Spot of co - transformants of either Bd0367 ( DgcA ; GGDEF ) response regulator domain or Bd3125 ( CdgA ; a degenerate GGDEF likely involved in c - di - GMP binding signalling ) with full Bd1971 , or N - terminal ( 1 - 145 ) , or C - terminal ( 145 - 400 ) portions of Bd1971 in either pUT18c or pKT25 as indicated . Positive control was co - transformants with pUT18c - zip pKT25 - zip , negative control was empty vectors . co - transformants were plated on LB - X - Gal medium with positive interaction resulting in blue colouration , which can be seen for of interactions in both plasmid combinations , but not for negative control . are representative of at least 4 independent experiments .

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Spot tests of co-transformants of either Bd0367 (DgcA; GGDEF protein) response regulator domain or Bd3125 (CdgA; a degenerate GGDEF protein likely involved in c-di-GMP binding and signalling) with full length Bd1971, or the N-terminal (1-145), or C-terminal (145-400) portions of Bd1971 in either pUT18c or pKT25 as indicated. Positive control was co-transformants with pUT18c-zip and pKT25-zip, negative control was the empty vectors. The co-transformants were plated on LB-X-Gal medium with positive interaction resulting in blue colouration, which can be seen for all of the interactions in both plasmid combinations, but not for the negative control. Images are representative of at least 4 independent experiments.


Confirmation of BTH interactions by β - Galactosidase activity assays . interaction is significantly stronger between Bd3125 N - terminal domain of Bd1971 than interaction with C - terminus of Bd1971 . response regulator domain of Bd0367 showed significant interaction with Bd1971 , but full Bd0367 did not . * p > 0 . 05 by Student ' s t - N / S - not significant . Data are from at least three independent experiments , error bars are standard error of

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

Confirmation of BTH interactions by β-Galactosidase activity assays. The interaction is significantly stronger between Bd3125 and the N-terminal domain of Bd1971 than the interaction with the C-terminus of Bd1971. The response regulator domain of Bd0367 showed significant interaction with Bd1971, but the full length Bd0367 did not. *p>0.05 by Student's t-test N/S- not significant. Data are from at least three independent experiments, error bars are the standard error of the mean


B . Blue native - PAGE loading of extracts from nrc2 / 3 / 4 knockout N . benthamiana after immunoprecipitation with anti - Flag antibody . Co - migration of Rpi - amr3 - HF AVRamr3 - V5 are indicated ( * , red ) . Same samples were loaded twice on one blue native - PAGE gel , transferred onto one , then was cut into two immunoblotted separately . GUS - V5 , β - glucuronidase fused with V5 tag . C . NRC2EEE - Myc does not alter association between Rpi - amr3 AVRamr3 . Samples of Fig 1B were SDS - boiled loaded on SDS - PAGE . Input samples were taken prior to immunoprecipitation to show expression of .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Blue native-PAGE loading of protein extracts from nrc2/3/4 knockout N. benthamiana plants after immunoprecipitation with anti-Flag antibody. Co-migration of Rpi-amr3-HF and AVRamr3-V5 are indicated (*, red). Same samples were loaded twice on one blue native-PAGE gel, transferred onto one membrane, and then the membrane was cut into two and immunoblotted separately. GUS-V5, β-glucuronidase fused with V5 tag. C. NRC2EEE-Myc does not alter association between Rpi-amr3 and AVRamr3. Samples of Fig 1B were SDS-boiled and loaded on SDS-PAGE. Input samples were taken prior to immunoprecipitation to show expression of all proteins.


A . NRC2EEE - Myc is oligomerized upon effector - dependent activation of Rpi - amr3 . lysates from Fig 1B , C were loaded on blue native - PAGE . SDS - boiled lysate samples serve as control for actual of NRC2EEE - Myc . Oligomerized NRC2EEE - Myc is indicated ( * , red ) . B . NRC2EEE - Myc oligomerizes upon effector - dependent activation of Rpi - amr1 . lysates from nrc2 / 3 / 4 knockout N . benthamiana were loaded on blue native - PAGE . Oligomerized NRC2EEE - Myc is indicated ( * , red ) .

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A. NRC2EEE-Myc is oligomerized upon effector-dependent activation of Rpi-amr3. Protein lysates from Fig 1B, C were loaded on blue native-PAGE. SDS-boiled protein lysate samples serve as control for actual size of NRC2EEE-Myc. Oligomerized NRC2EEE-Myc is indicated (*, red). B. NRC2EEE-Myc oligomerizes upon effector-dependent activation of Rpi-amr1. Protein lysates from nrc2/3/4 knockout N. benthamiana plants were loaded on blue native-PAGE. Oligomerized NRC2EEE-Myc is indicated (*, red).


C . Samples from Fig 2B were SDS - boiled loaded on SDS - PAGE . accumulation of Rpi - amr1 - Flag , NRC2EEE - Myc , AVRamr1 - V5 AVRamr3 - V5 are shown .

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C. Samples from Fig 2B were SDS-boiled and loaded on SDS-PAGE. Protein accumulation of Rpi-amr1-Flag, NRC2EEE-Myc, AVRamr1-V5 and AVRamr3-V5 are shown.


B . NRC2EEE Rpi - amr3 migration in absence of effector . N . benthamiana nrc2 / 3 / 4 knockout were transiently infiltrated with Rpi - amr3 - HF , NRC2EEE - Myc GUS - V5 followed by 2D - PAGE . NRC2EEE - Myc ( * , red ) Rpi - amr3 - HF ( * , blue ) is indicated . C . NRC2EEE Rpi - amr3 migration in presence of effector . N . benthamiana nrc2 / 3 / 4 knockout were transiently infiltrated with Rpi - amr3 - HF , NRC2EEE - Myc AVRamr3 - V5 followed by 2D - PAGE . NRC2EEE - Myc ( * , red ) Rpi - amr3 - HF ( * , blue ) is indicated .

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B. NRC2EEE and Rpi-amr3 migration in the absence of effector. N. benthamiana nrc2/3/4 knockout plants were transiently infiltrated with Rpi-amr3-HF, NRC2EEE-Myc and GUS-V5 followed by 2D-PAGE. NRC2EEE-Myc proteins (*, red) and Rpi-amr3-HF proteins (*, blue) is indicated. C. NRC2EEE and Rpi-amr3 migration in the presence of effector. N. benthamiana nrc2/3/4 knockout plants were transiently infiltrated with Rpi-amr3-HF, NRC2EEE-Myc and AVRamr3 -V5 followed by 2D-PAGE. NRC2EEE-Myc proteins (*, red) and Rpi-amr3-HF protein (*, blue) is indicated.


D . Re - localization of NRC2EEE - Myc upon effector - dependent activation of Rpi - amr1 . Lysates of nrc2 / 3 / 4 knockout transiently expressing Rpi - amr1 - Flag , NRC2EEE - Myc with AVRamr3 - V5 or AVRamr1 - V5 were fractionated into total ( T ) , soluble ( S ) , pellet ( P ) fractions . E . Re - localization of NRC2EEE - Myc upon effector - dependent activation of Rpi - amr3 . Lysates of nrc2 / 3 / 4 knockout transiently expressing Rpi - amr3 - HF , NRC2EEE - Myc with AVRamr3 - V5 or AVRamr1 - V5 were fractionated into total ( T ) , soluble ( S ) , pellet ( P ) fractions .

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D. Re-localization of NRC2EEE-Myc upon effector-dependent activation of Rpi-amr1. Lysates of nrc2/3/4 knockout plants transiently expressing Rpi-amr1-Flag, NRC2EEE-Myc with AVRamr3-V5 or AVRamr1-V5 were fractionated into total (T), soluble (S), and pellet (P) fractions. E. Re-localization of NRC2EEE-Myc upon effector-dependent activation of Rpi-amr3. Lysates of nrc2/3/4 knockout plants transiently expressing Rpi-amr3-HF, NRC2EEE-Myc with AVRamr3-V5 or AVRamr1-V5 were fractionated into total (T), soluble (S), and pellet (P) fractions.


A . P - loop of Rpi - amr3 is required for AVRamr3 - dependent HR in N . benthamiana . Representative leaf phenotype of HR ( hypersensitive response ) in wild - type N . benthamiana is shown . number of leaves tested occurrences of HR are indicated in parentheses . B . P - loop of NRC2 is required for AVRamr3 - dependent HR in N . benthamiana nrc2 / 3 / 4 knockout . Representative leaf phenotype of HR is shown . number of leaves tested occurrences of HR are indicated in parentheses .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. P-loop of Rpi-amr3 is required for AVRamr3-dependent HR in N. benthamiana. Representative leaf phenotype of HR (hypersensitive response) in wild-type N. benthamiana is shown. The number of leaves tested and occurrences of HR are indicated in parentheses. B. P-loop of NRC2 is required for AVRamr3-dependent HR in N. benthamiana nrc2/3/4 knockout plants. Representative leaf phenotype of HR is shown. The number of leaves tested and occurrences of HR are indicated in parentheses.


C . NRC2EEE - Myc requires functional P - loop of both Rpi - amr3 NRC2 for oligomerization . lysates from N . benthamiana nrc2 / 3 / 4 knockout expressing wild - type or P - loop mutants of Rpi - amr3 or NRC2EEE were loaded for blue native - PAGE . Membranes were immunoblotted with anti - Myc .

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C. NRC2EEE-Myc requires functional P-loop of both Rpi-amr3 and NRC2 for oligomerization. Protein lysates from N. benthamiana nrc2/3/4 knockout plants expressing wild-type or P-loop mutants of Rpi-amr3 or NRC2EEE were loaded for blue native-PAGE. Membranes were immunoblotted with anti-Myc.


B . lysates expressing NRC4AAA - Myc with Rpi - amr3 - HF or Rpi - amr1 - Flag in presence absence of cognate effector were loaded on blue native - PAGE . were transiently expressed in N . benthamiana nrc2 / 3 / 4 knockout . Non - specific bands are indicated with * .

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B. Protein lysates expressing NRC4AAA-Myc with Rpi-amr3-HF or Rpi-amr1-Flag in the presence and absence of cognate effector were loaded on blue native-PAGE. Proteins were transiently expressed in N. benthamiana nrc2/3/4 knockout plants. Non-specific bands are indicated with *.


B . NRC2EEE - Myc oligomerizes in response - dependent manner . lysates from N . benthamiana nrc2 / 3 / 4 knockout transiently expressing NRC2EEE - Myc , Rpi - amr3 - HF AVRamr3 alleles from P . infestans , P . capsici , P . parasitica were loaded on blue native - PAGE blotted for NRC2EEE ( anti - Myc ) . C . Recognition is correlated with interaction formation of AVRamr3 with Rpi - amr3 . extracts from Fig 5B were immunoprecipitated with anti - Flag antibody , separated on blue native - PAGE , AVRamr3 - GFP of P . infestans , P . capsici , P . parasitica were visualized .

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B. NRC2EEE-Myc oligomerizes in response-dependent manner. Protein lysates from N. benthamiana nrc2/3/4 knockout transiently expressing NRC2EEE-Myc, Rpi-amr3-HF and AVRamr3 alleles from P. infestans, P. capsici, and P. parasitica were loaded on blue native-PAGE and blotted for NRC2EEE (anti-Myc). C. Recognition is correlated with interaction and protein complex formation of AVRamr3 with Rpi-amr3. Protein extracts from Fig 5B were immunoprecipitated with anti-Flag antibody, separated on blue native-PAGE, and AVRamr3-GFP proteins of P. infestans, P. capsici, and P. parasitica were visualized.


( a ) HeLa lysates were immunoprecipitated with Atg16L1 antibody ( Atg16L1 ) or no antibody ( No Ab ) in control , subjected to SDS - PAGE ; were stained with SimplyBlue safe stain ( Invitrogen ) in accordance with manufacturer ' s protocol . Bands indicated in box were cut out digested with trypsin , identified by MALDI - TOF MS . HC , heavy chain .

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(a) HeLa cell lysates were immunoprecipitated with Atg16L1 antibody (Atg16L1) or no antibody (No Ab) in control, and subjected to SDS-PAGE; proteins were stained with SimplyBlue safe stain (Invitrogen) in accordance with the manufacturer's protocol. Bands indicated in the box were cut out and digested with trypsin, and identified by MALDI-TOF MS. HC, heavy chain.


( b ) HeLa transfected for 24 h with control vector , or wild - type or different deletion mutants ( residues 2 - 77 , 2 - 275 232 - 607 ) of Flag - Atg16L1 were immunoprecipitated with anti - Flag antibody ( Atg16L1 ) immunoblotted with anti - clathrin , anti - Flag anti - Atg5 antibodies . Clathrin interacts with N terminus of Atg16L1 , in a similar manner to Atg12 - Atg5 . Total lysates were run alongside as controls for input . FL , full - .

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(b) HeLa cells transfected for 24 h with control vector, or wild-type or different deletion mutants (residues 2-77, 2-275 and 232-607) of Flag-Atg16L1 were immunoprecipitated with anti-Flag antibody (Atg16L1) and immunoblotted with anti-clathrin, anti-Flag and anti-Atg5 antibodies. Clathrin interacts with the N terminus of Atg16L1, in a similar manner to Atg12-Atg5. Total lysates were run alongside as controls for protein input. FL, full-length.


( c ) HeLa lysates were immunoprecipitated ( IP ) with Atg16L1 antibody ( + Ab ) or no antibody ( no Ab ) immunoblotted with anti - clathrin , anti - AP2 anti - Atg16L1 antibodies . Total lysates ( TL ) were also blotted for input controls . Endogenous Atg16L1 interacts with clathrin heavy chain AP2 .

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(c) HeLa cell lysates were immunoprecipitated (IP) with Atg16L1 antibody (+Ab) or no antibody (no Ab) and immunoblotted with anti-clathrin, anti-AP2 and anti-Atg16L1 antibodies. Total cell lysates (TL) were also blotted for input controls. Endogenous Atg16L1 interacts with clathrin heavy chain and AP2.


( d ) HeLa transfected for 24 h with Flag - Atg16L1 increasing concentrations of Atg5 were immunoprecipitated ( IP ) with anti - Flag antibody ( Atg16L1 ) immunoblotted with anti - clathrin , anti - Flag anti - Atg5 antibodies . Atg5 overexpression neither interferes with nor increases clathrin - Atg16L1 binding . Uncropped of blots are shown in Supplementary Information , Fig . S9 .

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(d) HeLa cells transfected for 24 h with Flag-Atg16L1 and increasing concentrations of Atg5 were immunoprecipitated (IP) with anti-Flag antibody (Atg16L1) and immunoblotted with anti-clathrin, anti-Flag and anti-Atg5 antibodies. Atg5 overexpression neither interferes with nor increases clathrin-Atg16L1 binding. Uncropped images of blots are shown in Supplementary Information, Fig. S9.


( a ) HeLa transfected for 5 days with two rounds of control , epsin 1 ( eps . ) , clathrin heavy chain ( cla . ) , AP2 or AP1 siRNA were either left untreated or treated for 4 h with bafilomycin A1 ( + / - BafA1 ) , after which they were lysed for western blot with anti - LC3 anti - tubulin antibodies . Note that LC3 - I is often very faint compared with LC3 - II in HeLa under extraction conditions we use . However , this is not a problem because it is advisable to relate LC3 - II to tubulin / actin .

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

(a) HeLa cells transfected for 5 days with two rounds of control, epsin 1 (eps.), clathrin heavy chain (cla.), AP2 or AP1 siRNA were either left untreated or treated for 4 h with bafilomycin A1 (+/- BafA1), after which they were lysed for western blot analysis with anti-LC3 and anti-tubulin antibodies. Note that LC3-I is often very faint compared with LC3-II in HeLa cells under the protein extraction conditions we use. However, this is not a problem because it is advisable to relate LC3-II to tubulin/actin.


( b ) of LC3 - II to tubulin on knockdown ( KD ) of control , epsin , clathrin heavy chain , AP2 or AP1 , with or without bafilomycin A1 , quantified from three independent experiments ( two for epsin ) represented in graph . Asterisk , P 0 . 01 ; two asterisks , P 0 . 001 ; three asterisks , P 0 . 0001 .

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(b) Ratio of LC3-II to tubulin on knockdown (KD) of control, epsin, clathrin heavy chain, AP2 or AP1, with or without bafilomycin A1, quantified from three independent experiments (two for epsin) and represented in the graph. Asterisk, P 0.01; two asterisks, P 0.001; three asterisks, P 0.0001.


( c ) HeLa stably expressing GFP - mRFP - LC3 ( ref . 17 ) were transfected for 5 days with two rounds of control or clathrin heavy chain siRNA , during which they were either left untreated ( UT ) or treated with bafilomycin A1 ( BafA1 ) for last 15 h . were then fixed analysed on a Cellomics ArrayScan system . of autophagic vacuoles ( AV ) or autolysosomes ( AL ) per in different conditions is shown in graph . Asterisk , P 0 . 01 ; n . s . , not significant . n = 2 , 000 .

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

(c) HeLa cells stably expressing GFP-mRFP-LC3 (ref. 17) were transfected for 5 days with two rounds of control or clathrin heavy chain siRNA, during which they were either left untreated (UT) or treated with bafilomycin A1 (BafA1) for the last 15 h. Cells were then fixed and analysed on a Cellomics ArrayScan system. Quantification of autophagic vacuoles (AV) or autolysosomes (AL) per cell in the different conditions is shown in the graph. Asterisk, P 0.01; n.s., not significant. n = 2,000 cells.


( d ) HeLa transfected for 4 days with control , clathrin heavy chain , AP1 or AP2 siRNA were grown in Hanks balanced salt solution ( HBSS ) for 6 h , after which were fixed immunostained for endogenous LC3 . percentages of HeLa with more than 50 LC3 vesicles ( marked with arrows ) are quantified in graph . End . , endogenous . Three asterisks , P 0 . 0001 . n = 500 . error bars represent s . e . m . Scale bars , 10 μm . Uncropped of blots are shown in Supplementary Information , Fig . S9 .

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(d) HeLa cells transfected for 4 days with control, clathrin heavy chain, AP1 or AP2 siRNA were grown in Hanks balanced salt solution (HBSS) for 6 h, after which the cells were fixed and immunostained for endogenous LC3. The percentages of HeLa cells with more than 50 LC3 vesicles (marked with arrows) are quantified in the graph. End., endogenous. Three asterisks, P 0.0001. n = 500 cells. All error bars represent s.e.m. Scale bars, 10 μm. Uncropped images of blots are shown in Supplementary Information, Fig. S9.


( a - d ) HeLa transfected for 72 h with control , epsin 1 , clathrin heavy chain , AP2 or AP1 siRNA were treated with HBSS ( to induce autophagy ) for 6 h , after which they were fixed immunostained for endogenous Atg16L1 . Representative for control epsin 1 siRNA are in a , percentages of HeLa with Atg16L1vesicles are quantified in b ( for epsin 1 ) , c ( for clathrin ) d ( for AP1 AP2 ) . Three asterisks , P 0 . 0001 . Scale bars , 10 μm . ( Note that we used single siRNAs for experiments effects were confirmed with two independent sequences for clathrin heavy chain ) . n = 600 .

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(a-d) HeLa cells transfected for 72 h with control, epsin 1, clathrin heavy chain, AP2 or AP1 siRNA were treated with HBSS (to induce autophagy) for 6 h, after which they were fixed and immunostained for endogenous Atg16L1. Representative cells for the control and epsin 1 siRNA are in a, and percentages of HeLa cells with Atg16L1vesicles are quantified in b (for epsin 1), c (for clathrin) and d (for AP1 and AP2). Three asterisks, P 0.0001. Scale bars, 10 μm. (Note that we used single siRNAs for all experiments and the effects were confirmed with two independent sequences for clathrin heavy chain). n = 600 cells.


( e , f ) HeLa transfected for 24 h with Flag - tagged wild - type Atg16L1 or Atg16L1 deletion mutants 2 - 275 Atg16L1 or 232 - 607 Atg16L1 were fixed immunostained with anti - Flag antibody ( e ) ; proportions of with Flag - Atg16L1 vesicles are represented in f . Representative of Atg16L1 vesicles with different mutant constructs are shown in e . large boxes in each panel are higher magnifications of regions in smaller boxes . Three asterisks , P 0 . 0001 ; n . s . , not significant . n = 100 .

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(e, f) HeLa cells transfected for 24 h with Flag-tagged wild-type Atg16L1 or the Atg16L1 deletion mutants 2-275 Atg16L1 or 232-607 Atg16L1 were fixed and immunostained with anti-Flag antibody (e); the proportions of cells with Flag-Atg16L1 vesicles are represented in f. Representative images of Atg16L1 vesicles with the different mutant constructs are shown in e. The large boxes in each panel are higher magnifications of regions in the smaller boxes. Three asterisks, P 0.0001; n.s., not significant. n = 100 cells.


( g - i ) HeLa transfected with control , epsin 1 , clathrin heavy chain , AP2 or AP1 siRNA , as above , were transfected with GFP - Atg16L1 tomato LC3 for a further 24 h , after which were fixed . g shows representative for control , epsin 1 clathrin siRNA . of GFP - Atg16L1 ( green ) that co - localized ( yellow , marked by arrows ) with tomato LC3 vesicles ( red ) is quantified as shown in graph in h ( control , epsin 1 clathrin siRNA ) i ( control , AP2 AP1 siRNA ) . Three asterisks , P 0 . 0001 . n = 21 . Scale bars , 10 μm . error bars represent s . e . m .

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(g-i) HeLa cells transfected with control, epsin 1, clathrin heavy chain, AP2 or AP1 siRNA, as above, were transfected with GFP-Atg16L1 and tomato LC3 for a further 24 h, after which the cells were fixed. g shows representative cells for control, epsin 1 and clathrin siRNA. The percentage of GFP-Atg16L1 (green) that co-localized (yellow, marked by arrows) with tomato LC3 vesicles (red) is quantified as shown in the graph in h (control, epsin 1 and clathrin siRNA) and i (control, AP2 and AP1 siRNA). Three asterisks, P 0.0001. n = 21 cells. Scale bars, 10 μm. All error bars represent s.e.m.


( a ) HeLa transfected for 24 h with GFP - Atg16L1 were fixed to reveal Atg16L1 vesicles ( green ) . Scale bar , 10 μm .

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(a) HeLa cells transfected for 24 h with GFP-Atg16L1 were fixed to reveal the Atg16L1 vesicles (green). Scale bar, 10 μm.


( b ) HeLa transiently transfected for 20 h with GFP - Atg16L1 mRFP - GPi were cultured in starvation medium for a further 2 h . TIRF image with GFP - Atg16L1 ( green ) mRFP - GPi ( red ) is shown . Arrows indicate co - localization between Atg16L1 GPi . co - localization of GFP - Atg16L1 with mRFP - GPi is shown in graph . n = 2 . Scale bar , 5 μm .

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(b) HeLa cells transiently transfected for 20 h with GFP-Atg16L1 and mRFP-GPi were cultured in starvation medium for a further 2 h. TIRF image with GFP-Atg16L1 (green) and mRFP-GPi (red) is shown. Arrows indicate co-localization between Atg16L1 and GPi. The percentage co-localization of GFP-Atg16L1 with mRFP-GPi is shown in the graph. n = 2 cells. Scale bar, 5 μm.


( c ) HeLa transfected for 24 h with GFP - Atg16L1 were either left untreated or treated with 50 μM dynasore ( Sigma ) for 4 h processed for immunogold electron microscopy with anti - GFP antibodies ( 15 - nm gold particles ) anti - clathrin antibodies ( 10 - nm gold particles ) . Co - localization can be seen in boxed areas . of clathrin - coated structures ( CCS ) that were associated with Atg16L1 per 1 , 000 μm2 is shown in graph . Three asterisks , P 0 . 0001 . Scale bar , 100 nm . error bars represent s . e . m .

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(c) HeLa cells transfected for 24 h with GFP-Atg16L1 were either left untreated or treated with 50 μM dynasore (Sigma) for 4 h and processed for immunogold electron microscopy with anti-GFP antibodies (15-nm gold particles) and anti-clathrin antibodies (10-nm gold particles). Co-localization can be seen in the boxed areas. Quantification of clathrin-coated structures (CCS) that were associated with Atg16L1 per 1,000 μm2 is shown in the graph. Three asterisks, P 0.0001. Scale bar, 100 nm. All error bars represent s.e.m.


( a ) HeLa transfected for 24 h with GFP - Atg16L1 ( green ) CFP - LC3 ( blue ) were incubated with Alexa Fluor - 555 - conjugated cholera toxin subunit B ( red ) for 15 min at 4 ° C ( allowing toxin to bind to plasma ) . Then were incubated at 37 ° C ( allowing internalization of cholera toxin ) for 10 min fixed for confocal . Vesicles positive for both Atg16L1 cholera toxin are yellow ( also see high - magnification at right ) . Note that small Atg16L1 vesicles co - localizing with cholera toxin are negative for LC3 ( as marked with yellow arrows ) , Atg16L1 vesicles co - localizing with both cholera toxin LC3 ( marked with blue arrows ) are shown in magnified panels at right . Scale bar , 10 μm .

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(a) HeLa cells transfected for 24 h with GFP-Atg16L1 (green) and CFP-LC3 (blue) were incubated with Alexa Fluor-555-conjugated cholera toxin subunit B (red) for 15 min at 4 °C (allowing toxin to bind to the plasma membrane). Then cells were incubated at 37 °C (allowing internalization of cholera toxin) for 10 min and fixed for confocal analysis. Vesicles positive for both Atg16L1 and cholera toxin are yellow (also see high-magnification images at the right). Note that the small Atg16L1 vesicles co-localizing with cholera toxin are negative for LC3 (as marked with yellow arrows), and the Atg16L1 vesicles co-localizing with both cholera toxin and LC3 (marked with blue arrows) are shown in the magnified panels at the right. Scale bar, 10 μm.


( b ) HeLa transfected for 24 h with GFP - Atg16L1 ( green ) were incubated with Alexa Fluor - 555 - conjugated cholera toxin subunit B ( red ) as in a , after which they were fixed , immunostained for endogenous ( end . ) EEA1 ( blue ) analysed by confocal microscopy . Vesicles positive for Atg16L1 cholera toxin B are yellow ( arrows ) vesicles positive for both EEA1 cholera toxin are purple ( blue arrows ) are shown in high - magnification in middle . graph shows co - localization . Cho . tox . , cholera toxin . n = 20 . Scale bar , 10 μm .

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(b) HeLa cells transfected for 24 h with GFP-Atg16L1 (green) were incubated with Alexa Fluor-555-conjugated cholera toxin subunit B (red) as in a, after which they were fixed, immunostained for endogenous (end.) EEA1 (blue) and analysed by confocal microscopy. Vesicles positive for Atg16L1 and cholera toxin B are yellow (white arrows) and vesicles positive for both EEA1 and cholera toxin are purple (blue arrows) and are shown in the high-magnification images in the middle. The graph shows percentage co-localization. Cho. tox., cholera toxin. n = 20 cells. Scale bar, 10 μm.


( c ) HeLa treated with HBSS for 6 h were incubated with Alexa Fluor - 555 - labelled cholera toxin subunit B ( red ) as in a . were then fixed , immunostained for endogenous Atg16L1 ( green ) endogenous EEA1 ( blue ) . were analysed as in b ; graph shows of results . Scale bar , 10 μm . Arrows indicate Atg16L1 - cholera toxin co - localization ; arrowheads indicate EEA1 - cholera toxin co - localization . n = 20 .

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(c) HeLa cells treated with HBSS for 6 h were incubated with Alexa Fluor-555-labelled cholera toxin subunit B (red) as in a. Cells were then fixed, and immunostained for endogenous Atg16L1 (green) and endogenous EEA1 (blue). Cells were analysed as in b; the graph shows quantification of the results. Scale bar, 10 μm. Arrows indicate Atg16L1-cholera toxin co-localization; arrowheads indicate EEA1-cholera toxin co-localization. n = 20 cells.


( d ) HeLa treated with HBSS for 6 h were incubated with Alexa Fluor - 555 - labelled cholera toxin subunit B ( red ) as in a . were then fixed immunostained for endogenous Atg5 ( green ) or endogenous Atg12 ( green ) . Co - localization of Atg5 ( n = 18 ) or Atg12 vesicles ( n = 39 ) with cholera toxin is shown in yellow ( arrows ) is quantified in graph . Scale bar , 10 μm . error bars represent s . e . m .

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(d) HeLa cells treated with HBSS for 6 h were incubated with Alexa Fluor-555-labelled cholera toxin subunit B (red) as in a. Cells were then fixed and immunostained for endogenous Atg5 (green) or endogenous Atg12 (green). Co-localization of the Atg5 (n = 18 cells) or Atg12 vesicles (n = 39 cells) with cholera toxin is shown in yellow (arrows) and is quantified in the graph. Scale bar, 10 μm. All error bars represent s.e.m.


( a ) HeLa transfected for 24 h with Flag - tagged wild - type Atg16L1 or Atg16L1 deletion mutants 2 - 275 or 232 - 607 Atg16L1 , together with GFP - Atg5 CFP - LC3 , were fixed immunostained with anti - Flag antibody before confocal . Co - localization of wild - type or 2 - 275 Atg16L1 ( red ) with GFP - Atg5 ( green ) is marked by arrows in insets . Scale bars , 10 μm .

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(a) HeLa cells transfected for 24 h with Flag-tagged wild-type Atg16L1 or the Atg16L1 deletion mutants 2-275 or 232-607 Atg16L1, together with GFP-Atg5 and CFP-LC3, were fixed and immunostained with anti-Flag antibody before confocal analysis. Co-localization of wild-type or 2-275 Atg16L1 (red) with GFP-Atg5 (green) is marked by arrows in the insets. Scale bars, 10 μm.


( 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 .

(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 .

(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 .

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 .

(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 - .

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 .

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(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 ) .

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

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 ) .

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

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 ) .

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

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 ) .

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

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 ) .

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

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 ) .

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

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 ) .

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

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 ) .

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

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 ) .

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

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 ) .

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

(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 ) .

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

(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 ) .

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

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 ) .

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

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 ) .

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

(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 ) .

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

(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 ) .

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

(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 ) .

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

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 ) .

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

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 ) .

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

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 ) .

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

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 .

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

(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 ) .

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

(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 .

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

(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 ) .

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

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 ) .

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

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 ) .

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

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 ) .

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

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 ) .

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

(M) 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 ) .

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

(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 .

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

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 .

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

(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 ) .

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

(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 ) .

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

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 ) .

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

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 ) .

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

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 .

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

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 .

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

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 .

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

(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 ) .

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

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 ) .

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

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 ) .

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

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

(O) 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 ) .

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

(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 ) .

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

(B) 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 ) .

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

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 ) .

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

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 )

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

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 ) .

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

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).