆ SBD1 , or PHGDH ∆ RD mutants . WCL : whole - cell lysates , Mito : mitochondria . Tom20 was used as loading control .

O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Subcellular localization of PHGDH was determined by western blot in PLC cells overexpressing of Flag-tagged wild-type PHGDH, PHGDH∆SBD1, or PHGDH∆RD mutants. WCL: whole-cell lysates, Mito: mitochondria. Tom20 was used as loading control.


G Proteinase K protection assays were performed on purified mitochondria isolated from PLC and HepG2 cells . The outer mitochondrial membrane ( OMM ) protein Tom20 , the inner mitochondrial membrane ( IMM ) protein ANT2 , and the mitochondrial matrix ( MM ) protein TFAM were used as markers of the OMM , IMM , and MM , respectively .

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

G Proteinase K protection assays were performed on purified mitochondria isolated from PLC and HepG2 cells. The outer mitochondrial membrane (OMM) protein Tom20, the inner mitochondrial membrane (IMM) protein ANT2, and the mitochondrial matrix (MM) protein TFAM were used as markers of the OMM, IMM, and MM, respectively.


H Purified mitochondria of PLC or HepG2 cells were incubated with Na2CO3 ( pH 11 . 5 ) for 30 min at 4 ℃ . The soluble component ( Supernatant / Sup . ) and membrane component ( Pellet ) were separated by centrifugation prior to western blot analysis . COX2 , ATP8 , and ANT2 are markers for the membrane compotent ; CYTC and TFAM are markers for the soluble component .

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H Purified mitochondria of PLC or HepG2 cells were incubated with Na2CO3 (pH 11.5) for 30 min at 4 ℃. The soluble component (Supernatant/Sup.) and membrane component (Pellet) were separated by centrifugation prior to western blot analysis. COX2, ATP8, and ANT2 are markers for the membrane compotent; CYTC and TFAM are markers for the soluble component.


A The oxygen consumption rate ( OCR ) was measured by successive injections of oligomycin ( oligo ) , FCCP , and antimycin A / rotenone ( AA / Rot ) in PLC cells infected with viruses expressing nontargeting control ( NTC ) shRNA or shPHGDH . Basal respiration , ATP - linked respiration , and maximal respiration were analysed with the OCR curve .

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A The oxygen consumption rate (OCR) was measured by successive injections of oligomycin (oligo), FCCP, and antimycin A/rotenone (AA/Rot) in PLC cells infected with viruses expressing nontargeting control (NTC) shRNA or shPHGDH. Basal respiration, ATP-linked respiration, and maximal respiration were analysed with the OCR curve.


B Western blot analysis of mtDNA - encoded proteins in PLC cells stably expressing NTC or shPHGDH and empty vector ( EV ) or PLVX - PHGDH . β - Actin was used as the loading control .

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

B Western blot analysis of mtDNA-encoded proteins in PLC cells stably expressing NTC or shPHGDH and empty vector (EV) or PLVX-PHGDH. β-Actin was used as the loading control.


C PLC cells with endogenous PHGDH knockdown were further infected with viruses expressing EV or PLVX - PHGDH prior to western blot analysis of mtDNA - encoded proteins . β - Actin was used as the loading control .

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C PLC cells with endogenous PHGDH knockdown were further infected with viruses expressing EV or PLVX-PHGDH prior to western blot analysis of mtDNA-encoded proteins. β-Actin was used as the loading control.


D PLC cells expressing NTC or shPHGDH were treated with 100 μg / ml cycloheximide ( CHX ) for 20 min prior to puromycin treatment for 15 min . Purified mitochondrial lysates were subjected to a SUnSET assay with an anti - puromycin antibody to measure the actively translated polypeptide chains . Western blot analysis of PHGDH confirmed the knockdown efficiency of the PHGDH shRNAs . β - Actin was used as the loading control .

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D PLC cells expressing NTC or shPHGDH were treated with 100 μg/ml cycloheximide (CHX) for 20 min prior to puromycin treatment for 15 min. Purified mitochondrial lysates were subjected to a SUnSET assay with an anti-puromycin antibody to measure the actively translated polypeptide chains. Western blot analysis of PHGDH confirmed the knockdown efficiency of the PHGDH shRNAs. β-Actin was used as the loading control.


E SUnSET assays were performed in the indicated cell lines using whole - cell lysates .

E SUnSET assays were performed in the indicated cell lines using whole-cell lysates.


F PLC cells with or without PLVX - Flag - PHGDH overexpression were treated with DMSO or 50 μM chloramphenicol ( CAP ) in combination with 50 μM thiamphenicol ( TAP ) for 24 h prior to SUnSET assays . The assays were performed using whole - cell lysate .

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F PLC cells with or without PLVX-Flag-PHGDH overexpression were treated with DMSO or 50 μM chloramphenicol (CAP) in combination with 50 μM thiamphenicol (TAP) for 24 h prior to SUnSET assays. The assays were performed using whole-cell lysate.


G PLC cells with or without PLVX - PHGDH overexpression were treated with DMSO or 50 μM CAP in combination with 50 μM TAP for 24 h prior to western blot analysis of mtDNA - encoded proteins . β - Actin was used as the loading control .

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G PLC cells with or without PLVX-PHGDH overexpression were treated with DMSO or 50 μM CAP in combination with 50 μM TAP for 24 h prior to western blot analysis of mtDNA-encoded proteins. β-Actin was used as the loading control.


H The translation efficiency of mtDNA - encoded proteins was determined by a SUnSET assay in PLC cells with endogenous PHGDH knockdown that were further infected with viruses expressing EV , wild - type PHGDH , or the catalytically inactive PHGDH mutant PHGDHR236E as indicated . β - Actin was used as the loading control .

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H The translation efficiency of mtDNA-encoded proteins was determined by a SUnSET assay in PLC cells with endogenous PHGDH knockdown that were further infected with viruses expressing EV, wild-type PHGDH, or the catalytically inactive PHGDH mutant PHGDHR236E as indicated. β-Actin was used as the loading control.


I mtDNA - encoded proteins were analysed by western blotting in PLC cells overexpressing EV , wild - type PHGDH , or PHGDHR236E . β - Actin was used as the loading control .

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I mtDNA-encoded proteins were analysed by western blotting in PLC cells overexpressing EV, wild-type PHGDH, or PHGDHR236E. β-Actin was used as the loading control.


A HEK293T cells were transfected with Flag - tagged PHGDH in combination with HA - tagged EV , mtIF2 , mtIF3 , mtEFTu , mtEFG1 , mtEFG2 , or mtRRF . Co - immunoprecipitation ( Co - IP ) was performed with an anti - Flag antibody prior to western blot analysis .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 HEK293T cells were transfected with Flag-tagged PHGDH in combination with HA-tagged EV, mtIF2, mtIF3, mtEFTu, mtEFG1, mtEFG2, or mtRRF. Co-immunoprecipitation (Co-IP) was performed with an anti-Flag antibody prior to western blot analysis.


B HEK293T cells were transfected with HA - tagged PHGDHWT or PHGDHR236E in combination with Flag - EV or Flag - mtEFG2 , and Co - IP was performed with an anti - Flag antibody prior to western blot analysis .

B HEK293T cells were transfected with HA-tagged PHGDHWT or PHGDHR236E in combination with Flag-EV or Flag-mtEFG2, and Co-IP was performed with an anti-Flag antibody prior to western blot analysis.


C HEK293T cells were transfected with HA - tagged PHGDHWT or PHGDHR236E in combination with Flag - EV or Flag - ANT2 , and Co - IP was performed with an anti - Flag antibody prior to western blot analysis .

C HEK293T cells were transfected with HA-tagged PHGDHWT or PHGDHR236E in combination with Flag-EV or Flag-ANT2, and Co-IP was performed with an anti-Flag antibody prior to western blot analysis.


D HEK293T cells were transfected with HA - mtEFG2 in combination with Flag - EV or Flag - ANT2 , and Co - IP was performed with an anti - Flag antibody prior to western blot analysis .

D HEK293T cells were transfected with HA-mtEFG2 in combination with Flag-EV or Flag-ANT2, and Co-IP was performed with an anti-Flag antibody prior to western blot analysis.


E GST pull - down of His - PHGDHWT and His - PHGDHR236E by GST - ANT2 using proteins purified in E . coli , followed by western blot analysis with anti - His and anti - GST antibodies .

E GST pull-down of His-PHGDHWT and His-PHGDHR236E by GST-ANT2 using proteins purified in E. coli, followed by western blot analysis with anti-His and anti-GST antibodies.


F GST pull - down of His - mtEFG2 by GST - ANT2 using proteins purified in E . coli , followed by western blot analysis with anti - His and anti - GST antibodies .

O O O O O O O O O O O O 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 GST pull-down of His-mtEFG2 by GST-ANT2 using proteins purified in E. coli, followed by western blot analysis with anti-His and anti-GST antibodies.


G PLC cells stably expressing Flag - mtEFG2 and HA - PHGDH were further infected with viruses expressing NTC or shANT2 , and then cell lysates were immunoprecipitated with an anti - Flag antibody or IgG prior to western blot analysis .

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

G PLC cells stably expressing Flag-mtEFG2 and HA-PHGDH were further infected with viruses expressing NTC or shANT2, and then cell lysates were immunoprecipitated with an anti-Flag antibody or IgG prior to western blot analysis.


H PLC cells overexpressing PHGDH were further infected with viruses expressing NTC or shANT2 . The translation efficiency was measured by a SUnSET assay .

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

H PLC cells overexpressing PHGDH were further infected with viruses expressing NTC or shANT2. The translation efficiency was measured by a SUnSET assay.


I PLC cells overexpressing PLVX - PHGDH were further infected with viruses expressing NTC or shANT2 . The expression of mtDNA - encoded proteins was determined by western blotting . β - Actin was used as the loading control .

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

I PLC cells overexpressing PLVX-PHGDH were further infected with viruses expressing NTC or shANT2. The expression of mtDNA-encoded proteins was determined by western blotting. β-Actin was used as the loading control.


PLC cells overexpressing PHGDH were further infected with viruses expressing NTC or shmtEFG2 . The translation efficiency was measured by a SUnSET assay .

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

PLC cells overexpressing PHGDH were further infected with viruses expressing NTC or shmtEFG2. The translation efficiency was measured by a SUnSET assay.


K PLC cells overexpressing pSin - 3xFlag - PHGDH were further infected with viruses expressing NTC or shmtEFG2 . The expression of mtDNA - encoded proteins was determined by western blotting . β - Actin was used as the loading control .

K PLC cells overexpressing pSin-3xFlag-PHGDH were further infected with viruses expressing NTC or shmtEFG2. The expression of mtDNA-encoded proteins was determined by western blotting. β-Actin was used as the loading control.


A HEK293T cells were transfected with HA - mtEFG2 in combination with Flag - EV or Flag - mtRRF , and Co - IP was performed with an anti - Flag antibody prior to western blot analysis .

A HEK293T cells were transfected with HA-mtEFG2 in combination with Flag-EV or Flag-mtRRF, and Co-IP was performed with an anti-Flag antibody prior to western blot analysis.


B PLC cells stably expressing Flag - mtEFG2 and HA - mtRRF were further infected with viruses expressing NTC or shPHGDH , and then cell lysates were immunoprecipitated with anti - Flag antibody or IgG prior to western blot analysis .

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B PLC cells stably expressing Flag-mtEFG2 and HA-mtRRF were further infected with viruses expressing NTC or shPHGDH, and then cell lysates were immunoprecipitated with anti-Flag antibody or IgG prior to western blot analysis.


C PLC cells stably overexpressing mtEFG2 were further infected with viruses expressing NTC or shPHGDH . The translation efficiency was measured by a SUnSET assay .

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C PLC cells stably overexpressing mtEFG2 were further infected with viruses expressing NTC or shPHGDH. The translation efficiency was measured by a SUnSET assay.


D PLC cells stably overexpressing mtEFG2 were further infected with viruses expressing NTC or shPHGDH . mtDNA - encoded proteins were analysed by western blot . β - Actin was used as the loading control .

D PLC cells stably overexpressing mtEFG2 were further infected with viruses expressing NTC or shPHGDH. mtDNA-encoded proteins were analysed by western blot. β-Actin was used as the loading control.


E PLC cell lysates were immunoprecipitated with an anti - MRPL44 antibody or IgG prior to western blot analysis of MRPL44 , MRPL46 , MRPL48 , MRPS15 , MRPS16 , and MRPS35 .

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E PLC cell lysates were immunoprecipitated with an anti-MRPL44 antibody or IgG prior to western blot analysis of MRPL44, MRPL46, MRPL48, MRPS15, MRPS16, and MRPS35.


F Binding of mtDNA - encoded mRNA by endogenous MRPL44 ( representing mitochondrial ribosomes ) was evaluated by RNA immunoprecipitation ( RIP ) in PLC cells with PHGDH knockdown .

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F Binding of mtDNA-encoded mRNA by endogenous MRPL44 (representing mitochondrial ribosomes) was evaluated by RNA immunoprecipitation (RIP) in PLC cells with PHGDH knockdown.


G The same amount of mitochondria from PLC cells expressing NTC or shPHGDH treated with CAP and TAP were loaded onto the sucrose density gradient . After centrifugation , all fractions were subjected to western blot analysis of MRPS35 , MRPL44 , and MRPL48 proteins . RNA concentrations of ND6 were measured .

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G The same amount of mitochondria from PLC cells expressing NTC or shPHGDH treated with CAP and TAP were loaded onto the sucrose density gradient. After centrifugation, all fractions were subjected to western blot analysis of MRPS35, MRPL44, and MRPL48 proteins. RNA concentrations of ND6 were measured.


A PLC cells overexpressing EV , PHGDHWT , or PHGDHR236E were further infected with viruses expressing NTC or shmtEFG2 . The OCR was measured after successive injections of oligomycin ( oligo ) , FCCP , and antimycin A / rotenone ( AA / Rot ) . Basal respiration , ATP - linked respiration , and maximal respiration were analysed with the OCR curve .

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A PLC cells overexpressing EV, PHGDHWT, or PHGDHR236E were further infected with viruses expressing NTC or shmtEFG2. The OCR was measured after successive injections of oligomycin (oligo), FCCP, and antimycin A/rotenone (AA/Rot). Basal respiration, ATP-linked respiration, and maximal respiration were analysed with the OCR curve.


B PLC cells overexpressing EV , PHGDHWT , or PHGDHR236E were further infected with viruses expressing NTC or shmtEFG2 prior to western blot analysis . β - Actin was used as the loading control ( left ) . Cell growth curves were constructed by cell counting with trypan blue exclusion ( right ) .

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B PLC cells overexpressing EV, PHGDHWT, or PHGDHR236E were further infected with viruses expressing NTC or shmtEFG2 prior to western blot analysis. β-Actin was used as the loading control (left). Cell growth curves were constructed by cell counting with trypan blue exclusion (right).


C PLC cells overexpressing EV , PHGDHWT , or PHGDHR236E were further treated with H2O or tigecycline prior to western blot analysis . β - Actin was used as the loading control ( left ) . Cell growth curves were determined by cell counting with trypan blue exclusion ( right ) .

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C PLC cells overexpressing EV, PHGDHWT, or PHGDHR236E were further treated with H2O or tigecycline prior to western blot analysis. β-Actin was used as the loading control (left). Cell growth curves were determined by cell counting with trypan blue exclusion (right).


D - F PLC cells overexpressing EV or PHGDH were further infected with viruses expressing NTC or shmtEFG2 . Then , these cells were subcutaneously injected into male nude mice ( n = 5 in each group ) . Tumour sizes were measured beginning 14 days after inoculation ( D ) . At the end of the experiment , the tumours were excised ( E ) , and PHGDH and mtEFG2 expression in tumour lysates was analysed by western blotting ( F ) .

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D-F PLC cells overexpressing EV or PHGDH were further infected with viruses expressing NTC or shmtEFG2. Then, these cells were subcutaneously injected into male nude mice (n = 5 in each group). Tumour sizes were measured beginning 14 days after inoculation (D). At the end of the experiment, the tumours were excised (E), and PHGDH and mtEFG2 expression in tumour lysates was analysed by western blotting (F).


G - I PLC cells overexpressing EV or PHGDH were subcutaneously injected into male nude mice ( n = 5 in each group ) . Mice were intraperitoneally injected with 100 mg / kg tigecycline daily beginning 14 days after inoculation . Tumour sizes were measured beginning 14 days after inoculation ( G ) . At the end of the experiment , the tumours were excised ( H ) , and PHGDH expression in tumour lysates was analysed by western blotting ( I ) .

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G-I PLC cells overexpressing EV or PHGDH were subcutaneously injected into male nude mice (n = 5 in each group). Mice were intraperitoneally injected with 100 mg/kg tigecycline daily beginning 14 days after inoculation. Tumour sizes were measured beginning 14 days after inoculation (G). At the end of the experiment, the tumours were excised (H), and PHGDH expression in tumour lysates was analysed by western blotting (I).


A HERC3 increases the protein levels of active YAP , p - YAP ( S381 ) , YAP and TAZ . active - YAP , p - YAP ( S381 ) , p - YAP ( S127 ) , YAP , p - TAZ ( S89 ) , TAZ , HERC3 and β - Actin were detected by Western blotting with appropriate antibodies as indicated in MDA - MB - 231 cells stably expressing HERC3 - WT or HERC3 - CA . Ctrl , Control ; WT , wildtype of HERC3 ; CA , HERC3 - CA , a catalytically inactive mutant of HERC3 .

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A HERC3 increases the protein levels of active YAP, p-YAP (S381), YAP and TAZ. active-YAP, p-YAP (S381), p-YAP (S127), YAP, p-TAZ (S89), TAZ, HERC3 and β-Actin were detected by Western blotting with appropriate antibodies as indicated in MDA-MB-231 cells stably expressing HERC3-WT or HERC3-CA. Ctrl, Control; WT, wildtype of HERC3; CA, HERC3-CA, a catalytically inactive mutant of HERC3.


B HERC3 reverses the reduction in the active YAP and TAZ levels induced by serum starvation . MDA - MB - 231 cells stably expressing HERC3 or HERC3 - CA were cultured in medium with or without fetal bovine serum ( FBS ) for 1 h . Cells were harvested for Western blotting analysis with appropriate antibodies as indicated . Ctrl , Control ; WT , wildtype of HERC3 ; CA , HERC3 - CA , a catalytically inactive mutant of HERC3 .

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B HERC3 reverses the reduction in the active YAP and TAZ levels induced by serum starvation. MDA-MB-231 cells stably expressing HERC3 or HERC3-CA were cultured in medium with or without fetal bovine serum (FBS) for 1 h. Cells were harvested for Western blotting analysis with appropriate antibodies as indicated. Ctrl, Control; WT, wildtype of HERC3; CA, HERC3-CA, a catalytically inactive mutant of HERC3.


C Depletion of HERC3 decreases the steady - state levels of YAP / TAZ . Levels of active - YAP , p - YAP ( S381 ) , p - YAP ( S127 ) , YAP , p - TAZ ( S89 ) , TAZ , HERC3 and β - Actin were detected by Western blotting with appropriate antibodies as indicated in MDA - MB - 231 and SUM159 cells stably expressing shCtrl ( Control shRNA ) , sh - 1 and sh - 2 ( two shRNAs against HERC3 ) .

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C Depletion of HERC3 decreases the steady-state levels of YAP/TAZ. Levels of active-YAP, p-YAP (S381), p-YAP (S127), YAP, p-TAZ (S89), TAZ, HERC3 and β-Actin were detected by Western blotting with appropriate antibodies as indicated in MDA-MB-231 and SUM159 cells stably expressing shCtrl (Control shRNA), sh-1 and sh-2 (two shRNAs against HERC3).


E Depletion of HERC3 has no effects on mRNA levels of YAP / TAZ . Total mRNA levels of YAP / TAZ in control or HERC3 - deficient MDA - MB - 231 cells were analyzed by qRT - PCR using primers specific to YAP / TAZ . Data are shown as mean ± SEM ; n = 3 biological replicates . Statistical analysis was performed using two - tailed Student t test . n . s . , not significant .

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E Depletion of HERC3 has no effects on mRNA levels of YAP/TAZ. Total mRNA levels of YAP/TAZ in control or HERC3-deficient MDA-MB-231 cells were analyzed by qRT-PCR using primers specific to YAP/TAZ. Data are shown as mean ±SEM; n = 3 biological replicates. Statistical analysis was performed using two-tailed Student t test. n.s., not significant.


G Depletion of HERC3 attenuates YAP - or TAZ - induced 8xGTIIC - Luc reporter activity . HEK293T cells were transfected with expression plasmids for YAP or TAZ , shHERC3 or shCtrl , and reporter plasmids 8xGTIIC - Luc and Renilla - Luc as indicated . Relative luciferase activity was measured as described in the text . Western blots showing expression of indicated proteins were shown . H Depletion of HERC3 attenuates YAP - or TAZ - induced CTGF - Luc reporter activity . Cell transfection and luciferase assay were carried out as described in Panel G . Western blots showing expression of indicated proteins were shown . Data are shown as mean ± SEM ; n = 3 biological replicates . Statistical analysis was performed using two - tailed Student t test . * * P < 0 . 01 .

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G Depletion of HERC3 attenuates YAP- or TAZ-induced 8xGTIIC-Luc reporter activity. HEK293T cells were transfected with expression plasmids for YAP or TAZ, shHERC3 or shCtrl, and reporter plasmids 8xGTIIC-Luc and Renilla-Luc as indicated. Relative luciferase activity was measured as described in the text. Western blots showing expression of indicated proteins were shown. H Depletion of HERC3 attenuates YAP- or TAZ-induced CTGF-Luc reporter activity. Cell transfection and luciferase assay were carried out as described in Panel G. Western blots showing expression of indicated proteins were shown. Data are shown as mean ±SEM; n = 3 biological replicates. Statistical analysis was performed using two-tailed Student t test. **P < 0.01.


I Depletion of HERC3 attenuates transcription of YAP / TAZ target genes . Total mRNA levels of CTGF and ANKRD1 in control or HERC3 - deficient MDA - MB - 231 cells were analyzed by qRT - PCR using primers specific to the indicated target gene . Data are shown as mean ± SEM ; n = 3 biological replicates . Statistical analysis was performed using two - tailed Student t test . * * * P < 0 . 001 .

I Depletion of HERC3 attenuates transcription of YAP/TAZ target genes. Total mRNA levels of CTGF and ANKRD1 in control or HERC3-deficient MDA-MB-231 cells were analyzed by qRT-PCR using primers specific to the indicated target gene. Data are shown as mean ±SEM; n = 3 biological replicates. Statistical analysis was performed using two-tailed Student t test. ***P < 0.001.


A HERC3 and HERC3 - CA block β - TrCP - mediated degradation of TAZ . HEK293T cells were transfected with expression plasmids for HA - β - TrCP , Myc - HERC3 or Myc - HERC3 - CA , FLAG - TAZ or FLAG - GFP as indicated . Cells were harvested 24 h post - transfection . Protein levels were detected by Western blotting with appropriate antibodies as indicated .

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A HERC3 and HERC3-CA block β-TrCP-mediated degradation of TAZ. HEK293T cells were transfected with expression plasmids for HA-β-TrCP, Myc-HERC3 or Myc-HERC3-CA, FLAG-TAZ or FLAG-GFP as indicated. Cells were harvested 24 h post-transfection. Protein levels were detected by Western blotting with appropriate antibodies as indicated.


D HERC3 and HERC3 - CA extend the half - life of TAZ . Control or HERC3 - deficient MDA - MB - 231 cells were treated with cycloheximide ( CHX , 20 µM ) for 0 , 4 , 8 or 12 h before harvested . Left , Western blotting with appropriate antibodies . Right , Image J quantitation of YAP intensity and normalized to β - Actin at the indicated time points ( n = 3 biological replicates ) . Data are represented as the mean ± SEM ( right panel ) .

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D HERC3 and HERC3-CA extend the half-life of TAZ. Control or HERC3-deficient MDA-MB-231 cells were treated with cycloheximide (CHX, 20 µM) for 0, 4, 8 or 12 h before harvested. Left, Western blotting with appropriate antibodies. Right, Image J quantitation of YAP intensity and normalized to β-Actin at the indicated time points (n = 3 biological replicates). Data are represented as the mean ± SEM (right panel).


F Depletion of HERC3 decreases the half - life of YAP / TAZ . Control or HERC3 - deficient MDA - MB - 231 cells were treated with cycloheximide ( CHX , 20 µM ) for 0 , 2 , 4 or 6 h before harvested . Left , Western blotting with appropriate antibodies . Right , Image J quantitation of YAP intensity and normalized to β - Actin at the indicated time points ( n = 3 biological replicates ) . Data are represented as the mean ± SEM ( right panel ) .

F Depletion of HERC3 decreases the half-life of YAP/TAZ. Control or HERC3-deficient MDA-MB-231 cells were treated with cycloheximide (CHX, 20 µM) for 0, 2, 4 or 6 h before harvested. Left, Western blotting with appropriate antibodies. Right, Image J quantitation of YAP intensity and normalized to β-Actin at the indicated time points (n = 3 biological replicates). Data are represented as the mean ± SEM (right panel).


G Depletion of β - TrCP reverses shHERC3 - induced degradation of YAP / TAZ . Control or HERC3 - deficient MDA - MB - 231 were transfected with siCtrl or siβ - TrCP as indicated . Cells were harvested 48 h post - transfection . Protein levels were detected by Western blotting with appropriate antibodies as indicated .

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G Depletion of β-TrCP reverses shHERC3-induced degradation of YAP/TAZ. Control or HERC3-deficient MDA-MB-231 were transfected with siCtrl or siβ-TrCP as indicated. Cells were harvested 48 h post-transfection. Protein levels were detected by Western blotting with appropriate antibodies as indicated.


A HERC3 interacts with β - TrCP , but not YAP or TAZ . HEK293T cells were transfected with expression plasmids for FLAG - HERC3 , HA - YAP or HA - TAZ or HA - β - TrCP . Cells were harvested 24 h post - transfection and subjected to IP with FLAG antibody - conjugated agarose beads . Protein levels were detected by Western blotting with HA and FLAG antibodies . B HERC3 interacts with endogenous β - TrCP . MDA - MB - 231 cells stably expressing HERC3 or HERC3 - CA were harvested and subjected to IP with IgG or HA antibodies . Protein levels were detected by Western blotting with β - TRCP , HA and β - Actin antibodies .

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A HERC3 interacts with β-TrCP, but not YAP or TAZ. HEK293T cells were transfected with expression plasmids for FLAG-HERC3, HA-YAP or HA-TAZ or HA-β-TrCP. Cells were harvested 24 h post-transfection and subjected to IP with FLAG antibody-conjugated agarose beads. Protein levels were detected by Western blotting with HA and FLAG antibodies. B HERC3 interacts with endogenous β-TrCP. MDA-MB-231 cells stably expressing HERC3 or HERC3-CA were harvested and subjected to IP with IgG or HA antibodies. Protein levels were detected by Western blotting with β-TRCP, HA and β-Actin antibodies. C


H HERC3 blocks the interaction between β - TrCP and YAP . HEK293T cells were transfected with expression plasmids for FLAG - YAP , HA - β - TRCP , Myc - HERC3 and Myc - HERC3 - CA as indicated . Cells were harvested 24 h post - transfection and subjected to IP with FLAG antibody - conjugated agarose beads . Protein levels were detected by Western blotting with HA , FLAG and Myc antibodies .

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H HERC3 blocks the interaction between β-TrCP and YAP. HEK293T cells were transfected with expression plasmids for FLAG-YAP, HA-β-TRCP, Myc-HERC3 and Myc-HERC3-CA as indicated. Cells were harvested 24 h post-transfection and subjected to IP with FLAG antibody-conjugated agarose beads. Protein levels were detected by Western blotting with HA, FLAG and Myc antibodies.


L HERC3 displaces TAZ on β - TRCP . Increasing concentrations of purified HERC3 or HERC3 - CA proteins were added to the β - TrCP and TAZ complex , and followed by Western blotting analysis with indicated antibodies . The competitive binding assay in vitro was described in materials and methods .

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L HERC3 displaces TAZ on β-TRCP. Increasing concentrations of purified HERC3 or HERC3-CA proteins were added to the β-TrCP and TAZ complex, and followed by Western blotting analysis with indicated antibodies. The competitive binding assay in vitro was described in materials and methods.


A Depletion of HERC3 reduces cell proliferation . Control or HERC3 - deficient MDA - MB - 231 were analyzed for indicated days by CCK8 assay as described in the materials and methods . Data are shown as mean ± SEM ; n = 3 biological replicates .

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A Depletion of HERC3 reduces cell proliferation. Control or HERC3-deficient MDA-MB-231 were analyzed for indicated days by CCK8 assay as described in the materials and methods. Data are shown as mean ± SEM; n = 3 biological replicates.


C Depletion of HERC3 inhibits colony formation in MDA - MB - 231 cells . Control or HERC3 - deficient MDA - MB - 231 were analyzed by colony formation assay . Quantification of the results in colony formation assays was analyzed by Image J . D Depletion of HERC3 inhibits colony formation in SUM159 cells . Control or HERC3 - deficient SUM159 were analyzed by colony formation assay . Quantification of the results in colony formation assays was analyzed by Image J .

C Depletion of HERC3 inhibits colony formation in MDA-MB-231 cells. Control or HERC3-deficient MDA-MB-231 were analyzed by colony formation assay. Quantification of the results in colony formation assays was analyzed by Image J. D Depletion of HERC3 inhibits colony formation in SUM159 cells. Control or HERC3-deficient SUM159 were analyzed by colony formation assay. Quantification of the results in colony formation assays was analyzed by Image J.


Depletion of HERC3 attenuates cell migration . Control or HERC3 - deficient MDA - MB - 231 cells ( upper panel ) and SUM159 cells ( lower panel ) were analyzed by transwell migration assay . E , migrated were fixed in 4 % PFA and stained with 0 . 1 % crystal violet and photographed . data quantification of migrated MDA - MB - 231 cells ( left panel ) and SUM159 ( right panel ) cells counts by Image J .

Depletion of HERC3 attenuates cell migration. Control or HERC3-deficient MDA-MB-231 cells (upper panel) and SUM159 cells (lower panel) were analyzed by transwell migration assay. E, migrated were fixed in 4% PFA and stained with 0.1% crystal violet and photographed. data quantification of migrated MDA-MB-231 cells (left panel) and SUM159 (right panel) cells counts by Image J.


H RNAi - resistant HERC3 and YAP / TAZ mutants reverse the inhibitory effect of HERC3 depletion on colony formation . Ectopic expression of RNAi - resistant HERC3 - WT , HERC3 - CA and YAP / TAZ mutants in HERC3 - deficient MDA - MB - 231 cells were subjected to colony formation assay as described in Panel C .

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H RNAi-resistant HERC3 and YAP/TAZ mutants reverse the inhibitory effect of HERC3 depletion on colony formation. Ectopic expression of RNAi-resistant HERC3-WT, HERC3-CA and YAP/TAZ mutants in HERC3-deficient MDA-MB-231 cells were subjected to colony formation assay as described in Panel C.


I , J RNAi - resistant HERC3 and YAP / TAZ mutants reverse the inhibitory effect of HERC3 depletion on cell migration . I , ectopic expression of resistant variants of HERC3 - WT , HERC3 - CA and YAP / TAZ mutants in HERC3 - deficient MDA - MB - 231 cells were subjected to transwell migration assay . Scale bar , 100 μm . J , graphic representation of quantitated cell migration in Panel I .

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I, J RNAi-resistant HERC3 and YAP/TAZ mutants reverse the inhibitory effect of HERC3 depletion on cell migration. I, ectopic expression of resistant variants of HERC3-WT, HERC3-CA and YAP/TAZ mutants in HERC3-deficient MDA-MB-231 cells were subjected to transwell migration assay. Scale bar, 100 μm. J, graphic representation of quantitated cell migration in Panel I.


HERC3 knockdown inhibits breast tumorigenesis . Luciferase - harboring MDA - MB - 231 cells expressing shHERC3 or shCtrl were subcutaneously injected into 5 - week - old nude mice . Four weeks after injection , mice were monitored by bioluminescence using Xenogen IVIS imaging system . A , representative tumors expressing luciferase are indicated by radiance bar ( p / sec / cm2 / sr ) . B , quantitation of bioluminescence data . Data are shown as mean ± SEM ; n = 6 biological replicates . Statistical analysis was performed using two - tailed Student t test . * * P < 0 . 01 , * * * P < 0 . 001 .

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HERC3 knockdown inhibits breast tumorigenesis. Luciferase-harboring MDA-MB-231 cells expressing shHERC3 or shCtrl were subcutaneously injected into 5-week-old nude mice. Four weeks after injection, mice were monitored by bioluminescence using Xenogen IVIS imaging system. A, representative tumors expressing luciferase are indicated by radiance bar (p/sec/cm2/sr). B, quantitation of bioluminescence data. Data are shown as mean ±SEM; n = 6 biological replicates. Statistical analysis was performed using two-tailed Student t test. **P < 0.01, ***P < 0.001.


J , K shHERC3 attenuates breast cancer lung metastasis . Mice were injected with control or HERC3 - deficient MDA - MB - 231 cells . J , Bright - field images ( upper panel , scale bars , 2 mm ) and H & E staining ( lower panel , scale bars , 50 μm ) of the lungs from mice . K , number of lung metastasis nodules . Data are shown as mean ± SEM ; n = 5 biological replicates . Statistical analysis was performed using two - tailed Student t test . * * * P < 0 . 001 .

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J, K shHERC3 attenuates breast cancer lung metastasis. Mice were injected with control or HERC3-deficient MDA-MB-231 cells. J, Bright-field images (upper panel, scale bars, 2 mm) and H&E staining (lower panel, scale bars, 50 μm) of the lungs from mice. K, number of lung metastasis nodules. Data are shown as mean ±SEM; n = 5 biological replicates. Statistical analysis was performed using two-tailed Student t test. ***P < 0.001.


RNAi - resistant HERC3 and unphosphorylatable YAP - S381A can counteract the tumor suppressive effect of HERC3 depletion . HERC3 - deficient cells with or without ectopic expression of YAP - S381A , RNAi - resistant variants of HERC3 - WT or HERC3 - CA and control MDA - MB - 231 cells were performed subcutaneously injection as described in Panel A . L , tumors were dissected and photographed at four weeks after cell implantation . M , volume from all tumors was recorded .

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RNAi-resistant HERC3 and unphosphorylatable YAP-S381A can counteract the tumor suppressive effect of HERC3 depletion. HERC3-deficient cells with or without ectopic expression of YAP-S381A, RNAi-resistant variants of HERC3-WT or HERC3-CA and control MDA-MB-231 cells were performed subcutaneously injection as described in Panel A. L, tumors were dissected and photographed at four weeks after cell implantation. M, volume from all tumors was recorded.


HERC3 is highly expressed in breast cancer specimens . A , representative images of IHC staining of HERC3 in tumor samples of breast cancer specimens and their paired adjacent normal tissues . Scale bars , 50 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 O O O O

HERC3 is highly expressed in breast cancer specimens. A, representative images of IHC staining of HERC3 in tumor samples of breast cancer specimens and their paired adjacent normal tissues. Scale bars, 50 µm.


D HERC3 is correlated with poor prognosis in breast cancer patients . Kaplan - Meier curves for overall survival of breast cancer patients according to the HERC3 expression level ( upper left panel ) ( http : / / kmplot . com / analysis / index . php ? p = service & cancer = breast ) . HERC3 is negatively correlated with the overall survival ( upper right panel ) , the distant metastasis free survival ( lower left panel ) and the relapse free survival ( lower right panel ) in breast cancer patients . Microarray analysis was performed to examine the prognostic potential of HERC3 in breast cancer using the PrognoScan database with one cohort ( GSE7390 ) of 198 breast cancer specimens ( http : / / www . prognoscan . org )

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 HERC3 is correlated with poor prognosis in breast cancer patients. Kaplan-Meier curves for overall survival of breast cancer patients according to the HERC3 expression level (upper left panel) (http://kmplot.com/analysis/index.php?p=service&cancer=breast). HERC3 is negatively correlated with the overall survival (upper right panel), the distant metastasis free survival (lower left panel) and the relapse free survival (lower right panel) in breast cancer patients. Microarray analysis was performed to examine the prognostic potential of HERC3 in breast cancer using the PrognoScan database with one cohort (GSE7390) of 198 breast cancer specimens (http://www.prognoscan.org)


. E HERC3 expression is positively correlated with TAZ or active YAP expression in breast cancer patients . Representative images of IHC staining of HERC3 , TAZ and active YAP in tumor samples of breast cancer

O O O O O O O O O O O O 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 HERC3 expression is positively correlated with TAZ or active YAP expression in breast cancer patients. Representative images of IHC staining of HERC3, TAZ and active YAP in tumor samples of breast cancer patients


B . FPKM of the 7 LncRNAs identified in the RNA - seq with high expression in PGCs . The solid lines at each end of the box plot represent the maximum and minimum values , respectively . The three dotted lines in the middle of the violin diagram represent the 75 % percentile , the mean , and the 25 % percentile in turn .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. FPKM of the 7 LncRNAs identified in the RNA-seq with high expression in PGCs. The solid lines at each end of the box plot represent the maximum and minimum values, respectively. The three dotted lines in the middle of the violin diagram represent the 75% percentile, the mean, and the 25% percentile in turn.


A . Up : The schematic diagram of PGCs induced from chicken ESCs in vitro . Down : Morphological observation of the number of embryoid bodies in the in vitro PGCs induction model after LncBMP4overexpression and interference ; oeLncBMP4 indicates the overexpression of LncBMP4 ; shLncBMP4 indicates the interference of LncBMP4 ; BMP4 induction was regarded as the control . Scale bar : 60 µm ( 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 O O O O O O O O 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. Up: The schematic diagram of PGCs induced from chicken ESCs in vitro. Down: Morphological observation of the number of embryoid bodies in the in vitro PGCs induction model after LncBMP4overexpression and interference; oeLncBMP4 indicates the overexpression of LncBMP4; shLncBMP4 indicates the interference of LncBMP4; BMP4 induction was regarded as the control. Scale bar: 60 µm (n = 3 independent experiments).


B . The expression of reproductive marker genes ( Cvh and C - kit ) , LncBMP4 , and BMP4 were detected by qRT - PCR after LncBMP4 overexpression and interference , in vitro ( Data are shown as mean ± SEM , n = 3 independent experiments , * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 , one - way ANOVA ) .

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B. The expression of reproductive marker genes (Cvh and C-kit), LncBMP4, and BMP4 were detected by qRT-PCR after LncBMP4 overexpression and interference, in vitro (Data are shown as mean ± SEM, n = 3 independent experiments, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, one-way ANOVA) .


A . The results of IHC examining using anti - DDX4 antibody shows that the overexpression of LncBMP4 increases the number of PGCs in the genital ridge , and interference with LncBMP4 reduces the number of PGCs in the genital ridge . The number of PGCs was counted through the Segmentation module of Image J software . Scale bars : 200 μm ( top row ) , 40 μm ( bottom row ) ( Data are shown as mean ± SEM , n = 3 independent experiments , * p < 0 . 05 , * * p < 0 . 01 , * * * * p < 0 . 0001 , one - way ANOVA ) .

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

A. The results of IHC examining using anti-DDX4 antibody shows that the overexpression of LncBMP4 increases the number of PGCs in the genital ridge, and interference with LncBMP4 reduces the number of PGCs in the genital ridge. The number of PGCs was counted through the Segmentation module of Image J software. Scale bars:200 μm (top row),40 μm (bottom row) (Data are shown as mean ± SEM, n = 3 independent experiments, * p < 0.05, ** p < 0.01, **** p < 0.0001, one-way ANOVA).


B . The number of PGCs in the genital ridge , following the overexpression or interference of LncBMP4 , as determined by flow cytometry ( Data are shown as mean ± SEM , n = 3 independent experiments , * * * * p < 0 . 0001 , one - way ANOVA ) .

B. The number of PGCs in the genital ridge, following the overexpression or interference of LncBMP4, as determined by flow cytometry (Data are shown as mean ± SEM, n = 3 independent experiments, **** p < 0.0001, one-way ANOVA).


C . The number of PGCs labeled with PkH26 ( red ) that migrated to the genital ridge in a frozen section , the number of PGCs labeled with PkH26 ( red ) was counted using Image J software . Scale bar : 70 µm ( Data are shown as mean ± SEM , n = 6 independent experiments , * * p < 0 . 01 , * * * * p < 0 . 0001 , one - way ANOVA . ) .

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

C. The number of PGCs labeled with PkH26 (red) that migrated to the genital ridge in a frozen section, the number of PGCs labeled with PkH26 (red) was counted using Image J software. Scale bar: 70 µm (Data are shown as mean ± SEM, n = 6 independent experiments, ** p < 0.01, **** p < 0.0001, one-way ANOVA.).


D . The expression of reproductive marker genes ( Cvh and C - kit ) , LncBMP4 , and BMP4 were detected by qRT - PCR in the genital ridge after gga - mir - 12211 overexpression and interference in vitro ( Data are shown as mean ± SEM , n = 3 independent experiments , * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 , one - way ANOVA ) .

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

D. The expression of reproductive marker genes (Cvh and C-kit), LncBMP4, and BMP4 were detected by qRT-PCR in the genital ridge after gga-mir-12211 overexpression and interference in vitro (Data are shown as mean ± SEM, n =3 independent experiments, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, one-way ANOVA).


E . The efficiency of PGC differentiation was detected by IF , 4 d after induction , using C - KIT and CVH antibodies . Scale bar : 60 µm ( 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

E. The efficiency of PGC differentiation was detected by IF, 4 d after induction, using C-KIT and CVH antibodies. Scale bar: 60 µm (n = 3 independent experiments).


A . A luciferase reporting system was used to detect the regulatory effects of gga - mir - 12211 on the activity of BMP4 - 3 ' UTR ( Data are shown as mean ± SEM , n = 3 independent experiments , * p < 0 . 05 , * * * * p < 0 . 0001 , one - way ANOVA ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. A luciferase reporting system was used to detect the regulatory effects of gga-mir-12211 on the activity of BMP4-3'UTR (Data are shown as mean ± SEM, n =3 independent experiments, * p < 0.05, **** p < 0.0001, one-way ANOVA).


B . qRT - PCR was used to detect the regulatory effects of gga - mir - 12211 on LncBMP4 expression ( Data are shown as mean ± SEM , n = 3 independent experiments , * * * p < 0 . 001 , * * * * p < 0 . 0001 , one - way ANOVA ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. qRT-PCR was used to detect the regulatory effects of gga-mir-12211 on LncBMP4 expression (Data are shown as mean ± SEM, n =3 independent experiments, *** p < 0.001, **** p < 0.0001, one-way ANOVA).


Enrichment levels of m6A in LncBMP4 were detected by RIP - qPCR ( Data are shown as mean ± SEM , n = 3 independent experiments , * * * * p < 0 . 0001 , one - way ANOVA ) .

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

Enrichment levels of m6A in LncBMP4 were detected by RIP-qPCR (Data are shown as mean ± SEM, n =3 independent experiments, **** p < 0.0001, one-way ANOVA).


H and I . The effects of m6A modifications of LncBMP4 on the ceRNA system were detected by the luciferase reporting system ( Data are shown as mean ± SEM , n = 3 independent experiments , * p < 0 . 05 , * * * * p < 0 . 0001 , one - way ANOVA . ) .

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

H and I. The effects of m6A modifications of LncBMP4 on the ceRNA system were detected by the luciferase reporting system (Data are shown as mean ± SEM, n =3 independent experiments, * p < 0.05, **** p < 0.0001, one-way ANOVA.).


A . The influence of m6A on the stability of LncBMP4 was detected by qRT - PCR ( Data are shown as mean ± SEM , n = 3 independent experiments , * * * * p < 0 . 0001 , one - way ANOVA ) .

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

A. The influence of m6A on the stability of LncBMP4 was detected by qRT-PCR (Data are shown as mean ± SEM, n =3 independent experiments, **** p < 0.0001, one-way ANOVA).


D . The enrichment level of m6A , for LncBMP4 variants with the progressive accumulation of RRACH site mutations , was detected by RIP - qPCR ( Data are shown as mean ± SEM , n = 3 independent experiments , * p < 0 . 05 , * * p < 0 . 01 , one - way ANOVA ) .

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

D. The enrichment level of m6A, for LncBMP4 variants with the progressive accumulation of RRACH site mutations, was detected by RIP-qPCR (Data are shown as mean ± SEM, n =3 independent experiments, * p < 0.05, ** p < 0.01, one-way ANOVA).


E . The ability of LncBMP4 , modified with different levels of m6A , to bind gga - mir - 12211 was detected by the luciferase reporting system ( Data are shown as mean ± SEM , n = 3 independent experiments , * * p < 0 . 01 , * * * * p < 0 . 0001 , one - way ANOVA . ) .

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

E. The ability of LncBMP4, modified with different levels of m6A, to bind gga-mir-12211 was detected by the luciferase reporting system (Data are shown as mean ± SEM, n =3 independent experiments, ** p < 0.01, **** p < 0.0001, one-way ANOVA.).


B . qRT - PCR was used to detect the expression of related genes in each group , during induction ( Data are shown as mean ± SEM , n = 3 independent experiments , * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , one - way ANOVA ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. qRT-PCR was used to detect the expression of related genes in each group, during induction (Data are shown as mean ± SEM, n =3 independent experiments, * p < 0.05, ** p < 0.01, *** p < 0.001, one-way ANOVA).


D . The number of CVH - positive cells was determined by flow cytometry ( Data are shown as mean ± SEM , n = 3 independent experiments , * * p < 0 . 01 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 , one - way ANOVA . ) .

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

D. The number of CVH-positive cells was determined by flow cytometry (Data are shown as mean ± SEM, n =3 independent experiments, ** p < 0.01, ** p < 0.01, *** p < 0.001, **** p < 0.0001, one-way ANOVA.).


C . Effects of m6A on small peptide expression , assessed by western blot ( Up ) , Image J software was used for gray level analysis ( Down ) ( Data are shown as mean ± SEM , n = 3 independent experiments , * * * * p < 0 . 0001 , one - way ANOVA ) .

C. Effects of m6A on small peptide expression, assessed by western blot (Up), Image J software was used for gray level analysis(Down) (Data are shown as mean ± SEM, n =3 independent experiments, **** p < 0.0001, one-way ANOVA).


D and E . Western blot ( Up ) was used to detect gga - mir - 12211 and m6A affect the ability of LncBMP4 to translate EPC5 . Image J software was used for gray level analysis ( Down ) ( Data are shown as mean ± SEM , n = 3 independent experiments , * * * p < 0 . 001 , * * * * p < 0 . 0001 , one - way ANOVA . ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 and E. Western blot(Up) was used to detect gga-mir-12211 and m6A affect the ability of LncBMP4 to translate EPC5. Image J software was used for gray level analysis(Down) (Data are shown as mean ± SEM, n =3 independent experiments, *** p < 0.001,**** p < 0.0001, one-way ANOVA.).


A Transmigration of mouse neutrophils towards the chemokine CXCL - 1 through TNF - α - stimulated VE - cadherin WT ( WT ) or VE - cadherin Y731F ( Y731F ) primary endothelial cells , each transfected with control or SHP2 - specific siRNA . The transmigration rate is presented relative to that of WT cells transfected with control siRNA , set as 100 % . On the right , Western blot analysis of lysates from cells used in transmigration assays for the expression of SHP2 and α - tubulin .

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A Transmigration of mouse neutrophils towards the chemokine CXCL-1 through TNF-α-stimulated VE-cadherin WT (WT) or VE-cadherin Y731F (Y731F) primary endothelial cells, each transfected with control or SHP2-specific siRNA. The transmigration rate is presented relative to that of WT cells transfected with control siRNA, set as 100%. On the right, Western blot analysis of lysates from cells used in transmigration assays for the expression of SHP2 and α-tubulin.


B Transmigration of human neutrophils towards the chemokine IL - 8 through TNFα - stimulated human endothelial cells ( HUVEC ) , transfected with control or SHP2 - specific siRNA and were either not transduced ( - ) or transduced ( + ) with adenoviruses expressing human SHP2 ( Adv - SHP2 ) . The transmigration rate is presented relative to that of cells transfected with control siRNA , set as 100 % . On the right , Western blot analysis of lysates from cells used in transmigration assays for the expression of SHP2 and α - tubulin .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Transmigration of human neutrophils towards the chemokine IL-8 through TNFα-stimulated human endothelial cells (HUVEC), transfected with control or SHP2-specific siRNA and were either not transduced (-) or transduced (+) with adenoviruses expressing human SHP2 (Adv-SHP2). The transmigration rate is presented relative to that of cells transfected with control siRNA, set as 100%. On the right, Western blot analysis of lysates from cells used in transmigration assays for the expression of SHP2 and α-tubulin.


C Lysates of WT or Y731F primary endothelial cells were submitted to pulldowns with GST , GST ‐ SHP2 ‐ WT , or GST ‐ SHP2 ‐ DA . Pulldowns and total cell lysates were analyzed by Western blotting for VE - cadherin and GST . Graph below represents quantification of two independent experiments , presented as fold change of bound VE - cadherin .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Lysates of WT or Y731F primary endothelial cells were submitted to pulldowns with GST, GST‐SHP2‐WT, or GST‐SHP2‐DA. Pulldowns and total cell lysates were analyzed by Western blotting for VE-cadherin and GST. Graph below represents quantification of two independent experiments, presented as fold change of bound VE-cadherin.


TNFα - activated bEnd . 5 cells were incubated with mouse T cells for 0 - 30 min . SHP2 ( above ) or VE - cadherin ( below ) were immunoprecipitated ( IP ) from cell lysates and precipitates as well as total cell lysates were analyzed by immunoblotting for indicated antigens . Molecular weight markers are indicated in kDa . Graph below represents quantification of five independent experiments , presented as fold change of bound VE - cadherin .

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

TNFα-activated bEnd.5 cells were incubated with mouse T cells for 0-30 min. SHP2 (above) or VE-cadherin (below) were immunoprecipitated (IP) from cell lysates and precipitates as well as total cell lysates were analyzed by immunoblotting for indicated antigens. Molecular weight markers are indicated in kDa. Graph below represents quantification of five independent experiments, presented as fold change of bound VE-cadherin.


endothelial cells were stimulated with TNF - α for 17 h prior to adding leukocytes for indicated time points followed by immunoprecipitations and immunoblots of precipitates and endothelial cell lysates ( Iso = isotype control ) . ( A ) bEnd . 5 cells were incubated with antigen - stimulated mouse T cells for 0 - 15 min .

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

endothelial cells were stimulated with TNF-α for 17 h prior to adding leukocytes for indicated time points followed by immunoprecipitations and immunoblots of precipitates and endothelial cell lysates (Iso = isotype control). (A) bEnd.5 cells were incubated with antigen-stimulated mouse T cells for 0-15 min.


endothelial cells were stimulated with TNF - α for 17 h prior to adding leukocytes for indicated time points followed by immunoprecipitations and immunoblots of precipitates and endothelial cell lysates ( Iso = isotype control ) . ( B ) luEnd cells devoid of PECAM - 1 ( KO ) or re - transfected with PECAM - 1 ( RT ) were incubated with T cells for 30 min .

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

endothelial cells were stimulated with TNF-α for 17 h prior to adding leukocytes for indicated time points followed by immunoprecipitations and immunoblots of precipitates and endothelial cell lysates (Iso = isotype control). (B) luEnd cells devoid of PECAM-1 (KO) or re-transfected with PECAM-1 (RT) were incubated with T cells for 30 min.


endothelial cells were stimulated with TNF - α for 17 h prior to adding leukocytes for indicated time points followed by immunoprecipitations and immunoblots of precipitates and endothelial cell lysates ( Iso = isotype control ) . ( C ) HUVEC transfected with control or PECAM - 1 - specific siRNA were incubated with differentiated HL60 cells for 20 min .

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

endothelial cells were stimulated with TNF-α for 17 h prior to adding leukocytes for indicated time points followed by immunoprecipitations and immunoblots of precipitates and endothelial cell lysates (Iso = isotype control). (C) HUVEC transfected with control or PECAM-1-specific siRNA were incubated with differentiated HL60 cells for 20 min.


endothelial cells were stimulated with TNF - α for 17 h prior to adding leukocytes for indicated time points followed by immunoprecipitations and immunoblots of precipitates and endothelial cell lysates ( Iso = isotype control ) . ( D ) bEnd . 5 cells were pre - treated with anti - PECAM - 1 ( Mec13 . 3 ) or anti - endomucin antibodies for 30 min prior to incubation with T cells for 30 min .

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

endothelial cells were stimulated with TNF-α for 17 h prior to adding leukocytes for indicated time points followed by immunoprecipitations and immunoblots of precipitates and endothelial cell lysates (Iso = isotype control). (D) bEnd.5 cells were pre-treated with anti-PECAM-1 (Mec13.3) or anti-endomucin antibodies for 30 min prior to incubation with T cells for 30 min.


endothelial cells were stimulated with TNF - α for 17 h prior to adding leukocytes for indicated time points followed by immunoprecipitations above ) and immunoblots of precipitates and endothelial cell lysates ( Iso = isotype control ) . ( E ) HUVEC , siRNA depleted for endogenous PECAM - 1 , were transduced with wild - type PECAM - 1 ( PECAM - 1 WT ) or Y663 , 686F PECAM - 1 double mutant ( PECAM - 1 YF ) and incubated with HL60 - derived neutrophils for 20 min . On the right , immunoblot analysis of lysates from siRNA treated cells before PECAM - 1 re - expression .

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

endothelial cells were stimulated with TNF-α for 17 h prior to adding leukocytes for indicated time points followed by immunoprecipitations above) and immunoblots of precipitates and endothelial cell lysates (Iso = isotype control). (E) HUVEC, siRNA depleted for endogenous PECAM-1, were transduced with wild-type PECAM-1 (PECAM-1 WT) or Y663,686F PECAM-1 double mutant (PECAM-1 YF) and incubated with HL60-derived neutrophils for 20 min. On the right, immunoblot analysis of lysates from siRNA treated cells before PECAM-1 re-expression.


F Transmigration of human neutrophils towards IL - 8 through TNFα - stimulated HUVEC expressing PECAM - 1 WT or PECAM - 1 - Y663 , 686F . The transmigration rate is presented relative to that of PECAM - 1 WT expressing cells , set as 100 % .

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F Transmigration of human neutrophils towards IL-8 through TNFα-stimulated HUVEC expressing PECAM-1 WT or PECAM-1-Y663,686F. The transmigration rate is presented relative to that of PECAM-1 WT expressing cells, set as 100%.


Endocytosis of VE - cadherin in 17 h TNF - α treated HUVEC was induced by adding HL60 - derived neutrophils for 20 - 30 min at 37 ° C and monitored by the internalization of preincubated mAb BV6 , which was visualized in fixed cells with a secondary fluorescent antibody ( white ) . Total VE - cadherin was stained with another mAb ( red ) , nuclei stained with Hoechst ( white ) . ( A ) HUVEC had been transfected prior to the experiment with control or PECAM - 1 - specific siRNA . Cell lysates were analyzed for expression levels of PECAM - 1 and tubulin ( below ) . Relative VE - cadherin internalization efficiency is given on the right .

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Endocytosis of VE-cadherin in 17 h TNF-α treated HUVEC was induced by adding HL60-derived neutrophils for 20-30 min at 37°C and monitored by the internalization of preincubated mAb BV6, which was visualized in fixed cells with a secondary fluorescent antibody (white). Total VE-cadherin was stained with another mAb (red), nuclei stained with Hoechst (white). (A) HUVEC had been transfected prior to the experiment with control or PECAM-1-specific siRNA. Cell lysates were analyzed for expression levels of PECAM-1 and tubulin (below). Relative VE-cadherin internalization efficiency is given on the right.


Endocytosis of VE - cadherin in 17 h TNF - α treated HUVEC was induced by adding HL60 - derived neutrophils for 20 - 30 min at 37 ° C and monitored by the internalization of preincubated mAb BV6 , which was visualized in fixed cells with a secondary fluorescent antibody ( white ) . Total VE - cadherin was stained with another mAb ( red ) , nuclei stained with Hoechst ( white ) . ( B ) PECAM - 1 siRNA treated HUVEC were transduced with WT PECAM - 1 - EGFP or PECAM - 1 - Y663 , 686F - EGFP . Expression levels of transduced PECAM - 1 forms and of EGFP and VE - cadherin are shown in immunoblots ( on the right ) . Relative VE - cadherin internalization efficiency is given on the right .

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Endocytosis of VE-cadherin in 17 h TNF-α treated HUVEC was induced by adding HL60-derived neutrophils for 20-30 min at 37°C and monitored by the internalization of preincubated mAb BV6, which was visualized in fixed cells with a secondary fluorescent antibody (white). Total VE-cadherin was stained with another mAb (red), nuclei stained with Hoechst (white). (B) PECAM-1 siRNA treated HUVEC were transduced with WT PECAM-1-EGFP or PECAM-1-Y663,686F-EGFP. Expression levels of transduced PECAM-1 forms and of EGFP and VE-cadherin are shown in immunoblots (on the right). Relative VE-cadherin internalization efficiency is given on the right.


A Transmigration of mouse neutrophils towards CXCL - 1 through TNF - α - stimulated bEnd . 5 cells transfected with control or SHP2 - specific siRNA and pre - treated with anti - endomucin ( 7C7 . 1 ) or anti - PECAM - 1 ( Mec13 . 3 ) antibodies for 30 min at 37 ° C before addition of neutrophils . The transmigration rate is presented relative to that of control cells , set as 100 % .

A Transmigration of mouse neutrophils towards CXCL-1 through TNF-α-stimulated bEnd.5 cells transfected with control or SHP2-specific siRNA and pre-treated with anti-endomucin (7C7.1) or anti-PECAM-1 (Mec13.3) antibodies for 30 min at 37°C before addition of neutrophils. The transmigration rate is presented relative to that of control cells, set as 100%.


B Transmigration of mouse neutrophils towards CXCL - 1 through TNF - α - stimulated WT or Y731F primary mouse endothelial cells pre - treated with isotype - control or anti - PECAM - 1 ( 2H8 ) antibodies for 30 min at 37 ° C prior to addition of neutrophils . The transmigration rate is presented relative to that of control cells , set as 100 % .

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B Transmigration of mouse neutrophils towards CXCL-1 through TNF-α-stimulated WT or Y731F primary mouse endothelial cells pre-treated with isotype-control or anti-PECAM-1 (2H8) antibodies for 30 min at 37°C prior to addition of neutrophils. The transmigration rate is presented relative to that of control cells, set as 100%.


Extravasated leukocytes ( C ) in cremaster tissue from WT or Y731F VE - cadherin mice stimulated intrascrotally with IL - 1β and treated i . v . with isotype - control or anti - PECAM - 1 ( 2H8 ) antibodies for 4 h before intravital microscopy .

Extravasated leukocytes (C) in cremaster tissue from WT or Y731F VE-cadherin mice stimulated intrascrotally with IL-1β and treated i.v. with isotype-control or anti-PECAM-1 (2H8) antibodies for 4 h before intravital microscopy.


adherent leukocytes ( D ) in cremaster tissue from WT or Y731F VE - cadherin mice stimulated intrascrotally with IL - 1β and treated i . v . with isotype - control or anti - PECAM - 1 ( 2H8 ) antibodies for 4 h before intravital microscopy .

adherent leukocytes (D) in cremaster tissue from WT or Y731F VE-cadherin mice stimulated intrascrotally with IL-1β and treated i.v. with isotype-control or anti-PECAM-1 (2H8) antibodies for 4 h before intravital microscopy.


( D ) and rolling flux fraction of leukocytes ( E ) in cremaster tissue from WT or Y731F VE - cadherin mice stimulated intrascrotally with IL - 1β and treated i . v . with isotype - control or anti - PECAM - 1 ( 2H8 ) antibodies for 4 h before intravital microscopy .

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(D) and rolling flux fraction of leukocytes (E) in cremaster tissue from WT or Y731F VE-cadherin mice stimulated intrascrotally with IL-1β and treated i.v. with isotype-control or anti-PECAM-1 (2H8) antibodies for 4 h before intravital microscopy.


A HUVEC were transduced with increasing titers of adenovirus expressing human SHP2 . VE - cadherin was immunoprecipitated from cell lysates and precipitates as well as total cell lysates were analyzed by immunoblots for indicated antigens .

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A HUVEC were transduced with increasing titers of adenovirus expressing human SHP2. VE-cadherin was immunoprecipitated from cell lysates and precipitates as well as total cell lysates were analyzed by immunoblots for indicated antigens.


B bEnd . 3 cells were lysed and VE - cadherin or immature VE - cadherin was immunoprecipitated respectively with C5 or VD47 antibodies . Precipitates were blotted against pY731 - VE - cadherin , β - catenin , plakoglobin , p120 , α - catenin and VE - cadherin .

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B bEnd.3 cells were lysed and VE-cadherin or immature VE-cadherin was immunoprecipitated respectively with C5 or VD47 antibodies. Precipitates were blotted against pY731-VE-cadherin, β -catenin, plakoglobin, p120, α -catenin and VE-cadherin.


C VE - cadherin - catenin complex was immunoprecipitated from HUVEC lysates and was either left untreated ( no SDS ) or treated with 0 . 2 % SDS for 30 min at room temperature , followed by removal of SDS and addition of 2 μg recombinant human SHP2 for 30 min at 30 ° C . Precipitates were analyzed in immunoblots for pY731 - VE - cadherin , plakoglobin , β - catenin and VE - cadherin . D Quantification of pY731 blot signals of three independent experiments ( from C ) , with the signal intensity for untreated VE - cadherin set as 1 . Molecular weight markers are indicated in kDa .

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C VE-cadherin-catenin complex was immunoprecipitated from HUVEC lysates and was either left untreated (no SDS) or treated with 0.2% SDS for 30 min at room temperature, followed by removal of SDS and addition of 2 μg recombinant human SHP2 for 30 min at 30°C. Precipitates were analyzed in immunoblots for pY731-VE-cadherin, plakoglobin, β-catenin and VE-cadherin. D Quantification of pY731 blot signals of three independent experiments (from C), with the signal intensity for untreated VE-cadherin set as 1. Molecular weight markers are indicated in kDa.


A Transmigration of mouse neutrophils towards the chemokine CXCL - 1 through TNFα - stimulated WT or Y731F primary endothelial cells , either pre - treated with vehicle control ( DMSO ) or Ca2 + chelator ( MAPTAM ) for 30 min at 37 ° C prior to addition of neutrophils . The transmigration rate is presented relative to DMSO - treated endothelial cells expressing WT VE - cadherin .

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A Transmigration of mouse neutrophils towards the chemokine CXCL-1 through TNFα-stimulated WT or Y731F primary endothelial cells, either pre-treated with vehicle control (DMSO) or Ca2+ chelator (MAPTAM) for 30 min at 37°C prior to addition of neutrophils. The transmigration rate is presented relative to DMSO-treated endothelial cells expressing WT VE-cadherin.


B TNFα - activated bEnd . 5 cells were treated with DMSO or MAPTAM , prior to incubation with antigen - stimulated mouse T cells for 30 min . VE - cadherin was immunoprecipitated from cell lysates and precipitates as well as total cell lysates were analyzed by immunoblots for indicated antigens . Quantification of pY731 blot signals of five independent experiments are shown below , with the signal intensity for the control sample ( no T cells , vehicle - control ) set as 100 % .

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B TNFα-activated bEnd.5 cells were treated with DMSO or MAPTAM, prior to incubation with antigen-stimulated mouse T cells for 30 min. VE-cadherin was immunoprecipitated from cell lysates and precipitates as well as total cell lysates were analyzed by immunoblots for indicated antigens. Quantification of pY731 blot signals of five independent experiments are shown below, with the signal intensity for the control sample (no T cells, vehicle-control) set as 100%.


A Transmigration of mouse neutrophils towards the chemokine CXCL - 1 through TNFα - stimulated WT or Y731F primary endothelial cells , either pre - treated with vehicle control ( DMSO ) or Ca2 + chelator for 30 min at 37 ° C prior to addition of neutrophils . The transmigration rate is presented relative to DMSO - treated endothelial cells expressing WT VE - cadherin . C Transmigration assays treatment with Blebbistatin .

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A Transmigration of mouse neutrophils towards the chemokine CXCL-1 through TNFα-stimulated WT or Y731F primary endothelial cells, either pre-treated with vehicle control (DMSO) or Ca2+ chelator for 30 min at 37°C prior to addition of neutrophils. The transmigration rate is presented relative to DMSO-treated endothelial cells expressing WT VE-cadherin. C Transmigration assays treatment with Blebbistatin.


B TNFα - activated bEnd . 5 cells were treated with DMSO prior to incubation with antigen - stimulated mouse T cells for 30 min . VE - cadherin was immunoprecipitated from cell lysates and precipitates as well as total cell lysates were analyzed by immunoblots for indicated antigens . Quantification of pY731 blot signals of five independent experiments are shown below , with the signal intensity for the control sample ( no T cells , vehicle - control ) set as 100 % . treatment with Blebbistatin .

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B TNFα-activated bEnd.5 cells were treated with DMSO prior to incubation with antigen-stimulated mouse T cells for 30 min. VE-cadherin was immunoprecipitated from cell lysates and precipitates as well as total cell lysates were analyzed by immunoblots for indicated antigens. Quantification of pY731 blot signals of five independent experiments are shown below, with the signal intensity for the control sample (no T cells, vehicle-control) set as 100%. treatment with Blebbistatin.


E HUVEC transduced with lacZ or with SHP2 were either untreated ( - ) or treated ( + ) with thrombin for 30 min followed by immunoblotting either VE - cadherin immunoprecipitates or cell lysates for the indicated antigens on the right .

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E HUVEC transduced with lacZ or with SHP2 were either untreated (-) or treated (+) with thrombin for 30 min followed by immunoblotting either VE-cadherin immunoprecipitates or cell lysates for the indicated antigens on the right.


HUVEC were transfected with control or VE - cadherin - specific siRNA and transduced with VE - cad FL or VE - cad FL - TS . Cell lysates were immunoblotted for VE - cadherin and actin .

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HUVEC were transfected with control or VE-cadherin-specific siRNA and transduced with VE-cad FL or VE-cad FL-TS. Cell lysates were immunoblotted for VE-cadherin and actin.


C Immunofluorescence staining for VE - cadherin of HUVEC transfected with control or VE ‑ cadherin specific siRNA ( VE - cad kd ) and transduced with VE - cad FL or VE - cad FL - TS . D HUVEC were either transfected with control or VE - cadherin targeting siRNA and transduced with VE ‑ cad FL or VE - cad FL ‑ TS . The tension sensor was immunoprecipitated from cell lysates using an anti - GFP antibody . Precipitates as well as total cell lysates were analyzed by immunoblotting for indicated antigens . Molecular weight markers are indicated in kDa . E , Quantification of FRET efficiency ( percentage ) in junctions of HUVEC expressing VE - cad FL or VE - cad FL - TS , after thrombin stimulation ( 1 U / ml ) or under control conditions .

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C Immunofluorescence staining for VE-cadherin of HUVEC transfected with control or VE‑cadherin specific siRNA (VE-cad kd) and transduced with VE-cad FL or VE-cad FL-TS. D HUVEC were either transfected with control or VE-cadherin targeting siRNA and transduced with VE‑cad FL or VE-cad FL‑TS. The tension sensor was immunoprecipitated from cell lysates using an anti-GFP antibody. Precipitates as well as total cell lysates were analyzed by immunoblotting for indicated antigens. Molecular weight markers are indicated in kDa. E, Quantification of FRET efficiency (percentage) in junctions of HUVEC expressing VE-cad FL or VE-cad FL-TS, after thrombin stimulation (1 U/ml) or under control conditions.


A - C Quantification of FRET efficiency ( percentage ) in junctions of HUVEC expressing VE - cad FL ( A , B ) or VE - cad FL - TS ( C ) . HUVEC were stimulated 4h with TNFα prior to adding IL - 8 for 4 min , followed by flow with buffer alone ( A ) or together with PMNs ( B , C ) . The connected data points represent FRET efficiency of same junctions compared before and after addition of flow / PMNs .

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A-C Quantification of FRET efficiency (percentage) in junctions of HUVEC expressing VE-cad FL (A, B) or VE-cad FL-TS (C). HUVEC were stimulated 4h with TNFα prior to adding IL-8 for 4 min, followed by flow with buffer alone (A) or together with PMNs (B, C). The connected data points represent FRET efficiency of same junctions compared before and after addition of flow/PMNs.


D Quantification of FRET efficiency ( percentage ) in junctions of HUVEC expressing VE - cad FL or VE - cad FL - TS . Cells were either exposed to flow and PMNs or to flow alone , fixed using 4 % PFA and washed with PBS . FLIM measurements were performed at sites of transmigration ( PMNs ) or at junctions without PMNs ( Flow ) .

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D Quantification of FRET efficiency (percentage) in junctions of HUVEC expressing VE-cad FL or VE-cad FL-TS. Cells were either exposed to flow and PMNs or to flow alone, fixed using 4 % PFA and washed with PBS. FLIM measurements were performed at sites of transmigration (PMNs) or at junctions without PMNs (Flow).


E Representative images of HUVEC expressing VE - cad FL at a site of PMN transmigration ( left micrographs ) or without PMN ( right micrographs ) . Maximum intensity projection of a Z - stack of YPet fluorescence ( VE - cadherin in green ) and CellTracker DeepRed ( PMN in red ) is shown for the upper micrographs . The part of the PMN above the HUVEC monolayer is encircled in yellow and the part underneath in blue . The amplitude averaged lifetime of YPet per pixel ( FLIM measurement ) of the same cells is shown in the bottom micrographs .

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E Representative images of HUVEC expressing VE-cad FL at a site of PMN transmigration (left micrographs) or without PMN (right micrographs). Maximum intensity projection of a Z-stack of YPet fluorescence (VE-cadherin in green) and CellTracker DeepRed (PMN in red) is shown for the upper micrographs. The part of the PMN above the HUVEC monolayer is encircled in yellow and the part underneath in blue. The amplitude averaged lifetime of YPet per pixel (FLIM measurement) of the same cells is shown in the bottom micrographs.