( b ) HeLa transfected for 24 h with GFP - Atg16L1 were incubated for 15 min with horseradish peroxidase ( HRP ) - conjugated cholera toxin subunit B at 4 ° C . were then incubated for 10 min at 37 ° C , after which they were processed for double immunogold labelling with anti - HRP antibodies ( 15 - nm gold particles ) anti - GFP antibodies ( 10 - nm gold particles ) for electron microscopy . PM , plasma . Scale bar , 100 nm . Co - localization can be seen in boxed areas . n = 49 fields . error bars represent s . e . m .

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(b) HeLa cells transfected for 24 h with GFP-Atg16L1 were incubated for 15 min with horseradish peroxidase (HRP)-conjugated cholera toxin subunit B at 4 °C. The cells were then incubated for 10 min at 37 °C, after which they were processed for double immunogold labelling with anti-HRP antibodies (15-nm gold particles) and anti-GFP antibodies (10-nm gold particles) for electron microscopy. PM, plasma membrane. Scale bar, 100 nm. Co-localization can be seen in boxed areas. n = 49 fields. All error bars represent s.e.m.


( a ) HeLa transfected for 24 h with GFP - Atg16L1 ( green ) were incubated with CellMask Orange plasma stain for 5 min at 37 ° C , after which they were imaged immediately ( in an incubated chamber at 37 ° C ) . A time series after fusion of a CellMask Orange vesicle ( red ) with a GFP - Atg16L1 vesicle ( green ) is shown . Scale bar , 5 μm . A higher - magnification image showing co - localization ( yellow ) of GFP - Atg16L1 vesicle ( green ) with CellMask Orange - positive vesicle ( red ) , indicated by arrows , is also shown in top bottom panels .

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(a) HeLa cells transfected for 24 h with GFP-Atg16L1 (green) were incubated with CellMask Orange plasma membrane stain for 5 min at 37 °C, after which they were imaged immediately (in an incubated chamber at 37 °C). A time series after fusion of a CellMask Orange vesicle (red) with a GFP-Atg16L1 vesicle (green) is shown. Scale bar, 5 μm. A higher-magnification image showing co-localization (yellow) of GFP-Atg16L1 vesicle (green) with CellMask Orange-positive vesicle (red), indicated by arrows, is also shown in the top and bottom panels.


( b , c ) HeLa transfected with control , clathrin heavy chain , AP1 or AP2 siRNA for 72 h were subsequently transfected with GFP - Atg16L1 ( green ) together with siRNA for next 24 h . were then incubated for 15 min with Alexa Fluor - 555 - conjugated cholera toxin subunit B ( red ) at 4 ° C , followed by further incubation for 10 min at 37 ° C , after which were fixed for confocal ( b ) . co - localization ( yellow ) of GFP - Atg16L1 ( green ) with cholera toxin ( red ) in control or clathrin heavy - chain knockdown is shown is quantified in graphs in c . n = 25 . Three asterisks , P = 0 . 0002 for clathrin heavy - chain knockdown , P 0 . 0001 for AP2 knockdown . error bars represent s . e . m . Scale bar , 5 μm .

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(b, c) HeLa cells transfected with control, clathrin heavy chain, AP1 or AP2 siRNA for 72 h were subsequently transfected with GFP-Atg16L1 (green) together with the siRNA for the next 24 h. The cells were then incubated for 15 min with Alexa Fluor-555-conjugated cholera toxin subunit B (red) at 4 °C, followed by further incubation for 10 min at 37 °C, after which the cells were fixed for confocal analysis (b). The co-localization (yellow) of GFP-Atg16L1 (green) with cholera toxin (red) in control or clathrin heavy-chain knockdown is shown and is quantified in the graphs in c. n = 25 cells. Three asterisks, P = 0.0002 for clathrin heavy-chain knockdown, and P 0.0001 for AP2 knockdown. All error bars represent s.e.m. Scale bar, 5 μm.


( a ) HeLa transfected with two rounds of control , clathrin heavy chain or AP2 siRNA for 4 days were collected for immunoprecipitation ( IP ) with anti - Atg16L1 antibody . Western blot for total lysate ( bottom ) IP ( top ) were performed with anti - Atg16L1 anti - clathrin antibodies . Note decrease in Atg16L1 - clathrin interaction with AP2 knockdown . graph shows of Atg16L1 - clathrin or Atg16L1 - AP2 with knockdown of control , clathrin heavy chain or AP2 from two independent experiments .

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(a) HeLa cells transfected with two rounds of control, clathrin heavy chain or AP2 siRNA for 4 days were collected for immunoprecipitation (IP) with anti-Atg16L1 antibody. Western blot for total lysate (bottom) and IP (top) were performed with anti-Atg16L1 and anti-clathrin antibodies. Note the decrease in Atg16L1-clathrin interaction with AP2 knockdown. The graph shows quantification of the Atg16L1-clathrin or Atg16L1-AP2 with knockdown of control, clathrin heavy chain or AP2 from two independent experiments.


( b ) HeLa transfected for 24 h with Flag - tagged wild - type Atg16L1 , GFP - PLC ( PH ) together with empty vector ( control ) or dominant - negative dynamin II mutant ( K44A dynamin ; DN - Dyn ) were fixed analysed by confocal microscopy . Note co - localization of Atg16L1 with PLC ( PH ) at plasma ; this is quantified in graph at right . Asterisk , P 0 . 01 . n = 20 . Scale bars , 5 μm . DAPI , 4 ′ , 6 - diamidino - 2 - phenylindole .

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(b) HeLa cells transfected for 24 h with Flag-tagged wild-type Atg16L1, GFP-PLC(PH) together with empty vector (control) or dominant-negative dynamin II mutant (K44A dynamin; DN-Dyn) were fixed and analysed by confocal microscopy. Note the co-localization of Atg16L1 with PLC(PH) at the plasma membrane; this is quantified in the graph at the right. Asterisk, P 0.01. n = 20 cells. Scale bars, 5 μm. DAPI, 4′,6-diamidino-2-phenylindole.


( c ) HeLa treated for 5 h with HBSS alone or with HBSS containing 80 μM dynasore were collected for immunoprecipitation ( IP ) with anti - Atg16L1 antibody . Western blot analyses for total lysate ( TL ) IP were performed with anti - Atg16L1 anti - clathrin antibodies . Note strong Atg16L1 - clathrin interaction with dynasore treatment . graph shows from two independent experiments . error bars represent s . e . m .

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(c) HeLa cells treated for 5 h with HBSS alone or with HBSS containing 80 μM dynasore were collected for immunoprecipitation (IP) with anti-Atg16L1 antibody. Western blot analyses for total lysate (TL) and IP were performed with anti-Atg16L1 and anti-clathrin antibodies. Note the strong Atg16L1-clathrin interaction with dynasore treatment. The graph shows quantification from two independent experiments. All error bars represent s.e.m.


( b ) stably expressing WT or D620N GFP - VPS35 or otherwise transiently transfected with a panel of GFP - VPS35 constructs were lysed respective GFP - tagged recovered by immunoprecipitation ( IP ) .

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(b) Cells stably expressing WT or D620N GFP-VPS35 or otherwise transiently transfected with a panel of GFP-VPS35 constructs were lysed and the respective GFP-tagged protein recovered by immunoprecipitation (IP).


( c ) Untransfected HeLa or stably expressing VPS29 - GFP , GFP - VPS35 wild - type or GFP - VPS35 D620N were lysed lysates incubated with anti - VPS26 to IP retromer CSC .

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(c) Untransfected HeLa cells or cells stably expressing VPS29-GFP, GFP-VPS35 wild-type or GFP-VPS35 D620N were lysed and the lysates incubated with anti-VPS26 to IP the retromer CSC.


( d ) stably expressing VPS29 - GFP V90D mutant that cannot assemble with VPS35 were transiently transfected with mCherry - tagged WT VPS35 , D620N H675R constructs , along with an additional control of mCherry - FAM21 tail . were lysed 48 h post transfection , VPS29 - GFP V90D associated were recovered by anti - GFP native IP . Blots shown are representative of experiments replicated at least twice .

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(d) Cells stably expressing the VPS29-GFP V90D mutant that cannot assemble with VPS35 were transiently transfected with mCherry-tagged WT VPS35, D620N and H675R constructs, along with an additional control of mCherry-FAM21 tail. Cells were lysed 48 h post transfection, and VPS29-GFP V90D and associated proteins were recovered by anti-GFP native IP. Blots shown are representative of experiments replicated at least twice.


a ) expressing either WT GFP - VPS35 or D620N mutant were treated with siRNA to abolish expression of endogenous VPS35 . Each dish of was lysed in either HEPES lysis buffer ( H ) or PBS lysis buffer ( P ) . Following centrifugation , equal portions of each lysate were combined to generate a mixed lysate ( H / P ) . Each lysate was incubated with anti - GFP to recover respective GFP - tagged VPS35 .

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a) Cells expressing either WT GFP-VPS35 or the D620N mutant were treated with siRNA to abolish expression of endogenous VPS35. Each dish of cells was lysed in either HEPES lysis buffer (H) or PBS lysis buffer (P). Following centrifugation, equal portions of each lysate were combined to generate a mixed lysate (H/P). Each lysate was incubated with anti-GFP to recover the respective GFP-tagged VPS35 protein.


( b ) Similar to ( a ) , but only expressing GFP - VPS35 wild type were analysed , either mock treated or treated with siRNA to silence FKBP15 expression .

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(b) Similar to (a), but only cells expressing GFP-VPS35 wild type were analysed, either mock treated or treated with siRNA to silence FKBP15 expression.


( c ) stably expressing WT or D620N GFP - VPS35 were treated with indicated siRNAs , lysed immunoprecipitated as in ( a ) .

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(c) Cells stably expressing WT or D620N GFP-VPS35 were treated with indicated siRNAs, lysed and immunoprecipitated as in (a).


( a , b ) expressing either WT or D620N GFP - VPS35 were treated with siRNA to silence expression of endogenous VPS35 , fixed labelled for GFP FKBP15 ( a ) or FAM21 ( b ) . were imaged by microscopy . Scale bar , 20 μm .

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(a,b) Cells expressing either WT or D620N GFP-VPS35 were treated with siRNA to silence expression of endogenous VPS35, fixed and labelled for GFP and FKBP15 (a) or FAM21 (b). Cells were imaged by fluorescence microscopy. Scale bar, 20 μm.


( c ) stably expressing WT or D620N GFP - tagged VPS35 were treated with siRNA to abolish expression of endogenous VPS35 subsequently fixed labelled for GFP either VPS26 , FKBP15 or FAM21 . were imaged using Cellomics automated microscopy . Approximately 250 per well in four wells were analysed for each . data for FKBP15 FAM21 spot were normalized to GFP - VPS35 VPS26 signals . P0 . 0002 for both FKBP15 FAM21 spot in D620N compared with WT . Error bars indicate s . d .

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(c) Cells stably expressing WT or D620N GFP-tagged VPS35 were treated with siRNA to abolish expression of endogenous VPS35 and subsequently fixed and labelled for GFP and either VPS26, FKBP15 or FAM21. The cells were imaged using Cellomics automated fluorescence microscopy. Approximately 250 cells per well in four wells were analysed for each cell line. The data for FKBP15 and FAM21 spot intensity were normalized to GFP-VPS35 and VPS26 signals. P0.0002 for both FKBP15 and FAM21 spot intensity in D620N compared with WT. Error bars indicate s.d.


( d ) stably expressing either WT GFP - VPS35 or D620N mutant were treated with siRNA to silence expression of endogenous VPS35 . were permeabilized by flash freezing followed by rapid thawing centrifuged to separate supernatant ( S ) pellet ( P ) fractions .

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(d) Cells stably expressing either WT GFP-VPS35 or the D620N mutant were treated with siRNA to silence the expression of endogenous VPS35. Cells were permeabilized by flash freezing followed by rapid thawing and centrifuged to separate supernatant (S) and membrane pellet (P) fractions.


( e ) graph shows of - associated FKBP15 strumpellin is mean of three experiments . error bars indicate s . e . m . A representative blot of three experiments is shown , indicating efficacy of knockdown of endogenous VPS35 also further showing that such as CIMPR are detected only in pellet fraction .

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(e) The graph shows the percentage of membrane-associated FKBP15 and strumpellin and is the mean of three experiments. The error bars indicate s.e.m. A representative blot of three experiments is shown, indicating the efficacy of the knockdown of endogenous VPS35 and also further showing that membrane proteins such as the CIMPR are detected only in the pellet fraction.


a ) HeLa stably expressing GFP - VPS35 WT D620N were treated with bafilomycin A1 or DMSO vehicle control . Endogenous LC3 - II tubulin were examined by western blot . A representative experiment of six experiments is shown . ( b ) of representative experiment in triplicate shown in a , in which endogenous LC3 - II are normalized to tubulin expressed as a of in WT . * * * P = 7 . 86 × 10 − 6 ( DMSO ) 3 . 81 × 10 − 5 ( Baf ) by 2 - Student ' s t - .

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a) HeLa cells stably expressing GFP-VPS35 WT and D620N were treated with bafilomycin A1 or DMSO vehicle control. Endogenous LC3-II and tubulin levels were examined by western blot. A representative experiment of six experiments is shown. (b) Quantification of the representative experiment in triplicate shown in a, in which endogenous LC3-II levels are normalized to tubulin and expressed as a ratio of levels in WT. ***P=7.86 × 10−6 (DMSO) and 3.81 × 10−5 (Baf) by 2-tailed Student's t-test.


c ) HeLa stably expressing GFP - VPS35 WT D620N were transfected with mRFP - LC3 . number of LC3 vesicles was quantified by Cellomics automated microscopy . A representative experiment of three independent experiments is shown , with 344 ( WT ) 401 ( D620N ) analysed . * * * P0 . 0001 by 2 - Student ' s t - .

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c) HeLa cells stably expressing GFP-VPS35 WT and D620N were transfected with mRFP-LC3. The number of LC3 vesicles was quantified by Cellomics automated fluorescence microscopy. A representative experiment of three independent experiments is shown, with 344 (WT) and 401 (D620N) cells analysed. ***P0.0001 by 2-tailed Student's t-test.


( d ) GFP - VPS35 WT D620N - expressing were transfected with HA - Q74 immunostained for HA . of transfected with was counted by a blinded experimenter . shows mean of three experiments in triplicate with minimum 200 per replicate . * * * P = 0 . 00086 by 1 - Student ' s t - .

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(d) GFP-VPS35 WT and D620N-expressing cells were transfected with HA-Q74 and immunostained for HA. The percentage of transfected cells with aggregates was counted by a blinded experimenter. The quantification shows the mean of three experiments in triplicate with minimum 200 cells per replicate. ***P=0.00086 by 1-tailed Student's t-test.


( e ) Confocal representative of experiment described in d . Scale bar , 20 μm .

(e) Confocal images representative of the experiment described in d. Scale bar, 20 μm.


( f ) stably expressing WT D620N GFP - VPS35 were transfected with GFP - α - synuclein A53T GFP for 48 h analyzed by western blotting .

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(f) Cells stably expressing WT and D620N GFP-VPS35 were transfected with GFP-α-synuclein A53T and GFP for 48 h and analyzed by western blotting.


g ) of representative experiment in triplicate in f , in which level of α - synuclein was expressed as a to GFP . A representative experiment of two independent experiments is shown . * * P = 0 . 0047 by 2 - Student ' s t - .

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g) Quantification of the representative experiment in triplicate in f, in which the level of α-synuclein was expressed as a ratio to GFP. A representative experiment of two independent experiments is shown. **P=0.0047 by 2-tailed Student's t-test.


( h ) VPS35 was knocked down with two siRNA nucleotides in HeLa , were treated with bafilomycin A1 lysed as in a . A representative experiment is shown in

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(h) VPS35 was knocked down with two individual siRNA nucleotides in HeLa cells, and cells were treated with bafilomycin A1 and lysed as in a. A representative experiment is shown in triplicate


( i ) of three independent experiments in triplicate . * P = 0 . 026 ; other results non - significant by 2 - Student ' s t - .

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(i) Quantification of three independent experiments in triplicate. *P=0.026; other results non-significant by 2-tailed Student's t-test.


( j ) of CSC , including VPS35 , VPS26 VPS29 ) , were assessed upon VPS35 knockdown , confirming previous results that knockdown of one component destabilizes CSC . error bars indicate s . e . m .

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(j) Protein levels of CSC, including VPS35, VPS26 and VPS29), were assessed upon VPS35 knockdown, confirming previous results that knockdown of one component destabilizes the CSC. All error bars indicate s.e.m.


( a ) HeLa were transfected with pEGFP vector or GFP - FAM21 tail for 48 h subsequently treated with bafilomycin A1 as in Fig . 4 . Endogenous LC3 - II tubulin were assessed by western blot . ( b ) of representative experiment in triplicate shown in a , of two independent experiments . * P = 0 . 02 ( DMSO ) 0 . 08 ( Baf ) by 2 - Student ' s t - .

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(a) HeLa cells were transfected with pEGFP vector or GFP-FAM21 tail for 48 h and subsequently treated with bafilomycin A1 as in Fig. 4. Endogenous LC3-II and tubulin levels were assessed by western blot. (b) Quantification of the representative experiment in triplicate shown in a, of two independent experiments. *P=0.02 (DMSO) and 0.08 (Baf) by 2-tailed Student's t-test.


( c ) HeLa stably expressing GFP - VPS35 WT D620N were transfected with pEGFP vector or GFP - FAM21 , subsequently treated with bafilomycin A1 , lysed subjected to western blot as in a . ( d ) of representative experiment in triplicate shown in c , of two independent experiments . * * P = 0 . 008 by 2 - Student ' s t - .

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(c) HeLa cells stably expressing GFP-VPS35 WT and D620N were transfected with pEGFP vector or GFP-FAM21, and subsequently treated with bafilomycin A1, lysed and subjected to western blot as in a. (d) Quantification of the representative experiment in triplicate shown in c, of two independent experiments. **P=0.008 by 2-tailed Student's t-test.


( e ) were depleted of WASH1 , treated with bafilomycin A1 examined for endogenous LC3 - II , tubulin , WASH1 GAPDH . A representative experiment of six independent experiments is shown . ( f ) of representative experiment shown in e . * * P = 0 . 002 ( DMSO ) P = 0 . 0028 ( Baf ) by 2 - Student ' s t - .

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(e) Cells were depleted of WASH1, treated with bafilomycin A1 and examined for endogenous LC3-II, tubulin, WASH1 and GAPDH levels. A representative experiment of six independent experiments is shown. (f) Quantification of the representative experiment shown in e. **P=0.002 (DMSO) and P=0.0028 (Baf) by 2-tailed Student's t-test.


g ) HeLa depleted of WASH1 by siRNA were transfected with GFP - LC3 for 24 h . number of LC3 vesicles was quantified by Cellomics automated microscopy . A representative experiment of two independent experiments is shown , with 757 ( control ) 695 ( knockdown ) analysed . * * * P0 . 0001 by 2 - Student ' s t - .

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g) HeLa cells depleted of WASH1 by siRNA were transfected with GFP-LC3 for 24 h. The number of LC3 vesicles was quantified by Cellomics automated fluorescence microscopy. A representative experiment of two independent experiments is shown, with 757 (control) and 695 (knockdown) cells analysed. ***P0.0001 by 2-tailed Student's t-test.


( h ) HeLa depleted of FKBP15 were transfected with GFP - LC3 vesicles were quantified as in g . A representative experiment of two independent experiments in triplicate is shown , with 579 ( control ) 815 ( knockdown ) analysed in total .

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(h) HeLa cells depleted of FKBP15 were transfected with GFP-LC3 and vesicles were quantified as in g. A representative experiment of two independent experiments in triplicate is shown, with 579 (control) and 815 (knockdown) cells analysed in total.


( i ) HeLa depleted of FKBP15 were subsequently transfected with a GFP - Q74 construct for 24 h fixed in PFA . of transfected with was counted by a blinded experimenter . shows mean of two experiments , each in triplicate with min 220 counted per

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(i) HeLa cells depleted of FKBP15 were subsequently transfected with a GFP-Q74 construct for 24 h and fixed in PFA. The percentage of transfected cells with aggregates was counted by a blinded experimenter. The quantification shows the mean of two experiments, each in triplicate with min 220 cells counted per replicate


( j ) FKBP15 was depleted in HeLa as in e . A representative blot from three independent experiments in triplicate is shown . Error bars indicate s . e . m .

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(j) FKBP15 was depleted in HeLa cells as in e. A representative blot from three independent experiments in triplicate is shown. Error bars indicate s.e.m.


( a ) HeLa were immunostained for endogenous ATG9A VPS35 subjected to confocal microscopy . Magnified areas are shown on right of pictures . ( b ) HeLa were transfected with ATG9A - GFP for 24 h , subsequently fixed , immunostained for endogenous WASH1 subjected to confocal microscopy . ( c ) HeLa were transfected with ATG9A - GFP as in b , but immunostained instead for endogenous FAM21 .

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(a) HeLa cells were immunostained for endogenous ATG9A and VPS35 and subjected to confocal microscopy. Magnified areas are shown on the right of the pictures. (b) HeLa cells were transfected with ATG9A-GFP for 24 h, and subsequently fixed, immunostained for endogenous WASH1 and subjected to confocal microscopy. (c) HeLa cells were transfected with ATG9A-GFP as in b, but immunostained instead for endogenous FAM21.


( d ) HeLa stably expressing GFP - VPS35 WT D620N were depleted of endogenous VPS35 using 40 nM of siRNA , subsequently immunostained for TGN46 endogenous ATG9A subjected to confocal microscopy . ( e ) Colocalization between TGN ATG9A is expressed in terms of Pearson ' s Coefficient . n = 25 ( WT ) 33 ( D620N ) . Error bars represent s . e . m . * * P = 0 . 01 by 2 - Student ' s t - . Scale bars in ( a - d ) , 10 μm .

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(d) HeLa cells stably expressing GFP-VPS35 WT and D620N were depleted of endogenous VPS35 using 40 nM of siRNA, and subsequently immunostained for TGN46 and endogenous ATG9A and subjected to confocal microscopy. (e) Colocalization between TGN and ATG9A is expressed in terms of the Pearson's Coefficient. n=25 cells (WT) and 33 cells (D620N). Error bars represent s.e.m. and **P=0.01 by 2-tailed Student's t-test. Scale bars in (a-d), 10 μm.


( a ) HeLa depleted of WASH1 with two successive siRNA treatments were either starved in Hank ' s balanced salt solution for 1 h or kept in full medium . Following fixation , were immunostained for TGN46 , ATG9A VPS35 subjected to confocal microscopy . Representative pictures with magnified areas are shown . Scale bar , 10 μm . ( b ) Colocalization between TGN ATG9A is expressed in terms of Pearson ' s coefficient . Number of analysed : 38 ( control basal ) , 46 ( knockdown basal ) , 36 ( control starved ) , 53 ( knockdown starved ) . Error bars represent s . e . m . * * * P = 0 . 0005 ( basal ) P = 0 . 0004 ( starved ) by 2 - Student ' s t - .

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(a) HeLa cells depleted of WASH1 with two successive siRNA treatments were either starved in Hank's balanced salt solution for 1 h or kept in full medium. Following fixation, cells were immunostained for TGN46, ATG9A and VPS35 and subjected to confocal microscopy. Representative pictures with magnified areas are shown. Scale bar, 10 μm. (b) Colocalization between TGN and ATG9A is expressed in terms of the Pearson's coefficient. Number of cells analysed: 38 (control basal), 46 (knockdown basal), 36 (control starved), 53 (knockdown starved). Error bars represent s.e.m. and ***P=0.0005 (basal) and P=0.0004 (starved) by 2-tailed Student's t-test.


( a ) HeLa stably expressing GFP - VPS35 WT D620N were transfected with mRFP - LC3 for 24 h , immunostained for endogenous ATG9A , imaged by confocal microscopy .

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(a) HeLa cells stably expressing GFP-VPS35 WT and D620N were transfected with mRFP-LC3 for 24 h, immunostained for endogenous ATG9A, and imaged by confocal microscopy.


( b ) Colocalization is expressed in terms of Mander ' s coefficient M1 to indicate proportion of LC3 intensities that also contain ATG9A intensities . A representative experiment of three independent experiments is shown , in which at least 33 were analysed per condition . Error bars indicate s . e . m . , * * * P0 . 001 by 2 - Student ' s t - .

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(b) Colocalization is expressed in terms of Mander's coefficient M1 to indicate the proportion of LC3 intensities that also contain ATG9A intensities. A representative experiment of three independent experiments is shown, in which at least 33 cells were analysed per condition. Error bars indicate s.e.m., and ***P0.001 by 2-tailed Student's t-test.


( c ) HeLa depleted of WASH1 were transfected with GFP - LC3 for 24 h , immunostained for endogenous ATG9A , imaged by confocal microscopy .

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(c) HeLa cells depleted of WASH1 were transfected with GFP-LC3 for 24 h, immunostained for endogenous ATG9A, and imaged by confocal microscopy.


( d ) Colocalization expressed as M1 , as in b . A representative experiment of two independent experiments is shown , in which at least 24 were analysed per condition . Error bars indicate s . e . m . , * * * P0 . 001 by 2 - Student ' s t - . Scale bars in ( a , c ) , 10 μm . In b , d , colocalization was measured for entire of slice in each image . In order to ensure that results were not confounded by diffuse often observed in LC3 channel , analyses were repeated with nuclei excluded , of results remained statistically significant .

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(d) Colocalization expressed as M1, as in b. A representative experiment of two independent experiments is shown, in which at least 24 cells were analysed per condition. Error bars indicate s.e.m., and ***P0.001 by 2-tailed Student's t-test. Scale bars in (a,c), 10 μm. In b,d, colocalization was measured for the entire area of the cell slice in each image. In order to ensure that results were not confounded by diffuse nuclear staining often observed in the LC3 channel, the analyses were repeated with the nuclei excluded, and all of the results remained statistically significant.


( a ) SH - SY5Y were depleted of WASH1 , treated with bafilomycin A1 examined for LC3 - II , tubulin , WASH1 GAPDH . Blots shown are representative of two independent experiments in triplicate . ( b ) of representative experiment in triplicate shown in ( a ) , of two independent experiments . Error bars indicate s . e . m . * * P = 0 . 0058 by 1 - Student ' s t - .

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(a) SH-SY5Y cells were depleted of WASH1, treated with bafilomycin A1 and examined for LC3-II, tubulin, WASH1 and GAPDH levels. Blots shown are representative of two independent experiments in triplicate. (b) Quantification of the representative experiment in triplicate shown in (a), of two independent experiments. Error bars indicate s.e.m. **P=0.0058 by 1-tailed Student's t-test.


( c ) WASH1 - depleted SH - SY5Y were trypsinized , stained with propidium iodide analysed by flow cytometry . Living , not stained with propidium iodide , are shown as a of total . graph depicts a representative experiment in triplicate out of two independent experiments , in which at least 10 , 000 were analysed in each replicate . Error bars indicate s . e . m . * P = 0 . 045 by 1 - Student ' s t - .

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(c) WASH1-depleted SH-SY5Y cells were trypsinized, stained with propidium iodide and analysed by flow cytometry. Living cells, not stained with propidium iodide, are shown as a percentage of total cells. The graph depicts a representative experiment in triplicate out of two independent experiments, in which at least 10,000 cells were analysed in each replicate. Error bars indicate s.e.m. *P=0.045 by 1-tailed Student's t-test.


( A ) Expression in E . coli purification of human GS ( hGS ) . was separated by SDS - PAGE stained with Coomassie Blue dye . M : marker Precision Plus Dual Color Standard ( Biorad ) . From right Lanes 1 - 4 : E . coli Bl21 ( DE3 ) containing expression vector without ( lanes 1 2 ) with ( lanes 3 4 ) coding of hGS . Samples were taken immediately before ( lanes 1 3 ) 3 , 5 h later ( lanes 2 4 ) induction of expression with isopropil - β - D - 1 - tiogalattopiranoside ( IPTG ) 0 . 7mM . same number of was analyzed in each sample . Lane 5 : isolated purified inclusion bodies ( 4 μg ) . Adiacent boxed lane : Western Blotting of GS .

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(A) Expression in E. coli and purification of human GS (hGS). Protein was separated by SDS-PAGE and stained with Coomassie Blue dye. M: marker Precision Plus Protein Dual Color Standard (Biorad). From right Lanes 1-4: E. coli Bl21(DE3) cells containing the expression vector without (lanes 1 and 2) and with (lanes 3 and 4) the coding sequence of hGS. Samples were taken immediately before (lanes 1 and 3) and 3,5 h later (lanes 2 and 4) the induction of expression with isopropil-β-D-1-tiogalattopiranoside (IPTG) 0.7mM. The same number of bacteria was analyzed in each sample. Lane 5: isolated and purified inclusion bodies (4 μg). Adiacent boxed lane: Western Blotting analysis of GS.


Kinetic study of reaction catalyzed by recombinant hGS . reaction , started by adding ATP , was linear for at least 10 min at 25 ° C ( B ) .

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Kinetic study of the reaction catalyzed by recombinant hGS. The reaction, started by adding ATP, was linear for at least 10 min at 25°C (B).


Kinetic study of reaction catalyzed by recombinant hGS . Lineweaver - Burk plot reporting hGS activity at indicated concentrations of glutamate in absence ( ) , or in presence of glufosinate ( C ) Symbols - ( ) : 0 . 025 glufosinate ; or 0 . 050 mM of glufosinate ; or 0 . 065 mM of glufosinate ; 0 . 075 mM of glufosinate .

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Kinetic study of the reaction catalyzed by recombinant hGS. Lineweaver-Burk plot reporting the hGS activity at the indicated concentrations of glutamate in the absence (), or in the presence of glufosinate (C) Symbols- (): 0.025 glufosinate; or 0.050 mM of glufosinate; or 0.065 mM of glufosinate; 0.075 mM of glufosinate.


Kinetic study of reaction catalyzed by recombinant hGS . Lineweaver - Burk plot reporting hGS activity at indicated concentrations of glutamate in absence ( ) , or in presence of MSO Symbols - ( ) : 2 mM MSO ; ( ) 3 mM of MSO ; ( ) 4 mM of MSO ; ( ) 5 mM of

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Kinetic study of the reaction catalyzed by recombinant hGS. Lineweaver-Burk plot reporting the hGS activity at the indicated concentrations of glutamate in the absence (), or in the presence of MSO Symbols- (): 2 mM MSO ; () 3 mM of MSO ; () 4 mM of MSO ; () 5 mM of MSO


Kinetic study of reaction catalyzed by recombinant hGS . insets represent secondary plot of slopes of Lineweaver - Burk plot obtained at indicated concentrations of glufosinate ( C ) or MSO ( D ) used for determining inhibitor constant Ki . GS inhibition ( % ) in presence of increasing concentrations of glufosinate ( ) or MSO ( ) results from average of at least three independent experiments ( E ) . control value for uninhibited hGS activity is 0 . 51 ± 0 . 08 μmol / ( min x mg ) .

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Kinetic study of the reaction catalyzed by recombinant hGS. The insets represent the secondary plot of the slopes of Lineweaver-Burk plot obtained at the indicated concentrations of glufosinate (C) or MSO (D) used for determining the inhibitor constant Ki. GS inhibition (%) in presence of increasing concentrations of glufosinate () or MSO () results from the average of at least three independent experiments (E). The control value for uninhibited hGS activity is 0.51 ±0.08 μmol/(min x mg protein).


Evaluation of M1 markers in macrophages by real - time PCR . Fold change of TNFA , CD80 , CXCL9 , CXCL10 mRNA in IL10 , MSO - glufosinate ( 10 20µM ) - stimulated IL10 macrophages ( n = 3 ) . Evaluation of M2 markers in macrophages by real - time PCR . Fold change of MRC1 , MSR1 , mRNA in IL10 , MSO - glufosinate ( 10 20µM ) - stimulated IL10 macrophages ( n = 3 ) .

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Evaluation of M1 markers in macrophages by real-time PCR. Fold change of TNFA, CD80, CXCL9, and CXCL10 mRNA in IL10, MSO- and glufosinate (10 and 20µM)- stimulated IL10 macrophages (n=3). Evaluation of M2 markers in macrophages by real-time PCR. Fold change of MRC1, MSR1, mRNA in IL10, MSO- and glufosinate (10 and 20µM)- stimulated IL10 macrophages (n=3).


Evaluation of M2 markers in macrophages by real - time PCR . Fold change of CCL17 CCL18 mRNA in IL10 , MSO - glufosinate ( 10 20µM ) - stimulated IL10 macrophages ( n = 3 ) .

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Evaluation of M2 markers in macrophages by real-time PCR. Fold change of CCL17 and CCL18 mRNA in IL10, MSO- and glufosinate (10 and 20µM)- stimulated IL10 macrophages (n=3).


Evaluation of M1 markers in macrophages following HIF1α inhibition by real - time PCR . Fold change of TNFA , CXCL10 , CD86 mRNA in IL10 alone or glufosinate ( 10 20µM ) - acriflavine / glufosinate ( 10 20µM ) - IL10 macrophages ( n = 3 ) .

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Evaluation of M1 markers in macrophages following HIF1α inhibition by real-time PCR. Fold change of TNFA, CXCL10, CD86 mRNA in IL10 alone or glufosinate (10 and 20µM)- and acriflavine/glufosinate (10 and 20µM)- IL10 macrophages (n=3).


Evaluation of M1 markers in macrophages following HIF1α inhibition by real - time PCR . Fold change of CD80 CXCL9 mRNA in IL10 alone or glufosinate ( 10 20µM ) - acriflavine / glufosinate ( 10 20µM ) - IL10 macrophages ( n = 3 ) . Evaluation of M2 markers in macrophages following HIF1α inhibition by real - time PCR . Fold change of MRC1 , MSR1 , CCL18 mRNA in IL10 , glufosinate ( 10 20µM ) - acriflavine / glufosinate ( 10 20µM ) - IL10 macrophages ( n = 3 ) .

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Evaluation of M1 markers in macrophages following HIF1α inhibition by real-time PCR. Fold change of CD80 and CXCL9 mRNA in IL10 alone or glufosinate (10 and 20µM)- and acriflavine/glufosinate (10 and 20µM)- IL10 macrophages (n=3). Evaluation of M2 markers in macrophages following HIF1α inhibition by real-time PCR. Fold change of MRC1, MSR1, and CCL18 mRNA in IL10, glufosinate (10 and 20µM)- and acriflavine/glufosinate (10 and 20µM)- IL10 macrophages (n=3).


( Q ) of cancer motility through a matrigel coated in presence of IL10 , MSO / IL10 glufosinate ( 10 20µM ) - IL10 treated macrophages after 24 h of incubation ( n = 6 ) .

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(Q) Quantification of cancer cell motility through a matrigel coated membrane in presence of IL10, MSO/IL10 and glufosinate (10 and 20µM)- IL10 treated macrophages after 24 h of incubation (n=6).


( R ) Evaluation of capillary network formation in presence of macrophages pretreated for 24 h with IL10 or MSO / IL10 , glufosinate ( 10 20µM ) / IL10 after 4 h of incubation with HUVEC ( n = 6 ) .

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(R) Evaluation of the capillary network formation in presence of macrophages pretreated for 24 h with IL10 or MSO/IL10, and glufosinate (10 and 20µM)/IL10 after 4 h of incubation with HUVEC cells (n=6).


( S ) CD8 + T suppression by macrophages treated with IL10 or MSO / IL10 , glufosinate ( 10 20µM ) / IL10 for 24 h ( n = 4 ) . Proliferation was evaluated by reading radioactivity as cpm ( per minute ) , after incubation with 1 μCi / well tritiated thymidine . proliferation of T cultured without macrophages was used as control .

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(S) CD8+ T cell suppression by macrophages treated with IL10 or MSO/IL10, and glufosinate (10 and 20µM)/IL10 for 24 h (n=4). Proliferation was evaluated by reading radioactivity as cpm (counts per minute), after incubation with 1 μCi / well tritiated thymidine. The proliferation of T cells cultured without macrophages was used as control.


( T ) CD8 + T recruitment in a transwell system by macrophages treated with IL10 or MSO / IL10 , glufosinate ( 10 20µM ) / IL10 for 24 h versus macrophages treated with LPS / IFNγ ; migration of T cultured without macrophages ( Mφ - ) in presence of CXCL10 was used as positive control ( n = 4 ) .

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(T) CD8+ T cell recruitment in a transwell system by macrophages treated with IL10 or MSO/IL10, and glufosinate (10 and 20µM)/IL10 for 24 h versus macrophages treated with LPS/IFNγ; the migration of T cells cultured without macrophages (Mφ-) in presence of CXCL10 was used as positive control (n=4).


( U ) Representative image of Western blotting of HIF1α , REDD1 , 4E - BP1 , S6 P70S6K ( in their phosphorylated unphosphorilated form ) to mTOR activation in normoxic ( NRX ) hypoxic ( HYP ) IL10 macrophages treated with glufosinate ( 20µM ) , rapamycin ( 20nM ) a combination of both ( n = 3 ) .

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(U) Representative image of Western blotting analysis of HIF1α, REDD1, 4E-BP1, S6 and P70S6K (in their phosphorylated and unphosphorilated form) to test mTOR activation in normoxic (NRX) and hypoxic (HYP) IL10 macrophages treated with glufosinate (20µM), rapamycin (20nM) and a combination of both (n=3).


( V ) CD8 + T suppression by normoxic hypoxic IL10 macrophages treated with glufosinate ( 20µM ) , rapamycin a combination of both for 24 h ( n = 4 ) . Proliferation was evaluated by reading radioactivity as cpm ( per minute ) , after incubation with 1 μCi / well tritiated thymidine . proliferation of T cultured without macrophages was used as control .

(V) CD8+ T cell suppression by normoxic and hypoxic IL10 macrophages treated with glufosinate (20µM), rapamycin and a combination of both for 24 h (n=4). Proliferation was evaluated by reading radioactivity as cpm (counts per minute), after incubation with 1 μCi / well tritiated thymidine. The proliferation of T cells cultured without macrophages was used as control.


of glufosinate ( B ) in wet tissues of vehicle glufosinate - treated mice ( n = 8 ) , by liquid chromatography - mass spectrometry ( LC - MS ) .

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Quantification of glufosinate (B) levels in wet tumor tissues of vehicle and glufosinate-treated mice (n=8), by liquid chromatography-mass spectrometry (LC-MS).


of glutamine ( GLN ) ( C ) in wet tissues of vehicle glufosinate - treated mice ( n = 8 ) , by liquid chromatography - mass spectrometry ( LC - MS ) .

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Quantification of glutamine (GLN) (C) levels in wet tumor tissues of vehicle and glufosinate-treated mice (n=8), by liquid chromatography-mass spectrometry (LC-MS).


Subcutaneous LLC growth over time ( D ) ( pool of 2 independent experiments ; 10 mice per condition in total ) .

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Subcutaneous LLC tumor growth over the time (D) (pool of 2 independent experiments; 10 mice per condition in total).


number of lung metastatic nodules in vehicle glufosinate ( 10 20mg / kg ) treated mice ( E ) ( pool of 2 independent experiments ; 10 mice per condition in total ) .

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number of lung metastatic nodules in vehicle and glufosinate (10 and 20mg/kg) treated mice (E) (pool of 2 independent experiments; 10 mice per condition in total).


representative of F4 / 80 + CD11c + in lung metastasis ( F ) , F4 / 80 + CD206 + ( G ) infiltration in tumors of vehicle glufosinate - treated mice ( n = 6 ) .

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Quantification and representative images of F4/80+ CD11c+ cells in the lung metastasis (F), F4/80+ CD206+ (G) cells infiltration in tumors of vehicle and glufosinate-treated mice (n=6).


representative of F4 / 80 CD11c + ( H ) infiltration in tumors of vehicle glufosinate - treated mice ( n = 6 ) .

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Quantification and representative images of F4/80 CD11c+ (H) cells infiltration in tumors of vehicle and glufosinate-treated mice (n=6).


RT - PCR of M2 ( Ccl22 , Arg1 Ccl17 ) ( I ) markers in vehicle glufosinate treated mice ( n = 4 ) .

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RT-PCR quantification of M2 (Ccl22, Arg1 and Ccl17) (I) markers in vehicle and glufosinate treated mice (n=4).


RT - PCR of M1 ( Tnfa , Cxcl9 , Nos2 , Cd86 Cd80 ) ( J ) markers in vehicle glufosinate treated mice ( n = 4 ) .

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RT-PCR quantification of M1 (Tnfa, Cxcl9, Nos2, Cd86 and Cd80) (J) markers in vehicle and glufosinate treated mice (n=4).


of total of vessels ( A ) in vehicle glufosinate ( 20mg / kg ) treated mice ( n = 6 ) .

Quantification of total length of vessels (A) in vehicle and glufosinate (20mg/kg) treated mice (n=6).


of CD31 + vessel ( B ) of αSMA + pericyte - covered vessels over total number of CD31 + vessels ( with representative ) ( C ) in vehicle glufosinate ( 20mg / kg ) treated mice ( n = 6 ) .

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Quantification of CD31+ tumor vessel area (B) and of αSMA+ pericyte-covered vessels over the total number of CD31+ vessels (with representative images) (C) in vehicle and glufosinate (20mg/kg) treated mice (n=6).


( D ) representative of Lectin + vessel over total number of CD31 + vessels in vehicle glufosinate ( 20mg / kg ) treated mice ( n = 6 ) .

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(D) Quantification and representative images of Lectin+ tumor vessel area over the total number of CD31+ vessels in vehicle and glufosinate (20mg/kg) treated mice (n=6).


( E ) representative of PIMO + hypoxic areas in vehicle glufosinate ( 20mg / kg ) treated mice ( n = 6 ) .

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(E) Quantification and representative images of PIMO+ tumor hypoxic areas in vehicle and glufosinate (20mg/kg) treated mice (n=6).


FACS of TCRbeta + CD8 + cytotoxic T in tumors of vehicle glufosinate - treated mice ( F ) .

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FACS quantification of TCRbeta+ CD8+ cytotoxic T cells in tumors of vehicle and glufosinate-treated mice (F).


Evaluation of CD69 ( G ) CD25 ( H ) , expressed as Mean ( MFI ) , was determined by flow cytometry on surface of responders CD8 + T ( n = 4 ) .

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Evaluation of CD69 (G) and CD25 (H) proteins, expressed as Mean Fluorescence Intensity (MFI), was determined by flow cytometry on the surface of responders CD8+ T cells (n=4).


( I ) of secreted IFNγ in interstitial tumoral fluid of vehicle glufosinate - treated mice ( n = 6 ) .

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(I) Quantification of secreted IFNγ in interstitial tumoral fluid of vehicle and glufosinate-treated mice (n=6).


( B ) Evaluation of in vehicle glufosinate ( 20mg / kg ) treated mice ( n = 8 ) . ( C ) Representative of metastasis at beginning end of therapy . Evaluation of metastatic burden following glufosinate treatment by H & E ( n = 8 ) .

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(B) Evaluation of body weight in vehicle and glufosinate (20mg/kg) treated mice (n=8). (C) Representative images of metastasis at the beginning and end of therapy. Evaluation of the metastatic burden following glufosinate treatment by H&E staining (n=8).


( D ) Evaluation of lung total metastatic of lung metastatic lesions per by H & E in vehicle glufosinate ( 20mg / kg ) treated mice ( n = 8 ) .

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(D) Evaluation of lung total metastatic area and of lung metastatic lesions per size by H&E staining in vehicle and glufosinate (20mg/kg) treated mice (n=8).


( F ) of total metastatic in LLC - bearing mice preconditioned with IgG or anti - CSF1R antibody treated with vehicle or glufosinate ( 20mg / Kg ) .

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Quantification (F) of total metastatic area in LLC-tumor bearing mice preconditioned with IgG or anti-CSF1R antibody and treated with vehicle or glufosinate (20mg/Kg).


representative ( G ) of total metastatic in LLC - bearing mice preconditioned with IgG or anti - CSF1R antibody treated with vehicle or glufosinate ( 20mg / Kg ) .

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representative images (G) of total metastatic area in LLC-tumor bearing mice preconditioned with IgG or anti-CSF1R antibody and treated with vehicle or glufosinate (20mg/Kg).


Intradermal YUMM 1 . 7 ( CD90 . 1 + ) growth over time ( B ) in vehicle glufosinate ( 20mg / kg ) treated mice ( pool of 2 independent experiments ; 10 mice per condition in total ) .

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Intradermal YUMM 1.7 (CD90.1+) tumor growth over the time (B) in vehicle and glufosinate (20mg/kg) treated mice (pool of 2 independent experiments; 10 mice per condition in total).


number of metastatic lesions per lung ( C ) in vehicle glufosinate ( 20mg / kg ) treated mice ( pool of 2 independent experiments ; 10 mice per condition in total ) .

number of metastatic lesions per lung (C) in vehicle and glufosinate (20mg/kg) treated mice (pool of 2 independent experiments; 10 mice per condition in total).


( D ) Representative of metastatic lesions checked by DAB - H stain for CD90 . 1 positivity in vehicle glufosinate ( 20mg / kg ) treated mice ( n = 6 ) . Six per lung were analysed . Scale bar : 2mm . Zoomed scale bar : 50µm .

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(D) Representative images of metastatic lesions checked by DAB-H stain for CD90.1 positivity in vehicle and glufosinate (20mg/kg) treated mice (n=6). Six images per lung were analysed. Scale bar: 2mm. Zoomed area scale bar: 50µm.


( E , F ) FACS of M1 - like MHC class IIhigh ( E ) MHC class IIlow ( F ) CD11c + CD206 - TAMs over total number of F4 / 80 + in vehicle glufosinate ( 20mg / kg ) treated mice ( n = 6 ) .

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(E, F) FACS quantification of M1-like MHC class IIhigh (E) and MHC class IIlow (F) CD11c+ CD206- TAMs over the total number of F4/80+ cells in vehicle and glufosinate (20mg/kg) treated mice (n=6).


( G ) representative of CD11c + over total number of F4 / 80 + in vehicle glufosinate ( 20mg / kg ) treated mice ( n = 6 ) . Six per were analysed . Scale bars : 50 µm .

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(G) Quantification and representative images of CD11c+ cells over the total number of F4/80+ cells in vehicle and glufosinate (20mg/kg) treated mice (n=6). Six images per tumor were analysed. Scale bars: 50 µm.


FACS of M2 - like MHC class IIhigh CD11c - CD206 + TAMs over total number of F4 / 80 + in vehicle glufosinate ( 20mg / kg ) treated mice ( n = 6 ) .

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FACS quantification of M2-like MHC class IIhigh CD11c- CD206+ TAMs over the total number of F4/80+ cells in vehicle and glufosinate (20mg/kg) treated mice (n=6).


FACS of M2 - like MHC class IIlow ( I ) CD11c - CD206 + TAMs over total number of F4 / 80 + in vehicle glufosinate ( 20mg / kg ) treated mice ( n = 6 ) .

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FACS quantification of M2-like MHC class IIlow (I) CD11c- CD206+ TAMs over the total number of F4/80+ cells in vehicle and glufosinate (20mg/kg) treated mice (n=6).


( J ) representative of CD206 + over total number of F4 / 80 + in vehicle glufosinate ( 20mg / kg ) treated mice ( n = 6 ) . Six per were analysed . Scale bars : 50 µm .

(J) Quantification and representative images of CD206+ cells over the total number of F4/80+ cells in vehicle and glufosinate (20mg/kg) treated mice (n=6). Six images per tumor were analysed. Scale bars: 50 µm.


FACS of M1 - like MHC class IIhigh ( K ) TAMs over total number of F4 / 80 + in vehicle glufosinate ( 20mg / kg ) treated mice ( n = 6 ) .

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FACS quantification of M1-like MHC class IIhigh (K) TAMs over the total number of F4/80+ cells in vehicle and glufosinate (20mg/kg) treated mice (n=6).


FACS of M1 - like M2 - like MHC class IIlow ( L ) TAMs over total number of F4 / 80 + in vehicle glufosinate ( 20mg / kg ) treated mice ( n = 6 ) .

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FACS quantification of M1-like M2-like MHC class IIlow (L) TAMs over the total number of F4/80+ cells in vehicle and glufosinate (20mg/kg) treated mice (n=6).


( M ) of MHC - II , expressed as Mean ( MFI ) , on F4 / 80 + , was obtained by flow cytometry ( n = 6 ) .

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(M) Quantification of MHC-II protein, expressed as Mean Fluorescence Intensity (MFI), on F4/80+ cells, was obtained by flow cytometry (n=6).


( N ) RT - PCR of M2 ( Arg1 Cxcr4 ) M1 ( Cxcl9 Nos2 ) markers in vehicle glufosinate ( 20mg / kg ) treated mice ( n = 6 ) .

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(N) RT-PCR quantification of M2 (Arg1 and Cxcr4) and M1 (Cxcl9 and Nos2) markers in vehicle and glufosinate (20mg/kg) treated mice (n=6).


of total of vessels ( A ) Six per were analysed . Scale bar : 50µm .

Quantification of total length of vessels (A) Six images per tumor were analysed. Scale bar: 50µm.


CD31 + vessel ( B ) of αSMA + pericyte - covered vessels over total number of CD31 + vessels ( with representative ) ( C ) in vehicle glufosinate ( 20mg / kg ) treated mice ( n = 6 ) . Six per were analysed . Scale bar : 50µm .

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CD31+ tumor vessel area (B) and of αSMA+pericyte-covered vessels over the total number of CD31+ vessels (with representative images) (C) in vehicle and glufosinate (20mg/kg) treated mice (n=6). Six images per tumor were analysed. Scale bar: 50µm.


( D ) representative of CA9 + hypoxic areas in vehicle glufosinate ( 20mg / kg ) treated mice ( n = 6 ) . Six per were analysed . Scale bar : 50µm .

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(D) Quantification and representative images of CA9+ tumor hypoxic areas in vehicle and glufosinate (20mg/kg) treated mice (n=6). Six images per tumor were analysed. Scale bar: 50µm.


( E , F ) FACS of CD8 + cytotoxic T ( E ) CD69 + CD8 + T ( F ) over total number of CD45 + in vehicle or glufosinate ( 20mg / kg ) treated mice ( n = 6 ) .

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(E, F) FACS quantification of CD8+ cytotoxic T cells (E) and CD69+ CD8+ T cells (F) over the total number of CD45+ in vehicle or glufosinate (20mg/kg) treated mice (n=6).


Evaluation of 4T1 growth over time ( B ) , in vehicle glufosinate ( 10mg / kg ) treated mice ( pool of 2 independent experiments ; 10 mice per condition in total ) .

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Evaluation of 4T1 tumor growth over the time (B) , in vehicle and glufosinate (10mg/kg) treated mice (pool of 2 independent experiments; 10 mice per condition in total).


mice ( C ) in vehicle glufosinate ( 10mg / kg ) treated mice ( pool of 2 independent experiments ; 10 mice per condition in total ) .

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mice body weight (C) in vehicle and glufosinate (10mg/kg) treated mice (pool of 2 independent experiments; 10 mice per condition in total).


number of metastatic nodules ( D ) , in vehicle glufosinate ( 10mg / kg ) treated mice ( pool of 2 independent experiments ; 10 mice per condition in total ) .

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number of metastatic nodules (D), in vehicle and glufosinate (10mg/kg) treated mice (pool of 2 independent experiments; 10 mice per condition in total).


Total metastatic ( E ) in vehicle glufosinate ( 10mg / kg ) treated mice ( n = 8 ) . Six per lung were analysed . Scale bar : 2mm .

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Total metastatic area (E) in vehicle and glufosinate (10mg/kg) treated mice (n=8). Six images per lung were analysed. Scale bar: 2mm.


number of lung metastatic lesions per by H & E ( F ) with representative ( G ) in vehicle glufosinate ( 10mg / kg ) treated mice ( n = 8 ) . Six per lung were analysed . Scale bar : 2mm .

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number of lung metastatic lesions per size by H&E staining (F) with representative images (G) in vehicle and glufosinate (10mg/kg) treated mice (n=8). Six images per lung were analysed. Scale bar: 2mm.


( H , I ) of F4 / 80 + macrophage density ( H ) , of F4 / 80 + CD206 + infiltration in lung metastasis representative ( I ) , in vehicle glufosinate - treated mice ( n = 6 ) . Six per lung were analysed . Scale bar : 20µm .

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(H, I) Quantification of F4/80+ macrophage density (H), of F4/80+ CD206+ cells infiltration in the lung metastasis and representative images (I), in vehicle and glufosinate-treated mice (n=6). Six images per lung were analysed. Scale bar: 20µm.


GFP + CTCs amount in blood , expressed as GFP expression , was determined by qPCR ( n = 8 ) ( K ) .

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GFP+ CTCs amount in blood, expressed as GFP expression levels, was determined by qPCR (n=8) (K).


Blood was drawn from treated untreated - bearing mice at time of resection plated in culture dishes . formation of colonies was traced over time . Graph shows number of GFP + colonies ( n = 8 ) ( L ) .

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Blood was drawn from treated and untreated tumor-bearing mice at the time of tumor resection and plated in culture dishes. The formation of tumor cell colonies was traced over time. Graph shows the number of GFP+ tumor cell colonies (n=8) (L).


After treatment resection overall survival studies were performed until natural of post - surgical mice ( n = 11 ) ( M ) .

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After treatment and tumor resection overall survival studies were performed until the natural death of the post-surgical mice (n=11) (M).


( O ) GFP + cancer amount in lungs , expressed as GFP expression , was determined by qPCR in vehicle glufosinate ( 10mg / kg ) treated mice ( n = 8 ) . results were normalized to vehicle group .

(O) GFP+ cancer cells amount in lungs, expressed as GFP expression levels, was determined by qPCR in vehicle and glufosinate (10mg/kg) treated mice (n=8). The results were normalized to the vehicle group.


( A ) Time course evaluation of murine during treatment in vehicle glufosinate ( 10 20 mg / kg ) treated mice ( pool of 2 independent experiments ; 10 mice per condition in total ) .

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(A) Time course evaluation of murine body weight during treatment in vehicle and glufosinate (10 and 20 mg/kg) treated mice (pool of 2 independent experiments; 10 mice per condition in total).


( B ) Blood of WBC RBC in vehicle glufosinate ( 10 20 mg / kg ) treated mice ( n = 8 ) .

(B) Blood counts of WBC and RBC in vehicle and glufosinate (10 and 20 mg/kg) treated mice (n=8).


Evaluation of liver toxicity following glufosinate treatment . of liver glufosinate ( C ) in serum of vehicle glufosinate ( 10 20 mg / kg ) treated mice ( n = 8 ) .

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Evaluation of liver toxicity following glufosinate treatment. Quantification of liver glufosinate (C) in serum of vehicle and glufosinate (10 and 20 mg/kg) treated mice (n=8).


Evaluation of liver toxicity following glufosinate treatment . hepatotoxicity markers ALT ( Alanine Aminotransferase ) AST ( Aspartate aminotransferase ) ( D ) in serum of vehicle glufosinate ( 10 20 mg / kg ) treated mice ( n = 8 ) .

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Evaluation of liver toxicity following glufosinate treatment. hepatotoxicity markers ALT (Alanine Aminotransferase) and AST (Aspartate aminotransferase) (D) in serum of vehicle and glufosinate (10 and 20 mg/kg) treated mice (n=8).


Evaluation of neuronal toxicity following glufosinate treatment . of brain glufosinate ( E )

Evaluation of neuronal toxicity following glufosinate treatment. Quantification of brain glufosinate (E)


Evaluation of neuronal toxicity following glufosinate treatment . grip strength for front paws ( triangular bar ) ( F ) , as an indicator of development of tactile allodynia in vehicle glufosinate - treated mice ( n = 4 ) , before treatment ( baseline ) , after 1 day ( acute Toxicity ) after 15 days ( chronic toxicity ) ( pool of 2 independent experiments ; 10 mice per condition in total ) .

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Evaluation of neuronal toxicity following glufosinate treatment. grip strength for front paws (triangular bar) (F), as an indicator of the development of tactile allodynia in vehicle and glufosinate-treated mice (n=4), before the treatment (baseline), after 1 day (acute Toxicity) and after 15 days (chronic toxicity) (pool of 2 independent experiments; 10 mice per condition in total).


Evaluation of neuronal toxicity following glufosinate treatment . grip strength for paws ( rectangle grid ) ( G ) , of latency to fall off an accelerating rotarod ( H ) , of mean paw withdrawal force that caused ' response in von Frey ( left hindpaw , I ; right hindpaw , J ) as an indicator of development of tactile allodynia in vehicle glufosinate - treated mice ( n = 4 ) , before treatment ( baseline ) , after 1 day ( acute Toxicity ) after 15 days ( chronic toxicity ) ( pool of 2 independent experiments ; 10 mice per condition in total ) .

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Evaluation of neuronal toxicity following glufosinate treatment. grip strength for all paws (rectangle grid) (G), of the latency to fall off an accelerating rotarod (H), and of the mean paw withdrawal force that caused animals' response in the von Frey test (left hindpaw, I; right hindpaw, J) as an indicator of the development of tactile allodynia in vehicle and glufosinate-treated mice (n=4), before the treatment (baseline), after 1 day (acute Toxicity) and after 15 days (chronic toxicity) (pool of 2 independent experiments; 10 mice per condition in total).


C - terminal domains of IFT52 IFT46 co - purify with IFT81460 - C / 74460 - C / 27 / 251 - 136 ( C ) SEC fractions indicated on right with 1 - 3 were analysed by SDS - PAGE stained with Coomassie to evaluate composition .

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The C-terminal domains of IFT52 and IFT46 co-purify with the IFT81460-C/74460-C/27/251-136 protein complex (C) The SEC fractions indicated on the right with 1-3 were analysed by SDS-PAGE and stained with Coomassie to evaluate the protein composition.


C - terminal domains of IFT52 IFT46 but not with a that is missing residues 623 - C of IFT81 615 - C of IFT74 ( D ) . SEC fractions indicated on right with 1 - 3 were analysed by SDS - PAGE stained with Coomassie to evaluate composition .

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The C-terminal domains of IFT52 and IFT46 but not with a protein complex that is missing residues 623-C of IFT81 and 615-C of IFT74 (D). The SEC fractions indicated on the right with 1-3 were analysed by SDS-PAGE and stained with Coomassie to evaluate the protein composition.


( G ) Coomassie stained SDS PAGE gel of a GST - pulldown using CrIFT88His - GST / 70 / 521 - 430 / 46 immobilized on GST beads as bait CrIFT811 - 387 - GFP / 74135 - 420 used as prey . band highlighted by an * corresponds to pulled down CrIFT74135 - 420 . CrIFT811 - 387 - GFP runs on top of IFT70 is not visible in pull - down .

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(G) Coomassie stained SDS PAGE gel of a GST-pulldown using the CrIFT88His-GST/70/521-430/46 complex immobilized on GST beads as bait and the CrIFT811-387-GFP/74135-420 complex used as prey. The band highlighted by an * corresponds to pulled down CrIFT74135-420. CrIFT811-387-GFP runs on top of IFT70 and is not visible in the pull-down.


( B ) Pull - down experiments with purified IFT80Venus immobilized on GFP - binder beads either IFT172 or IFT172968 - C . IFT80 pulls down full IFT172 but not truncated version lacking N - terminal 967 residues .

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(B) Pull-down experiments with purified IFT80Venus immobilized on GFP-binder beads and either IFT172 or IFT172968-C. IFT80 pulls down full length IFT172 but not the truncated version lacking the N-terminal 967 residues.


( F ) GST tagged CrIFT1721 - 968 pulls down CH - domain of CrIFT571 - 234 but not mutated CH - domain of CrIFT57 where E77 K84 residues were replaced by alanines .

(F) GST tagged CrIFT1721-968 pulls down the CH-domain of CrIFT571-234 but not the mutated CH-domain of CrIFT57 where E77 and K84 residues were replaced by alanines.


( A ) Serum of miR - 483 - 3p / - 5p in IPAH ( n = 139 ) HC ( n = 95 ) measured by qPCR . data are fold change normalized to averaged level of HC . Data information : Values are expressed as median ± interquartile range . Statistical : Mann Whitney U ( A )

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(A) Serum levels of miR-483-3p/-5p in IPAH patients (n = 139) and HC (n = 95) measured by qPCR. The data are fold change normalized to the averaged level of HC. Data information: Values are expressed as median ± interquartile range. Statistical test: Mann Whitney U test (A)


( B ) ROC curve with sensitivity specificity of serum of miR - 483 - 3p / - 5p for differentiating IPAH from HCs at diagnosis ( IPAH , n = 139 ; HC , n = 95 ) . Data information : Values are expressed as median ± interquartile range .

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(B) ROC curve with sensitivity and specificity of serum levels of miR-483-3p/-5p for differentiating IPAH patients from HCs at diagnosis (IPAH, n = 139; HC, n = 95). Data information: Values are expressed as median ± interquartile range.


( C ) of miR - 483 - 3p / - 5p associated with PAH risk in 3 groups . IPAH were divided into a low risk group ( Low ) an intermediate plus high risk group ( Inter & high ) according to World Symposium on Pulmonary Hypertension 2018 [ 14 ] . Data information : Values are expressed as median ± interquartile range . Statistical : Kruskal - Wallis U ( C ) .

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(C) Levels of miR-483-3p/-5p associated with PAH risk in 3 groups. IPAH patients were divided into a low risk group (Low) and an intermediate plus high risk group (Inter&high) according to the World Symposium on Pulmonary Hypertension 2018 [14]. Data information: Values are expressed as median ± interquartile range. Statistical test: Kruskal-Wallis U test (C).


( D ) of miR - 483 - 3p were inversely correlated with serum of ET - 1 ( IPAH , n = 118 ; HC , n = 95 ) IL - 6 ( IPAH , n = 112 ; HC , n = 93 ) . Data information : Values are expressed as median ± interquartile range .

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(D) Levels of miR-483-3p were inversely correlated with serum levels of ET-1 (IPAH, n = 118; HC, n = 95) and IL-6 (IPAH, n = 112; HC, n = 93). Data information: Values are expressed as median ± interquartile range.


( A ) CD144 - enriched EVs were isolated from serum of IPAH ( n = 37 ) HCs ( n = 34 ) . of miR - 483 - 3p / - 5p were measured by qPCR . Data information : Values are expressed as median ± interquartile range . Statistical : Mann Whitney U .

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

(A) CD144-enriched EVs were isolated from serum of IPAH patients (n = 37) and HCs (n = 34). The levels of miR-483-3p/-5p were measured by qPCR. Data information: Values are expressed as median ± interquartile range. Statistical test: Mann Whitney U test.


PAECs were transfected with miR - 483 - 3p / - 5p mimic or scramble for 36 hr before isolation then analyzed by - seq . Data are results from two biological repeats . ( C ) Heat map comparison of log2 fold changes of indicated genes . Data information : Values are expressed as median ± interquartile range . Statistical : Mann Whitney U .

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

PAECs were transfected with miR-483-3p/-5p mimic or scramble RNA for 36 hr before RNA isolation and then analyzed by RNA-seq. Data are results from two biological repeats. (C) Heat map comparison of log2 fold changes of the indicated genes. Data information: Values are expressed as median ± interquartile range. Statistical test: Mann Whitney U test.


PAECs were infected with Lenti - pre - miR - 483 or Lenti - null for 24 hr . Expression of miR - 483 - 3p / - 5p measured by qPCR Data information : Values are expressed as mean ± from 3 independent experiments . Statistical : t - ( * P < 0 . 05 between indicated

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

PAECs were infected with Lenti-pre-miR-483 or Lenti-null for 24 hr. Expression levels of miR-483-3p/-5p measured by qPCR Data information: Values are expressed as mean ± SEM from 3 independent experiments. Statistical test: t-test (*P < 0.05 between the indicated groups


PAECs were infected with Lenti - pre - miR - 483 or Lenti - null for 24 hr . Expression of TGF - β , TGFBR2 , β - catenin , CTGF , IL - 1β , ET - 1 mRNA were measured by qPCR Data information : Values are expressed as mean ± from 3 independent experiments . Statistical : t - ( * P < 0 . 05 between indicated

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

PAECs were infected with Lenti-pre-miR-483 or Lenti-null for 24 hr. Expression levels of TGF-β, TGFBR2, β-catenin, CTGF, IL-1β, and ET-1 mRNA were measured by qPCR Data information: Values are expressed as mean ± SEM from 3 independent experiments. Statistical test: t-test (*P < 0.05 between the indicated groups


PAECs were infected with Lenti - pre - miR - 483 or Lenti - null for 24 hr . Expression of TGF - β , TGFBR2 , β - catenin , CTGF , IL - 1β , ET - 1 were measured by Western blot Data information : Values are expressed as mean ± from 3 independent experiments . Statistical : t - ( * P < 0 . 05 between indicated

PAECs were infected with Lenti-pre-miR-483 or Lenti-null for 24 hr. Expression levels of TGF-β, TGFBR2, β-catenin, CTGF, IL-1β, and ET-1 protein were measured by Western blot Data information: Values are expressed as mean ± SEM from 3 independent experiments. Statistical test: t-test (*P < 0.05 between the indicated groups


( E ) Bovine aortic ECs were transfected with a luciferase reporter fused with 3 ' UTR of TGF - β ( Luc - TGF - β WT ) , TGFBR2 ( Luc - TGFBR2 WT ) , IL - 1β ( Luc - IL - 1β WT ) or ET - 1 ( Luc - ET - 1 WT ) or a binding site mutation ( Luc - TGF - β Mut , Luc - TGFBR2 Mut , Luc - IL - 1β Mut , ET - 1 - Mut ) , then infected with Lenti - pre - miR - 483 for additional 36 hr . Luciferase activity was measured . Data information : Values are expressed as mean ± from 3 independent experiments . Statistical : t - ( * P < 0 . 05 between indicated

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

(E) Bovine aortic ECs were transfected with a luciferase reporter fused with the 3'UTR of TGF-β (Luc-TGF-β WT), TGFBR2 (Luc-TGFBR2 WT), IL-1β (Luc-IL-1β WT) or ET-1 (Luc-ET-1 WT) or a binding site mutation (Luc-TGF-β Mut, Luc-TGFBR2 Mut, Luc-IL-1β Mut, ET-1-Mut), then infected with Lenti-pre-miR-483 for additional 36 hr. Luciferase activity was measured. Data information: Values are expressed as mean ± SEM from 3 independent experiments. Statistical test: t-test (*P < 0.05 between the indicated groups


PAECs were infected with Lenti - pre - miR - 483 or Lenti - null for 36 hr . Ago1 - or Ago2 - associated miRNAs mRNAs were enriched by immunoprecipitation with anti - Ago1 or anti - Ago2 . of miR - 483 - 3p / - 5p mRNA were detected by qPCR normalized to those of Ago1 or Ago2 . Data information : Values are expressed as mean ± from 3 independent experiments . Statistical : t - ( * P < 0 . 05 between indicated

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

PAECs were infected with Lenti-pre-miR-483 or Lenti-null for 36 hr. The Ago1- or Ago2-associated miRNAs and mRNAs were enriched by immunoprecipitation with anti-Ago1 or anti-Ago2. Levels of miR-483-3p/-5p mRNA were detected by qPCR and normalized to those of Ago1 or Ago2 protein. Data information: Values are expressed as mean ± SEM from 3 independent experiments. Statistical test: t-test (*P < 0.05 between the indicated groups


PAECs were infected with Lenti - pre - miR - 483 or Lenti - null for 36 hr . Ago1 - or Ago2 - associated miRNAs mRNAs were enriched by immunoprecipitation with anti - Ago1 or anti - Ago2 . of TGF - β , TGFBR2 , β - catenin , CTGF , IL - 1β , ET - 1 mRNA were detected by qPCR normalized to those of Ago1 or Ago2 . Data information : Values are expressed as mean ± from 3 independent experiments . Statistical : t - ( * P < 0 . 05 between indicated

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

PAECs were infected with Lenti-pre-miR-483 or Lenti-null for 36 hr. The Ago1- or Ago2-associated miRNAs and mRNAs were enriched by immunoprecipitation with anti-Ago1 or anti-Ago2. Levels of TGF-β, TGFBR2, β-catenin, CTGF, IL-1β, and ET-1 mRNA were detected by qPCR and normalized to those of Ago1 or Ago2 protein. Data information: Values are expressed as mean ± SEM from 3 independent experiments. Statistical test: t-test (*P < 0.05 between the indicated groups


( A ) Lung ECs were isolated from EC - miR - 483 transgenic rats ( Tg ) their wild - type ( WT ) , n = 3 in each group . MiR - 483 - 3p / - 5p were measured by qPCR . Data information : Values are expressed as mean ± .

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

(A) Lung ECs were isolated from EC- miR-483 transgenic rats (Tg) and their wild-type littermates (WT), n = 3 in each group. MiR-483-3p/-5p levels were measured by qPCR. Data information: Values are expressed as mean ± SEM.


( B ) Serum from WT Tg rats ( n = 6 ) were collected . MiR - 483 - 3p - 5p associated with CD144 were enriched by immunoprecipitation with anti - CD144 . of miR - 483 - 3p / - 5p were measured by qPCR . Data information : Values are expressed as mean ± .

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

(B) Serum from WT and Tg rats (n = 6) were collected. MiR-483-3p and -5p associated with CD144 were enriched by immunoprecipitation with anti-CD144. Levels of miR-483-3p/-5p were measured by qPCR. Data information: Values are expressed as mean ± SEM.


Lung ECs isolated from miR - 483 - Tg rat WT were exposed to normoxia or hypoxia ( 0 . 2 % O2 ) for 24 hr . Proliferation of ECs were measured by flow cytometry Data information : Values are expressed as mean ± . Data are representative of three independent experiments Statistical : t - ( * P < 0 . 05 vs . respective controls or between indicated

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

Lung ECs isolated from miR-483-Tg rat and WT littermates were exposed to normoxia or hypoxia (0.2% O2) for 24 hr. Proliferation of ECs were measured by flow cytometry Data information: Values are expressed as mean ± SEM. Data are representative of three independent experiments Statistical test: t-test (*P < 0.05 vs. respective controls or between the indicated groups


Lung ECs isolated from miR - 483 - Tg rat WT were exposed to normoxia or hypoxia ( 0 . 2 % O2 ) for 24 hr . migration of ECs were measured by wound healing Data information : Values are expressed as mean ± . Data are representative of three independent experiments Statistical : t - ( * P < 0 . 05 vs . respective controls or between indicated

Lung ECs isolated from miR-483-Tg rat and WT littermates were exposed to normoxia or hypoxia (0.2% O2) for 24 hr. migration of ECs were measured by wound healing assay Data information: Values are expressed as mean ± SEM. Data are representative of three independent experiments Statistical test: t-test (*P < 0.05 vs. respective controls or between the indicated groups


Tg WT rats were injected with saline or MCT . Expression of TGF - β , TGFBR2 , β - catenin , CTGF , IL - 1β , ET - 1 mRNA in lung tissues were measured by qPCR Western blot , respectively ( E ) . Data information : Values are expressed as mean ± .

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

Tg and WT rats were injected with saline or MCT. Expression levels of TGF-β, TGFBR2, β-catenin, CTGF, IL-1β, and ET-1 mRNA and protein in lung tissues were measured by qPCR and Western blot, respectively (E). Data information: Values are expressed as mean ± SEM.


Tg WT rats were injected with saline or MCT . of miR - 483 - 3p / - 5p in serum lung tissues were measured by qPCR ( F ) . Data information : Values are expressed as mean ± .

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

Tg and WT rats were injected with saline or MCT. Levels of miR-483-3p/-5p in serum and lung tissues were measured by qPCR (F). Data information: Values are expressed as mean ± SEM.


Tg WT rats were injected with saline or MCT . Ago1 - or Ago2 - associated miRNAs ( G ) were enriched from isolated lung tissues by immunoprecipitation with anti - Ago1 or anti - Ago2 quantified by qPCR ( n = 3 × 3 , samples were pooled from three for each , 3 independent experiments [ a total of 9 ] were performed ) . Data information : Values are expressed as mean ± .

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

Tg and WT rats were injected with saline or MCT. Ago1- or Ago2-associated miRNAs (G) were enriched from the isolated lung tissues by immunoprecipitation with anti-Ago1 or anti-Ago2 and quantified by qPCR (n = 3×3, samples were pooled from three animals for each assay, and 3 independent experiments [a total of 9 animals] were performed). Data information: Values are expressed as mean ± SEM.


Tg WT rats were injected with saline or MCT . Ago1 - or Ago2 - associated mRNAs ( H ) were enriched from isolated lung tissues by immunoprecipitation with anti - Ago1 or anti - Ago2 quantified by qPCR ( n = 3 × 3 , samples were pooled from three for each , 3 independent experiments [ a total of 9 ] were performed ) . Data information : Values are expressed as mean ± .

Tg and WT rats were injected with saline or MCT. Ago1- or Ago2-associated mRNAs (H) were enriched from the isolated lung tissues by immunoprecipitation with anti-Ago1 or anti-Ago2 and quantified by qPCR (n = 3×3, samples were pooled from three animals for each assay, and 3 independent experiments [a total of 9 animals] were performed). Data information: Values are expressed as mean ± SEM.


EC - miR - 483 - Tg WT rats were injected with saline or MCT . of mPAP for indicated groups ( 7 rats per group ) Data information : Values are expressed as mean ± . Statistical : t - ( * P < 0 . 05 vs . respective controls or between indicated groups ) .

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

EC-miR-483-Tg and WT rats were injected with saline or MCT. Analysis of mPAP for the indicated groups (7 rats per group) Data information: Values are expressed as mean ± SEM. Statistical test: t-test (*P < 0.05 vs. respective controls or between the indicated groups).


EC - miR - 483 - Tg WT rats were injected with saline or MCT . of RV hypertrophy [ RV / ( LV + S ) ] for indicated groups ( 7 rats per group ) Data information : Values are expressed as mean ± . Statistical : t - ( * P < 0 . 05 vs . respective controls or between indicated groups ) .

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

EC-miR-483-Tg and WT rats were injected with saline or MCT. Analysis of RV hypertrophy [RV/(LV+S)] for the indicated groups (7 rats per group) Data information: Values are expressed as mean ± SEM. Statistical test: t-test (*P < 0.05 vs. respective controls or between the indicated groups).


Vascularization evaluated by H & E of pulmonary arteries ( C ) . Data information : Values are expressed as mean ± . Statistical : t - ( * P < 0 . 05 vs . respective controls or between indicated groups ) . Scale bar : 20 µm ( C )

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

Vascularization evaluated by H&E staining of pulmonary arteries (C). Data information: Values are expressed as mean ± SEM. Statistical test: t-test (*P < 0.05 vs. respective controls or between the indicated groups). Scale bar: 20 µm (C)


Vascular integrity evaluated by intravenous injection of Evans blue dye ( D ) . Data information : Values are expressed as mean ± . Statistical : t - ( * P < 0 . 05 vs . respective controls or between indicated groups ) . Scale bar : 0 . 5 cm ( D ) .

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

Vascular integrity evaluated by intravenous injection of Evans blue dye (D). Data information: Values are expressed as mean ± SEM. Statistical test: t-test (*P < 0.05 vs. respective controls or between the indicated groups). Scale bar: 0.5 cm (D).


EC - miR - 483 - Tg rats WT were injected with SU5416 then exposed to hypoxia for 3 had reoxygenation for 2 or injected with DMSO exposed to normoxia for 5 . of mPAP ( E ) for indicated groups . Data information : Values are expressed as mean ± . Statistical : t - ( * P < 0 . 05 vs . respective controls or between indicated groups ) .

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

EC-miR-483-Tg rats and WT littermates were injected with SU5416 and then exposed to hypoxia for 3 weeks and had reoxygenation for 2 weeks or injected with DMSO and exposed to normoxia for 5 weeks. Analysis of mPAP (E) for the indicated groups. Data information: Values are expressed as mean ± SEM. Statistical test: t-test (*P < 0.05 vs. respective controls or between the indicated groups).


EC - miR - 483 - Tg rats WT were injected with SU5416 then exposed to hypoxia for 3 had reoxygenation for 2 or injected with DMSO exposed to normoxia for 5 . of RV hypertrophy [ RV / ( LV + S ) ] ( F ) for indicated groups . Data information : Values are expressed as mean ± . Statistical : t - ( * P < 0 . 05 vs . respective controls or between indicated groups ) .

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

EC-miR-483-Tg rats and WT littermates were injected with SU5416 and then exposed to hypoxia for 3 weeks and had reoxygenation for 2 weeks or injected with DMSO and exposed to normoxia for 5 weeks. Analysis of RV hypertrophy [RV/(LV+S)] (F) for the indicated groups. Data information: Values are expressed as mean ± SEM. Statistical test: t-test (*P < 0.05 vs. respective controls or between the indicated groups).


qPCR of miR - 483 - 3p / - 5p in serum from PH ( MCT - treated ) rats . Data information : Values are expressed as mean ± . Statistical : t - ( * P < 0 . 05 vs . respective controls or between indicated

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

qPCR analysis of miR-483-3p/-5p in serum from PH (MCT-treated) rats. Data information: Values are expressed as mean ± SEM. Statistical test: t-test (*P < 0.05 vs. respective controls or between the indicated groups


qPCR of miR - 483 - 3p / - 5p in lung tissue from PH ( MCT - treated ) rats . Data information : Values are expressed as mean ± . Statistical : t - ( * P < 0 . 05 vs . respective controls or between indicated

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

qPCR analysis of miR-483-3p/-5p in lung tissue from PH (MCT-treated) rats. Data information: Values are expressed as mean ± SEM. Statistical test: t-test (*P < 0.05 vs. respective controls or between the indicated groups


( D ) mRNA of TGF - β , TGFBR2 , β - catenin , CTGF , IL - 1β , ET - 1 measured by Western blot qPCR , respectively ( n = 3 × 3 , samples were pooled from three for each , 3 independent experiments [ a total of 9 ] were performed ) . Data information : Values are expressed as mean ± . Statistical : t - ( * P < 0 . 05 vs . respective controls or between indicated

O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O B-MEASURED_VAR O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O B-MEASURED_VAR O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Protein and mRNA levels of TGF-β, TGFBR2, β-catenin, CTGF, IL-1β, and ET-1 measured by Western blot and qPCR, respectively (n = 3×3, samples were pooled from three animals for each assay, and 3 independent experiments [a total of 9 animals] were performed). Data information: Values are expressed as mean ± SEM. Statistical test: t-test (*P < 0.05 vs. respective controls or between the indicated groups


of mPAP ( E ) for indicated groups ( 7 rats in each group ) . Data information : Values are expressed as mean ± . Statistical : t - ( * P < 0 . 05 vs . respective controls or between indicated

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

Analysis of mPAP (E) for the indicated groups (7 rats in each group). Data information: Values are expressed as mean ± SEM. Statistical test: t-test (*P < 0.05 vs. respective controls or between the indicated groups


of RV hypertrophy [ RV / ( LV + S ) ] for indicated groups ( 7 rats in each group ) . Data information : Values are expressed as mean ± . Statistical : t - ( * P < 0 . 05 vs . respective controls or between indicated

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

Analysis of RV hypertrophy [RV/(LV+S)] for the indicated groups (7 rats in each group). Data information: Values are expressed as mean ± SEM. Statistical test: t-test (*P < 0.05 vs. respective controls or between the indicated groups


of systemic blood pressure ( SBP ) ( G ) for indicated groups ( 7 rats in each group ) . Data information : Values are expressed as mean ± . Statistical : t - ( * P < 0 . 05 vs . respective controls or between indicated

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

Analysis of systemic blood pressure (SBP) (G) for the indicated groups (7 rats in each group). Data information: Values are expressed as mean ± SEM. Statistical test: t-test (*P < 0.05 vs. respective controls or between the indicated groups


( H ) RV hypertrophy shown by H & E RV / ( LV + S ) . Data information : Values are expressed as mean ± . Statistical : t - ( * P < 0 . 05 vs . respective controls or between indicated groups Scale bar : 0 . 5 cm ( H )

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) RV hypertrophy shown by the H&E staining and RV/(LV+S) quantification. Data information: Values are expressed as mean ± SEM. Statistical test: t-test (*P < 0.05 vs. respective controls or between the indicated groups Scale bar: 0.5 cm (H)