( C ) RPE - 1 hTERT p53 - / - BRCA1 - / - Cas9 ( WT ) or their isogenic RAP80 - / - counterparts expressing indicated GFP fusion were processed 1 h post - irradiation ( 10 Gy ) for immunofluorescence using GFP antibodies against BRCA1 γH2AX . A minimum of 100 per replicate were analyzed bars represent mean ± S . D ( n = 3 biological replicates ) . Representative are shown in Figure EV3B . ( D ) RPE - 1 hTERT p53 - / - BRCA1 - / - Cas9 or their isogenic RAP80 - / - counterparts expressing indicated GFP fusion were processed 1 h post - irradiation ( 10 Gy ) for immunofluorescence using GFP antibodies against RAD51 γH2AX . A minimum of 100 per replicate were analyzed bars represent mean ± S . D ( n = 3 biological replicates ) . Representative are shown in Figure EV3C . Scale bar is 5 μm in ( B ) .

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(C) RPE-1 hTERT p53-/- BRCA1-/- Cas9 cells (WT) or their isogenic RAP80-/- counterparts expressing the indicated GFP fusion proteins were processed 1 h post-irradiation (10 Gy) for immunofluorescence using GFP and antibodies against BRCA1 and γH2AX. A minimum of 100 cells per replicate were analyzed and the bars represent the mean ± S.D (n=3 biological replicates). Representative micrographs are shown in Figure EV3B. (D) RPE-1 hTERT p53-/- BRCA1-/- Cas9 cells or their isogenic RAP80-/- counterparts expressing the indicated GFP fusion proteins were processed 1 h post-irradiation (10 Gy) for immunofluorescence using GFP and antibodies against RAD51 and γH2AX. A minimum of 100 cells per replicate were analyzed and the bars represent the mean ± S.D (n=3 biological replicates). Representative micrographs are shown in Figure EV3C. Scale bar is 5 μm in (B).


( A , B ) MDA - MB - 436 transduced with lentivirus expressing indicated HA fusions were irradiated ( 10 Gy ) 3 h later were pulsed with EdU to label S phase 20 min prior to fixation . were processed for EdU labeling immunofluorescence using HA BRCA1 antibodies . Shown in ( A ) is quantitation of a minimum of 100 EdU + per replicate bars represent mean ± S . D ( n = 3 biological replicates ) . Representative are shown in ( B ) .

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(A, B) MDA-MB-436 cells transduced with lentivirus expressing the indicated HA fusions were irradiated (10 Gy) and 3 h later were pulsed with EdU to label S phase cells 20 min prior to fixation. Cells were processed for EdU labeling and immunofluorescence using HA and BRCA1 antibodies. Shown in (A) is the quantitation of a minimum of 100 EdU+ cells per replicate and the bars represent the mean ± S.D (n= 3 biological replicates). Representative micrographs are shown in (B).


MDA - MB - 436 transduced with lentivirus expressing indicated HA fusions were irradiated ( 10 Gy ) 3 h later were pulsed with EdU to label S phase 20 min prior to fixation . were processed for EdU labeling immunofluorescence using HA RAD51 antibodies . Representative are shown in ( D ) .

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MDA-MB-436 cells transduced with lentivirus expressing the indicated HA fusions were irradiated (10 Gy) and 3 h later were pulsed with EdU to label S phase cells 20 min prior to fixation. Cells were processed for EdU labeling and immunofluorescence using HA and RAD51 antibodies. Representative micrographs are shown in (D).


( E ) Clonogenic survival assays using indicated dose of olaparib MDA - MB - 436 transduced with lentivirus expressing indicated HA fusions . Data is shown as mean ± S . D ( n = 3 biological replicates ) . Representative clonogenic are shown in Figure EV5D .

O O O O O O O O O O O O O O O O O O O O O O O O O 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) Clonogenic survival assays using the indicated dose of olaparib and MDA-MB-436 cells transduced with lentivirus expressing the indicated HA fusions. Data is shown as the mean ± S.D (n=3 biological replicates). Representative clonogenic images are shown in Figure EV5D.


SDS - PAGE of SecYEG - ND sample after - exclusion chromatography . Asterisks indicate translocon - enriched fractions used for forming RNC FtsQ : SecYEG - ND . Lipid - loaded " empty " nanodiscs elute at larger volumes so can be separated .

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SDS-PAGE of SecYEG-ND sample after size-exclusion chromatography. Asterisks indicate translocon-enriched fractions used for forming the RNC FtsQ:SecYEG-ND complex. Lipid-loaded "empty" nanodiscs elute at larger volumes and so can be separated.


Representative cryo - EM micrograph of RNC FtsQ : SecYEG - ND . Exemplary ribosomes are encircled . Examples of two - dimensional classes of imaged particles . RNC : nanodisc assemblies can be seen at different view angles .

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Representative cryo-EM micrograph of RNC FtsQ:SecYEG-ND. Exemplary individual ribosomes are encircled. Examples of two-dimensional classes of imaged particles. RNC:nanodisc assemblies can be seen at different view angles.


Western - blot against hemagglutinin tag within nascent chain FtsQC40 reveals a cross - linking product of ~ 80 kDa in presence of nanodisc - reconstituted SecYC282EG SecYC283EG , presumably assigned to FtsQxSecY adduct . double band for FtsQC40 likely indicates incomplete cleavage of hexa - histidine tag by 3C protease .

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Western-blot against the hemagglutinin tag within the nascent chain FtsQC40 reveals a cross-linking product of ~80 kDa in presence of nanodisc-reconstituted SecYC282EG and SecYC283EG, presumably assigned to FtsQxSecY adduct. The double band for FtsQC40 likely indicates the incomplete cleavage of the hexa-histidine tag by 3C protease.


In - gel imaging of SecYCF488A reveals a cross - linking product of ~ 80 kDa assigned to FtsQC40xSecYC283 adduct ( " SecYxFtsQ " ) . Bands for covalently cross - linked SecY dimer ( " SecYxSecY " ) , adducts of SecY ribosomal ( " SecYxRiboP " ) are indicated . nascent chain - specific adduct " SecYxFtsQ " does not depend on presence of PE lipids .

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

In-gel fluorescence imaging of SecYCF488A reveals a cross-linking product of ~80 kDa assigned to the FtsQC40xSecYC283 adduct ("SecYxFtsQ"). Bands for covalently cross-linked SecY dimer ("SecYxSecY"), and adducts of SecY and ribosomal proteins ("SecYxRiboP") are indicated. The nascent chain-specific adduct "SecYxFtsQ" does not depend on the presence of PE lipids.


FtsQ : SecY cross - linking is position specific . FtsQ TMH C - terminal residues 40 - 42 could be cross - linked to lateral gate ( SecY residue 283 ) , while no adduct could be detected at upstream position 35 .

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

FtsQ:SecY cross-linking is position specific. FtsQ TMH C-terminal residues 40-42 could be cross-linked to the lateral gate (SecY residue 283), while no adduct could be detected at the upstream position 35.


C . from whole extracts culture supernatants of PAKΔretS , derivative tssA1 mutant ( ΔA1 ) ( both carrying pBBR plasmid ) complemented ΔA1 mutant carrying pBBR - tssA1 ( ΔA1 + A1 ) were analysed by Western blot . Polyclonal antibodies directed against TssA1 ( A1 ) , Hcp1 , VgrG1a Tse3 were used . anti - VgrG1a antibody detects VgrG1a , VgrG1b VgrG1c , as indicated on left . polymerase ( RNAP ) was monitored using monoclonal antibody directed against its β - subunit .

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

C. Proteins from whole cell extracts and culture supernatants of PAKΔretS, the derivative tssA1 mutant (ΔA1) (both carrying the pBBR plasmid) and the complemented ΔA1 mutant carrying pBBR-tssA1 (ΔA1+A1) were analysed by Western blot. Polyclonal antibodies directed against TssA1 (A1), Hcp1, VgrG1a and Tse3 were used. The anti-VgrG1a antibody detects VgrG1a, VgrG1b and VgrG1c, as indicated on the left. Cytoplasmic RNA polymerase (RNAP) was monitored using monoclonal antibody directed against its β-subunit.


A . BTH experiments showing that TssA1 is able to self - interact in vivo . A graphical representation of β - galactosidase activity from co - transformants of E . coli DHM1 producing TssA1 ( A1 ) fused to adenylate cyclase T25 or adenylate cyclase T18 subunits is shown . values shown on y axis correspond to activity in Miller units . In each case , average activity from two independent experiments is shown error bars indicate standard deviation ( S . D . ) . Experiments were carried out in quadruplicate .

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

A. BTH experiments showing that TssA1 is able to self-interact in vivo. A graphical representation of β-galactosidase activity from co-transformants of E. coli DHM1 cells producing TssA1 (A1) fused to the adenylate cyclase T25 or adenylate cyclase T18 subunits is shown. The values shown on the y axis correspond to the activity in Miller units. In each case, average activity from two independent experiments is shown and error bars indicate the standard deviation (S.D.). Experiments were carried out in quadruplicate.


B . In vitro cross - linking experiments of purified His6 - TssA1 . About 30 μg of purified His6 - TssA1 was cross - linked ( 30 min , 1h 2h ) at room using increasing amount of ethylene glycol - bis ( succinimidylsuccinate ) EGS ( 2 mM 5 mM ) where indicated . Western blot of cross - linked products using an anti - His6 monoclonal antibody is shown . cross - linked species are highlighted with stars with corresponding oligomeric state indicated on right ( 1x = monomer ; 2x = dimer ; 6x = hexamer ) . Molecular markers ( kDa ) are indicated on left .

O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-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 O O O O 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. In vitro cross-linking experiments of purified His6-TssA1. About 30 μg of purified His6-TssA1 was cross-linked (30 min, 1h and 2h) at room temperature using increasing amount of ethylene glycol-bis(succinimidylsuccinate) EGS (2 mM and 5 mM) where indicated. Western blot analysis of cross-linked products using an anti-His6 monoclonal antibody is shown. The cross-linked species are highlighted with stars with the corresponding oligomeric state indicated on the right (1x = monomer; 2x = dimer; 6x = hexamer). Molecular weight markers (kDa) are indicated on the left.


C . of oligomeric state of TssA1 by AUC . Sedimentation data of His6 - TssA1 ( 0 . 56 mg / ml ) recorded at a rotor speed of 20 , 000 rpm is shown . left panel shows sedimentation boundary fits for clarity , only every third scan is shown in fitted data plots . experimental absorbance data are shown as black circles , whereas boundary fits are shown as grey lines . right panel shows - distribution c ( s ) , obtained from fitting scan boundaries using SEDFIT , revealing a dodecameric peak ( Mr ~ 479 kDa ) at S20 , w value of 16 . 6 S with a minor peak ( Mr ~ 1 MDa ) at S20 , w value of 29 . 01 S .

O O O O O O B-MEASURED_VAR O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Analysis of the oligomeric state of TssA1 by AUC. Sedimentation data of His6-TssA1 (0.56 mg/ml) recorded at a rotor speed of 20,000 rpm is shown. The left panel shows the sedimentation boundary fits and for clarity, only every third scan is shown in the fitted data plots. The experimental absorbance data are shown as black circles, whereas the boundary fits are shown as grey lines. The right panel shows the size-distribution analysis c(s), obtained from fitting the scan boundaries using SEDFIT, revealing a dodecameric peak (Mr ~ 479 kDa) at S20,w value of 16.6 S with a minor peak (Mr ~1 MDa) at S20,w value of 29.01 S.


A . A representative micrograph of TssA1 ( left panel ) showing discrete ring - shaped particles with a diameter of ~ 260 Å . rotationally symmetrised average of data set ( inset panel ) presents two distinct rings ( numbered ) , disclosing layered nature of TssA1 . Representative class averages of TssA1 ( right panel ) demonstrating wide range of conformations taken by ring - shaped structures . Scale bars are 500 Å in left panel , 130 Å in inset 100 Å in right panels , respectively .

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A. A representative micrograph of the TssA1 complex (left panel) showing discrete ring-shaped particles with a diameter of ~260 Å. The rotationally symmetrised average of the data set (inset panel) presents two distinct rings (numbered), disclosing the layered nature of the TssA1 complex. Representative class averages of the TssA1 complex (right panel) demonstrating the wide range of conformations taken by the ring-shaped structures. Scale bars are 500 Å in the left panel, 130 Å in the inset and 100 Å in the right panels, respectively.


B . Double MSA classification : ( i ) representative orientation class averages show TssA1 rings arranged into four - fold ( 4x ) , five - fold ( 5x ) six - fold ( 6x ) symmetry - like complexes ( indicated in red , blue green , respectively ) ; ( ii ) sub - class averages obtained from sub - classification of corresponding orientation class ; ( iii ) calculated difference showing some or black peaks ( coloured circles ) at outer ring region . Scale bar is 100 Å .

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B. Double MSA classification: (i) representative orientation class averages show TssA1 rings arranged into four-fold (4x), five-fold (5x) and six-fold (6x) symmetry-like complexes (indicated in red, blue and green, respectively); (ii) sub-class averages obtained from sub-classification of the corresponding orientation class; and (iii) calculated difference images showing some white or black peaks (coloured circles) at the outer ring region. Scale bar is 100 Å.


D . SEC - MALS of purified His6 - TssA11 - 245 . TssA11 - 245 elution peak is shown corresponding mass is indicated . molar mass differential refractive index ( RI ) are plotted versus elution volume . Data were analysed using ASTRA method ( Slotboom , Duurkens et al . , 2008 ) .

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D. SEC-MALS analysis of the purified His6-TssA11-245. The TssA11-245 elution peak is shown and the corresponding mass is indicated. The molar mass and the differential refractive index (RI) are plotted versus the elution volume. Data were analysed using the ASTRA method (Slotboom, Duurkens et al., 2008).


A . Co - purification experiments using His6 - TssA1 untagged TssB1C1 . Western blot of Ni - NTA outputs using antibodies directed against His6 - tag ( upper panel ) TssB1 ( lower panel ) . I , Input ; FT , Flow through ; W , Wash E , Elution .

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A. Co-purification experiments using His6-TssA1 and untagged TssB1C1. Western blot analysis of the Ni-NTA outputs using antibodies directed against the His6-tag (upper panel) and TssB1 (lower panel). I, Input; FT, Flow through; W, Wash and E, Elution.


B . SEC elution fraction of co - purified His6 - TssA1 TssB1C1 . SDS - PAGE showing presence of three His6 - TssA1 ( A1 ) , TssB1 ( B1 ) TssC1 ( C1 ) . Molecular markers ( kDa ) are indicated on left .

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B. SEC elution fraction of the co-purified His6-TssA1 and TssB1C1. SDS-PAGE showing the presence of the three proteins His6-TssA1 (A1), TssB1 (B1) and TssC1 (C1). Molecular weight markers (kDa) are indicated on the left.


C . Representative of immunogold - labelled His6 - TssA1 / TssB1C1 . presence of gold particles is indicated with arrows . Two close - up views of TssB1C1 sheath displaying gold particles at one extremity are shown ( inset panels ) . Scale bars are 2 , 000 Å 500 Å for 1 2 - 6 , respectively .

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C. Representative micrographs of the immunogold-labelled complex His6-TssA1/TssB1C1. The presence of gold particles is indicated with white arrows. Two close-up views of TssB1C1 sheath displaying gold particles at one extremity are shown (inset panels). Scale bars are 2,000 Å and 500 Å for images 1 and 2-6, respectively.


D . BTH experiment showing interaction between TssA1 ( A1 ) Hcp1 . A graphical representation of β - galactosidase activity from E . coli DHM1 producing indicated fused to adenylate cyclase T25 or adenylate cyclase T18 subunits is shown . values shown on y axis correspond to activity in Miller units . In each case , average activity from two independent experiments is shown error bars indicate standard deviation ( S . D . ) . Experiments were carried out in triplicate .

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 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

D. BTH experiment showing interaction between TssA1 (A1) and Hcp1. A graphical representation of β-galactosidase activity from E. coli DHM1 cells producing the indicated proteins fused to the adenylate cyclase T25 or adenylate cyclase T18 subunits is shown. The values shown on the y axis correspond to the activity in Miller units. In each case, average activity from two independent experiments is shown and error bars indicate the standard deviation (S.D.). Experiments were carried out in triplicate.


B . Cartoon representations of dodecameric gp6 _ 334C structure ( PDB code 3H3W ) . Chains A B of gp6 dimers are shown in magenta green respectively , conserved blocks mapped onto assembled dodecameric gp6 structure are shown in orange . A close up view showing conserved blocks present in regions of gp6 involved in ring assembly , is shown on right .

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B. Cartoon representations of the dodecameric gp6_334C structure (PDB code 3H3W). Chains A and B of gp6 dimers are shown in magenta and green respectively, and the conserved blocks mapped onto the assembled dodecameric gp6 structure are shown in orange. A close up view showing the conserved blocks present in regions of gp6 involved in ring assembly, is shown on the right.


A microscopy showing presence / absence of TssBC sheaths ( seen as foci ) in ∆ B1 : : B1 - sfGFP ( top panel ) , ∆ B1 ∆ A1 : : B1 - sfGFP , ∆ B1 ∆ E1 : : B1 - sfGFP ( middle panels ) in complemented strains ∆ B1 ∆ A1 : : B1 - sfGFP + A1 ∆ B1 ∆ E1 : : B1 - sfGFP + E1 ( bottom panels ) . Scale bars are 2 μm . B . of TssB1 - sfGFP foci of diffuse in various P . aeruginosa backgrounds imaged in panel A . Values are normalized to total number of ( % ) . Data were obtained from three independent experiments , with values from representative replicates being shown .

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

A Fluorescence microscopy images showing the presence/absence of TssBC sheaths (seen as fluorescent foci) in the ∆B1::B1-sfGFP (top panel), ∆B1∆A1::B1-sfGFP, ∆B1∆E1::B1-sfGFP cells (middle panels) and in the complemented strains ∆B1∆A1::B1-sfGFP + A1 and ∆B1∆E1::B1-sfGFP + E1 (bottom panels). Scale bars are 2 μm. B. Quantification of TssB1-sfGFP foci and of diffuse fluorescence in various P. aeruginosa backgrounds imaged in panel A. Values are normalized to the total number of bacteria (%). Data were obtained from three independent experiments, with images and values from representative replicates being shown.


BTH screen for interactions between TssA1 T6SS components of interest . A graphical representation ( bottom ) of β - galactosidase activity from E . coli DHM1 producing indicated fused to adenylate cyclase T25 or adenylate cyclase T18 subunits of corresponding spots on X - gal LB agar plates ( top ) are shown . combination of T25 / T18 fusion are indicated as 25 or 18 followed by name of fused T6SS . T6SS are abbreviated as follows : A1 = TssA1 , E1 = TssE1 , F1 = TssF1 , G1 = TssG1 K1 = TssK1 . values shown on y axis correspond to activity in Miller units . In each case , average activity from two independent experiments is shown error bars indicate standard deviation ( S . D . ) . Experiments were carried out in triplicate .

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

BTH screen for interactions between TssA1 and T6SS components of interest. A graphical representation (bottom) of β-galactosidase activity from E. coli DHM1 cells producing the indicated proteins fused to the adenylate cyclase T25 or adenylate cyclase T18 subunits and images of corresponding spots on X-gal LB agar plates (top) are shown. The combination of T25/T18 fusion proteins are indicated as 25 or 18 followed by the name of the fused T6SS protein. T6SS proteins are abbreviated as follows: A1=TssA1, E1=TssE1, F1=TssF1, G1=TssG1 and K1=TssK1. The values shown on the y axis correspond to the activity in Miller units. In each case, average activity from two independent experiments is shown and error bars indicate the standard deviation (S.D.). Experiments were carried out in triplicate.


B . Cartoon representation of gp6 / gp8 baseplate subcomplex of T4 phage . gp6 ring structure ( PDB code 3H3W ) is shown using same colour code described in figure 5 . chains A B of gp8 dimers ( PDB code 1PDM ) are shown in blue yellow , respectively . conserved blocks mapped onto dimeric structure of gp8 are shown in orange . A close up view of gp8 structure ( bottom ) shows that conserved blocks are involved in dimerization interaction with gp6 .

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

B. Cartoon representation of the gp6/gp8 baseplate subcomplex of the T4 phage. The gp6 ring structure (PDB code 3H3W) is shown using the same colour code described in figure 5. The chains A and B of the gp8 dimers (PDB code 1PDM) are shown in blue and yellow, respectively. The conserved blocks mapped onto the dimeric structure of gp8 are shown in orange. A close up view of the gp8 structure (bottom) shows that the conserved blocks are involved in dimerization and interaction with gp6.


A . Structure of sheath / baseplate of bacteriophage T4 , adapted from Kostyuchenko et al . ( Kostyuchenko , Leiman et al . , 2003 ) . A colour code with corresponding gp is provided . proposed corresponding homolog in T6SS is indicated with 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

A. Structure of the sheath/baseplate complex of the bacteriophage T4, adapted from Kostyuchenko et al. (Kostyuchenko, Leiman et al., 2003). A colour code with corresponding gp protein is provided. The proposed corresponding homolog in the T6SS is indicated with parentheses.


I Correlation between peptide - spectrum matches ( PSMs ) of identified their fold change calculated by SAINTexpress .

I Correlation between peptide-spectrum matches (PSMs) of identified proteins and their fold change calculated by SAINTexpress.


A Pulldown Western blot validated interaction between viral ORF3a its interactors , VPS11 CLCC1 , binding of S to SPCS2 . HEK293T with SFB - tagged bait expression were collected lysed . lysates were subjected to pulldown using S - beads . Western blot was conducted with indicated antibodies . transfected with vector or construct encoding control genes were included as controls in these experiments .

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A Pulldown and Western blot analysis validated the interaction between the viral protein ORF3a and its interactors, VPS11 and CLCC1, and the binding of S protein to SPCS2. HEK293T cells with SFB-tagged bait expression were collected and lysed. Cell lysates were subjected to pulldown assay using S-protein beads. Western blot analysis was conducted with the indicated antibodies. Cells transfected with vector or construct encoding control genes were included as controls in these experiments.


B Immunostaining of localization . U2OS were transfected with construct encoding ORF3a . were fixed stained with indicated antibodies . green signal is CLCC1 or lysosome marker LAMP1 , red signal is flag ( for SFB - ORF3a ) , blue signal indicates DAPI / nuclei . Scale bar : 10 µm .

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B Immunostaining analysis of protein localization. U2OS cells were transfected with construct encoding ORF3a. Cells were fixed and stained with the indicated antibodies. The green signal is CLCC1 or lysosome marker LAMP1, the red signal is flag (for SFB-ORF3a), and the blue signal indicates DAPI/nuclei. Scale bar: 10 µm.


B Comparison of SARS - CoV - 2 - host interaction to genetic screening with CRISPR technique . Two different MOIs ( MOI 0 . 01 MOI 0 . 3 ) were used in these screens we compared our results with both of these datasets . Blue dots are identified genes from genetic screening . Orange dots are highlighted overlapped genes between our - host interaction study results from genetic screening . Top five overlapped genes are indicated .

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B Comparison of SARS-CoV-2 virus-host interaction to the genetic screening with CRISPR technique. Two different MOIs (MOI 0.01 and MOI 0.3) were used in these screens and we compared our results with both of these datasets. Blue dots are the identified genes from the genetic screening. Orange dots are the highlighted overlapped genes between our virus-host interaction study and the results from the genetic screening. Top five overlapped genes are indicated.


C Analyzing number of HCIPs with expression changes identified by single - sequencing using from COVID - 19 .

C Analyzing the number of HCIPs with RNA expression changes identified by single-cell RNA sequencing using cells from COVID-19 patients.


D Pulldown Western blot validation of interaction between human coronavirus NSP1 human PYCR1 / PYCR2 . transfected with vector or construct encoding GFP were included as controls in these experiments .

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D Pulldown and Western blot validation of the interaction between human coronavirus NSP1 proteins and the human proteins PYCR1/PYCR2. Cells transfected with vector or construct encoding GFP were included as controls in these experiments.


C Heat map of N HCIPs identified by SFB - tandem affinity purification . N HCIPs were compared among seven human coronaviruses using preys ' spectral . Three areas of heat map are manually selected , enlarged labeled as ( 1 ) , ( 2 ) , ( 3 ) , which are enriched by N of seven human coronaviruses . Functional characterization for each is shown with red circles below enlarged .

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C Heat map of the N protein HCIPs identified by SFB-tandem affinity purification. N protein HCIPs were compared among seven human coronaviruses using the preys' spectral counts. Three areas of the heat map are manually selected, enlarged and labeled as (1), (2), and (3), which are enriched by all N proteins of the seven human coronaviruses. Functional characterization for each protein is shown with the red circles below the enlarged images.


A Preys identified showing significant enrichment by N specifically from one or more human coronaviruses . PSM , peptide - spectrum match .

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A Preys identified showing significant enrichment by N proteins specifically from one or more human coronaviruses. PSM, peptide-spectrum match.


B Pulldown Western blot validation of interaction between N G3BP1 / G3BP2 . transfected with vector or construct encoding GFP or N from different human coronaviruses were compared in these experiments .

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B Pulldown and Western blot validation of the interaction between N protein and G3BP1/G3BP2. Cells transfected with vector or construct encoding GFP or N protein from different human coronaviruses were compared in these experiments.


C , D Effect of SARS - CoV - 2 N on stress granule formation . A549 ( C ) MCF10A ( D ) were transfected with pinducer20 - N of SARS - CoV - 2 . are treated with sodium arsenite but with or without Dox to induce N expression . were fixed stained with indicated antibodies . green signal is G3BP1 , red signal is HA ( for N ) , blue signal indicates DAPI / nuclei . Scale bar : 10 µm .

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C,D Effect of SARS-CoV-2 N protein on stress granule formation. A549 (C) and MCF10A (D) cells were transfected with pinducer20-N protein of SARS-CoV-2. All the cells are treated with sodium arsenite but with or without Dox to induce N protein expression. Cells were fixed and stained with the indicated antibodies. The green signal is G3BP1, the red signal is HA (for N protein), and the blue signal indicates DAPI/nuclei. Scale bar: 10 µm.


E of stress granule formation in A549 MCF10A . Total of 30 were counted a student ' s t was used for statistical ( * * * , p < 0 . 001 ) . Box limits represent 25th percentile 75th percentile ; horizontal represents median . Whiskers display min . to max . values .

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E Quantification of the stress granule formation in A549 and MCF10A cells. Total of 30 cells were counted and a student's t test was used for the statistical analysis (***, p<0.001). Box limits represent 25th percentile and 75th percentile; horizontal line represents median. Whiskers display min. to max. values.


( A ) Electron micrograph of an with abundant double - spherules ( DMSs ) . DMVs ( asterisks ) are interspersed surrounding DMS cluster . ( B ) Slice through a tomogram ( left ) corresponding surface - rendered model ( right ) of a representative containing different types of MERS - CoV - induced modifications : DMSs ( orange ) , convoluted membranes ( CM , blue ) DMVs ( yellow lilac , outer inner membranes ) . model also highlights ER membranes ( green ) a vesicle ( silver ) containing new virions ( pink ) . ( See also S1 Video ) . ( C ) Comparison of DMSs virions ( arrowheads in left right panels , respectively ) in enlarged views of tomographic slices from regions boxed in ( B ) . DMSs are similar in but distinct in appearance from newly - formed MERS - CoV particles . ( D ) Whisker plots of distribution of DMSs ( n = 58 ) , virions ( n = 28 ) DMVs ( n = 109 ) , as measured from tomograms . DMSs virions have a comparable ( median diameter , 80 nm ) , while median diameter of DMVs is 247 nm . ( E ) Models tomographic slices through an open ( left ) closed ( right ) DMS . Both types of DMSs are connected with CM . In open DMSs , both inner outer membranes ( blue orange , respectively ) are continuous with CM . Two ~ 8 - nm apart slices through reconstruction are shown . For closed DMSs only outer is connected to CM , while inner seems to define a closed compartment .

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(A) Electron micrograph of an area with abundant double-membrane spherules (DMSs). DMVs (asterisks) are interspersed and surrounding the DMS cluster. (B) Slice through a tomogram (left) and corresponding surface-rendered model (right) of a representative area containing the different types of MERS-CoV-induced membrane modifications: DMSs (orange), convoluted membranes (CM, blue) and DMVs (yellow and lilac, outer and inner membranes). The model also highlights ER membranes (green) and a vesicle (silver) containing new virions (pink). (See also S1 Video). (C) Comparison of DMSs and virions (arrowheads in left and right panels, respectively) in enlarged views of tomographic slices from the regions boxed in (B). The DMSs are similar in size but distinct in appearance from newly-formed MERS-CoV particles. (D) Whisker plots of the size distribution of DMSs (n=58), virions (n=28) and DMVs (n=109), as measured from the tomograms. DMSs and virions have a comparable size (median diameter, 80 nm), while the median diameter of the DMVs is 247 nm. (E) Models and tomographic slices through an open (left) and closed (right) DMS. Both types of DMSs are connected with the CM. In open DMSs, both the inner and outer membranes (dark blue and orange, respectively) are continuous with CM. Two ~8-nm apart slices through the reconstruction are shown. For closed DMSs only the outer membrane is connected to CM, while the inner membrane seems to define a closed compartment.


( F ) Gallery of tomographic slices highlighting connections between different elements of MERS - CoV RO , of these with ER . These include CM - ER ( black arrowheads ) , DMV - ER ( arrowheads ) , CM - DMV ( grey arrowheads ) , CM - DMS ( grey arrowhead ) connections . Scale bars , 250 nm ( A , B ) , 100 nm ( C - F ) .

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(F) Gallery of tomographic slices highlighting membrane connections between different elements of the MERS-CoV RO, and of these with the ER. These include CM-ER (black arrowheads), DMV-ER (white arrowheads), CM-DMV (dark grey arrowheads), and CM-DMS (light grey arrowhead) connections. Scale bars, 250 nm (A, B), and 100 nm (C -F).


alpha - CoVs . 2D - EM from 100 - nm thick sections of different mammal infected with ( A ) SARS - CoV ( MOI 10 , 9 hpi ) , ( B ) MHV ( MOI 10 , 8 hpi ) ( C ) HCoV - 229E ( MOI 5 , 24 hpi ) . Both beta - CoVs ( A , B ) alpha - CoV ( C ) induce modifications that include not only DMVs ( asterisks ) CM , but also DMSs ( arrowheads ) . Scale bars , 250 nm .

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alpha-CoVs.2D-EM images from 100-nm thick sections of different mammal cells infected with (A) SARS-CoV (MOI 10, 9 hpi), (B) MHV (MOI 10, 8 hpi) and (C) HCoV-229E (MOI 5, 24 hpi). Both beta-CoVs (A,B) and the alpha-CoV (C) induce membrane modifications that include not only DMVs (asterisks) and CM, but also DMSs (white arrowheads). Scale bars, 250 nm.


( A ) Overview of an infected in which regions with different - induced modifications are annotated in yellow ( DMVs ) , blue ( CM ) orange ( DMSs ) . Several densely - labelled regions containing DMVs , but not other - induced structures are apparent . A close - up of one of these regions ( boxed ) is shown in ( B ) .

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(A) Overview of an infected cell in which regions with different virus-induced modifications are annotated in yellow (DMVs), blue (CM) and orange (DMSs). Several densely-labelled regions containing DMVs, but not the other virus-induced structures are apparent. A close-up of one of these regions (boxed area) is shown in (B).


( C , D ) Distribution of autoradiography signal around DMVs ( nDMVs = 36 , see Material Methods for selection criteria details ) . data is plotted ( C ) as a histogram , or ( D ) normalized by radius to DMV centre to account for increase in screened . Scale bars , ( A ) 5 μm , ( B ) 500 nm .

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(C, D) Distribution of the autoradiography signal around DMVs (nDMVs = 36, see Material and Methods for selection criteria and details). The data is plotted (C) as a histogram, or (D) normalized by the radius to the DMV centre to account for the increase in the screened area. Scale bars, (A) 5 μm, (B) 500 nm.


( A ) of amount of radioactive label incorporated into as a function of labelling time in SARS - CoV - infected Vero E6 ( MOI 10 ) , as measured by scintillation counting on isolated from . label was provided simultaneously to samples at 6 hpi .

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(A) Analysis of the amount of radioactive label incorporated into RNA as a function of the labelling time in SARS-CoV-infected Vero E6 cells (MOI 10), as measured by scintillation counting on the RNA isolated from the cells. The label was provided simultaneously to all the samples at 6 hpi.


( B - D ) EM detection by autoradiography . ( B ) Overview of a SARS - CoV - infected Vero E6 ( MOI 10 ) were labelled for 20 min just before fixation at 7 hpi , then processed for EM autoradiography . Autoradiography grains accumulate in RO regions . N , nucleus ; m , mitochondria ; VCR , virion - containing regions ; LD , lipid droplets . Scale bar , 1 μm .

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(B-D) EM detection by autoradiography. (B) Overview of a SARS-CoV-infected Vero E6 cell (MOI 10) were labelled for 20 min just before fixation at 7 hpi, and then processed for EM autoradiography. Autoradiography grains accumulate in the RO regions. N, nucleus; m, mitochondria; VCR, virion-containing regions; LD, lipid droplets. Scale bar, 1 μm.


( C , D ) of autoradiography signal per subcellular structure ( see also S1 Table ) . Labelling densities relative labelling indexes ( RLI ) in different subcellular regions of ( C ) Vero E6 infected with SARS - CoV ( MOI 10 ) or ( D ) Huh7 infected with MERS - CoV ( MOI 5 ) . Radioactively - labelled uridine was provided for indicated periods of time immediately before fixation at 7 hpi 12 hpi , respectively . Control mock - infected are excluded from RLI plots , as RLI comparisons between conditions require same number of classes ( subcellular regions ) these lack ROs virions .

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(C, D) Quantification of the autoradiography signal per subcellular structure (see also S1 Table). Labelling densities and relative labelling indexes (RLI) in different subcellular regions of (C) Vero E6 cells infected with SARS-CoV (MOI 10) or (D) Huh7 cells infected with MERS-CoV (MOI 5). Radioactively-labelled uridine was provided for the indicated periods of time immediately before fixation at 7 hpi and 12 hpi, respectively. Control mock-infected cells are excluded from the RLI plots, as RLI comparisons between conditions require the same number of classes (subcellular regions) and these cells lack ROs and virions.


( A ) Overview of a cluster of MERS - CoV - induced modifications in Huh7 prepared as described in Fig . 4 . Some DMSs are boxed in orange while regions with CM are encircled in blue . In comparison with densely - labelled surrounding DMVs , these regions are relatively devoid of autoradiography signal .

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(A) Overview of a cluster of MERS-CoV-induced membrane modifications in Huh7 cells prepared as described in Fig. 4. Some DMSs are boxed in orange while regions with CM are encircled in blue. In comparison with the densely-labelled surrounding DMVs, these regions are relatively devoid of autoradiography signal.


( B ) distribution of autoradiography grains on CM was not homogeneous ( nCM = 9 ) , label was predominantly found close to boundaries of CM , as expected if signal arises from surrounding DMVs .

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(B) The distribution of autoradiography grains on CM was not homogeneous (nCM=9), and label was predominantly found close to the boundaries of the CM, as expected if the signal arises from the surrounding DMVs.


( C - E ) of label around / on DMSs ( see Materials Methods for selection criteria details ) . ( C ) Enlargements of DMS areas boxed in ( A ) .

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(C-E) Analysis of the label around/on the DMSs (see Materials and Methods for selection criteria and details). (C) Enlargements of the DMS areas boxed in (A).


( C - E ) of label around / on DMSs ( see Materials Methods for selection criteria details ) . Most DMSs were devoid of signal those who contained label were close to labelled DMVs ( D ) ( nDMS = 105 ) .

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(C-E) Analysis of the label around/on the DMSs (see Materials and Methods for selection criteria and details). Most DMSs were devoid of signal and those who contained label were close to labelled DMVs (D) (nDMS = 105).


( C - E ) of label around / on DMSs ( see Materials Methods for selection criteria details ) . ( E ) distribution of signal around DMSs shows an increase in amount of autoradiography grains with from DMS centre , as expected from a random distribution ( nDMSs = 54 ) . Scale bars , ( A ) 500 nm , ( C ) 100 nm .

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(C-E) Analysis of the label around/on the DMSs (see Materials and Methods for selection criteria and details). (E) The distribution of signal around DMSs shows an increase in the amount of autoradiography grains with the distance from the DMS centre, as expected from a random distribution (nDMSs = 54). Scale bars, (A) 500 nm, (C) 100 nm.


IEM detection of viral markers in MERS - CoV - infected . ( A - G ) Immunogold labeling of thawed cryo - sections of MERS - CoV - infected Huh7 ( 12 hpi ) for detection of indicated viral . ( A - C ) Structural were detected on virions ( black arrowheads ) , for M S , also on Golgi cisterna . While regions containing DMS ( arrowheads ) CM labelled for N ( D ) nsp3 ( G ) , M S were not detected in these areas . ( H - I ) Immunogold labeling of dsRNA in HPF - FS samples of MERS - CoV - infected Huh7 ( 13 hpi ) . label accumulated on DMVs , which could be easily detected in this type of samples ( grey arrowheads ) , while regions with CM DMSs , which appeared as areas among DMV clusters , where devoid of dsRNA signal . G , Golgi ; m , mitochondria . Scale bars , 250 nm .

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IEM detection of viral markers in MERS-CoV-infected cells.(A-G) Immunogold labeling of thawed cryo-sections of MERS-CoV-infected Huh7 cells (12 hpi) for the detection of the indicated viral proteins. (A-C) Structural proteins were detected on virions (black arrowheads) and, for the M and S proteins, also on Golgi cisterna. While regions containing DMS (white arrowheads) and CM labelled for the N protein (D) and nsp3 (G), the M and S protein were not detected in these areas. (H-I) Immunogold labeling of dsRNA in HPF-FS samples of MERS-CoV-infected Huh7 cells (13 hpi). The label accumulated on DMVs, which could be easily detected in this type of samples (grey arrowheads), while the regions with CM and DMSs, which appeared as dark areas among the DMV clusters, where devoid of dsRNA signal. G, Golgi complex; m, mitochondria. Scale bars, 250 nm.


( A ) Netrin - 1 ( NTN1 ) gene expression profiling from 6 adults control brains using microarray data from Allen Human Brain Atlas .

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(A) Netrin-1 (NTN1) gene expression profiling from 6 adults control brains using microarray data from the Allen Human Brain Atlas.


( B ) Reporter gene expression in midbrain ( substantia nigra ( SN ) ventral tegmental ( VTA ) ) of netrin - 1 + / lacZ + adult ( P60 ) mice incubated with S - Gal . ( Scale bar , 2000 µm ) . Two coronal sections of mice midbrain are shown from more anterior is shown in upper panel .

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(B) Reporter gene expression in the midbrain (substantia nigra (SN) and ventral tegmental area (VTA)) of netrin-1+/lacZ+ adult (P60) mice incubated with S-Gal. (Scale bar, 2000 µm). Two coronal sections of the mice midbrain are shown from the more anterior is shown in the upper panel.


( C ) Netrin - 1 localization in SN of adult rat . ( Scale bar , 200 µm ) . Upper panel , confocal image of SN . Higher magnification of SNpc ( bottoms panels ) . Netrin - 1 ( red ) , Tyrosine hydroxylase ( TH ) ( green ) .

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(C) Netrin-1 protein localization in the SN of adult rat. (Scale bar, 200 µm). Upper panel, confocal image of the SN. Higher magnification of the SNpc (bottoms panels). Netrin-1 (red), Tyrosine hydroxylase (TH) (green).


( D ) Localization of netrin - 1 in adult rat striatum . ( Scale bar , 600µm ) . Left panel , confocal image of rat striatum . Netrin - 1 ( red ) , Dopamine transporter ( DAT ) ( green ) . Right panels , higher magnification .

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(D) Localization of netrin-1 protein in the adult rat striatum. (Scale bar, 600µm). Left panel, confocal image of the rat striatum. Netrin-1 (red), Dopamine transporter (DAT) (green). Right panels, higher magnification.


( A ) Representative of dopamine neurons ( TH - positive ) in substantia nigra ( SN ) ( left panel ) in SN striatum ( right panel top , bottom respectively ) by immunofluorescence , six after intranigral injection of control ( CTL ) ( AAV6 - GFP ) or Cre ( AAV6 - GFP Cre ) adenovirus vectors in Netrin - 1fl / fl mice ( left panel : green - GFP , red - TH ; right panel : Cy - 5 - TH ) ( Scale bar , 1000 μm ) . Filled arrows indicate SN TH dopamine neurons .

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(A) Representative images of dopamine neurons (TH-positive) in the substantia nigra (SN) (left panel) and in the SN and striatum (right panel top, and bottom respectively) by immunofluorescence, six weeks after the intranigral injection of control (CTL) (AAV6-GFP) or Cre (AAV6-GFP Cre) adenovirus vectors in Netrin-1fl/fl mice (left panel: green-GFP, red-TH; right panel: Cy-5-TH) (Scale bar, 1000 μm). Filled arrows indicate SN TH dopamine neurons.


( B ) Representative of TH netrin - 1 stainings by immunofluorescence ( scale bar , 2000 μm ) six after intranigral injection of CTL adenovirus vector ( left panel ) or Cre adenovirus vector ( right panel ) , in midbrain ( substantia nigra ( SN ) ventral tegmental ( VTA ) of Netrin - 1fl / fl mice .

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(B) Representative images of TH and netrin-1 stainings by immunofluorescence (scale bar, 2000 μm) six weeks after the intranigral injection of CTL adenovirus vector (left panel) or Cre adenovirus vector (right panel), in the midbrain (substantia nigra (SN) and ventral tegmental area (VTA) of Netrin-1fl/fl mice.


( C ) of of TH - positive neurons , in substantia nigra ( SN ) ( left ) , fibres , in striatum ( right ) , six after adenovirus vector injection , in Netrin - 1fl / fl mice . Bars error bars represent mean ± . Statistical significance was determined by an unpaired t - . * P < 0 . 05 ; * * P < 0 . 01 . N = 3 each group .

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(C) Quantification of fluorescence intensity of TH-positive neurons, in the substantia nigra (SN) (left), and fibres, in the striatum (right), six weeks after adenovirus vector injection, in Netrin-1fl/fl mice. Bars and error bars represent the mean ± SEM. Statistical significance was determined by an unpaired t-test. *P < 0.05; **P< 0.01. N=3 each group.


( D ) Terminal deoxynucleotidyl transferase ( TdT ) dUTP Nick - End Labelling ( TUNEL ) reactivity in SN after Cre control adenovirus vector injection , in Netrin - 1fl / fl mice ( Scale bar , 50μm ) . apoptotic index is expressed as a of TUNEL positive neurons out of total number of TH - positive neurons . Bars error bars represent mean ± . Statistical significance was determined by an unpaired t - . * * P < 0 . 01 . N = 3 each group .

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(D) Terminal deoxynucleotidyl transferase (TdT) dUTP Nick-End Labelling (TUNEL) reactivity in the SN after Cre and control adenovirus vector injection, in Netrin-1fl/fl mice (Scale bar, 50μm). The apoptotic index is expressed as a percentage of TUNEL positive neurons out of the total number of TH-positive neurons. Bars and error bars represent the mean ± SEM. Statistical significance was determined by an unpaired t-test. **P< 0.01. N=3 each group.


( E ) Motor behavior assays at six after treatment . N = 8 / group . Bars error bars represent mean ± . Statistical significance was determined by an unpaired t - . * P < 0 .

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(E) Motor behavior assays at six weeks after treatment. N = 8 animals/group. Bars and error bars represent the mean ± SEM. Statistical significance was determined by an unpaired t-test. *P < 0.05


( F ) Immunoblot of SN lysates from netrin - 1 wild type ( WT ) netrin - 1 conditional KO mice ( left upper panel ) . Densitometry band of receptor over full receptor ( lower right panel ) . We randomly selected 2 mice from each group for immunoblot . N = 3 independent experiments . Bars error bars represent mean ± . Statistical significance was determined by an unpaired t - . * P < 0 . 05 ; P * * < 0 . 01 .

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(F) Immunoblot of SN lysates from netrin-1 wild type (WT) and netrin-1 conditional KO mice (left upper panel). Densitometry band quantification of cleaved receptor over full length receptor ratio (lower right panel). We randomly selected 2 mice from each group for immunoblot analysis. N=3 independent experiments. Bars and error bars represent the mean ± SEM. Statistical significance was determined by an unpaired t-test. *P < 0.05; P**< 0.01.


( A ) Representative of TH + dopamine neurons by immunofluorescence , in SN striatum ( Str ) 6 after CTL ( AAV6 - GFP ) ; Cre ( AAV6 - GFP Cre ) ; sh DCC ( AAV6 - GFP Cre + Sh - DCC ) shUNC5B ( AAV6 GFP Cre + Sh - UNC5B ) injection into SN of Netrin - 1fl / fl mice . ( Scale bar , 2000 µm ) . ( B ) of TH in SN ( upper bar graph ) striatum ( Str ) of Netrin - 1fl / fl mice ( lower bar graph ) . Data are shown as mean + . Statistical significance was determined by an unpaired t - . N = 3 each group . Tukey post hoc after one - way ANOVA * P < 0 . 05 , * * P < 0 . 01 , N . S . , not significant .

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(A) Representative images of TH+ dopamine neurons and fibers staining by immunofluorescence, in the SN and the striatum (Str) 6 weeks after CTL (AAV6-GFP); Cre (AAV6-GFP Cre); sh DCC (AAV6-GFP Cre + Sh-DCC) and shUNC5B (AAV6 GFP Cre + Sh-UNC5B) injection into the SN of Netrin-1fl/fl mice. (Scale bar, 2000 µm). (B) Quantification of TH fluorescent intensity in the SN (upper bar graph) and striatum (Str) of Netrin-1fl/fl mice (lower bar graph). Data are shown as mean + SEM. Statistical significance was determined by an unpaired t-test. N = 3 each group. Tukey post hoc analysis after one-way ANOVA *P < 0.05, **P <0.01, N.S., not significant.


( C ) DCC depletion mitigates netrin - 1 depletion - induced apoptosis in Netrin - 1fl / fl mice . TH - positive loss was assessed by TUNEL in SN . Upper panel , TUNEL ( red ) TH ( Cy5 - ) ( Scale bar , 50 µm ) . Apoptotic index ( bar graph , bottom panel ) expressed as a of TUNEL positive neurons out of total number of TH - positive neurons . N = 3 each group . Bars error bars represent mean ± . Statistical significance was determined using a one - way ANOVA followed by post hoc Tukey for multiple group comparison . * P < 0 . 05 ; P * * < 0 . 01 .

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(C) DCC depletion mitigates netrin-1 depletion-induced apoptosis in Netrin-1fl/fl mice. TH-positive cell loss was assessed by TUNEL assay in the SN. Upper panel, TUNEL (red) and TH (Cy5-white) (Scale bar, 50 µm). Apoptotic index (bar graph, bottom panel) expressed as a percentage of TUNEL positive neurons out of the total number of TH-positive neurons. N = 3 each group. Bars and error bars represent the mean ± SEM. Statistical significance was determined using a one-way ANOVA followed by post hoc Tukey test for multiple group comparison. *P < 0.05; P**< 0.01.


( D ) Immunoblot of SN lysates ( upper panel ) corresponding ( bottom panel ) . of band ( bottom ) . Mouse N = 2 . N = 3 independent immunoblot . Bars error bars represent mean ± . Statistical significance was determined by an unpaired t - . * P < 0 . 05 ; P * * < 0 . 01 .

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(D) Immunoblot of SN lysates (upper panel) and corresponding quantification (bottom panel). Quantification of band intensity (bottom). Mouse N=2. N=3 independent immunoblot analysis. Bars and error bars represent the mean ± SEM. Statistical significance was determined by an unpaired t-test. *P < 0.05; P**< 0.01.


( E ) Motor behaviour at six after treatment . N = 8 / group . Bars error bars represent mean ± . Statistical significance was determined using a one - way ANOVA followed by post hoc Tukey for multiple group comparison . * P < 0 . 05 ; N . S . , not significant .

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(E) Motor behaviour tests at six weeks after treatment. N = 8 animals/group. Bars and error bars represent the mean ± SEM. Statistical significance was determined using a one-way ANOVA followed by post hoc Tukey test for multiple group comparison. *P < 0.05; N.S., not significant.


( B ) Amphetamine - induced rotations at two six after lesion . values Mean ± SD are shown , n = 11 in each group . Tukey post hoc after two - way RM ANOVA , * P < 0 . 05 compared to WT .

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(B) Amphetamine-induced rotations at two and six weeks after lesion. Individual values and Mean ± SD are shown, n=11 animals in each group. Tukey post hoc analysis after two-way RM ANOVA, *P < 0.05 compared to WT.


( C ) Optical density of TH - positive fibres in striatum . values mean ± SD , n = 10 , unpaired t - , * P < 0 . 05 .

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(C) Optical density of TH-positive fibres in the striatum. Individual values and mean ± SD, n= 10 animals, unpaired t-test, *P < 0.05.


( D ) TH - positive in SN . values mean ± SD , n = 10 , unpaired t - .

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(D) TH-positive cell count in the SN. Individual values and mean ± SD, n= 10 animals, unpaired t-test.


( F ) TH by immunohistochemistry ( Scale bar , 2000µm ) in SN ( left ) striatum ( right ) of WT SNCA Tg mice , 6 days after treatment .

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(F) TH staining by immunohistochemistry (Scale bar, 2000µm) in the SN (left) and striatum (right) of WT and SNCA Tg mice, 6 days after treatment.


( G ) TH - positive bar graph . experiments were repeated 3 times . We randomly selected 2 mice from each group for immunohistochemistry TH positive . Bars error bars represent mean ± . Statistical significance was determined using a one - way ANOVA followed by post hoc Bonferroni for multiple group comparison . * * P < 0 . 01 ; N . S . , not significant .

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(G) TH-positive cell count bar graph. All experiments were repeated 3 times. We randomly selected 2 mice from each group for immunohistochemistry TH positive cell analysis. Bars and error bars represent the mean ± SEM. Statistical significance was determined using a one-way ANOVA followed by post hoc Bonferroni test for multiple group comparison. **P< 0.01; N.S., not significant.


Motor behavioural assays ( Rotarod rod ( H ) N = 8 each group . Bars error bars represent mean ± . Statistical significance was determined using a one - way ANOVA followed by post hoc Bonferroni for multiple group comparison . * P < 0 . 05 ; * * P < 0 . 01 .

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Motor behavioural assays (Rotarod rod test (H) N = 8 animals each group. Bars and error bars represent the mean ± SEM. Statistical significance was determined using a one-way ANOVA followed by post hoc Bonferroni test for multiple group comparison. *P<0.05;**P< 0.01.


Motor behavioural assays Cylinder ( I ) , Grid ( J ) ) N = 8 each group . Bars error bars represent mean ± . Statistical significance was determined using a one - way ANOVA followed by post hoc Bonferroni for multiple group comparison . * P < 0 . 05 ; * * P < 0 . 01 .

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Motor behavioural assays Cylinder test (I), Grid test (J)) N = 8 animals each group. Bars and error bars represent the mean ± SEM. Statistical significance was determined using a one-way ANOVA followed by post hoc Bonferroni test for multiple group comparison. *P<0.05;**P< 0.01.


( B ) Cumulative amphetamine - induced rotations . Progression of amphetamine - induced rotations over time ( right panel ) . Mean + SD are shown , n = 11 - 12 in each group . Tukey post hoc after two - way RM ANOVA , * * * * P < 0 . 0001 compared to initial rotation score at week 2 . Comparison of treatment ' s effect between groups ( left panel ) . Mean + SD are shown , n = 11 - 12 in each group . Tukey post hoc after two - way RM ANOVA , * P < 0 . 05 , * * P < 0 . 01 compared to vehicle group .

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(B) Cumulative amphetamine-induced ipsilateral rotations. Progression of amphetamine-induced ipsilateral rotations over time (right panel). Mean + SD are shown, n=11-12 animals in each group. Tukey post hoc analysis after two-way RM ANOVA, **** P <0.0001 compared to the initial rotation score at week 2. Comparison of the treatment's effect between groups (left panel). Mean + SD are shown, n=11-12 animals in each group. Tukey post hoc analysis after two-way RM ANOVA, *P < 0.05, **P < 0.01 compared to vehicle group.


( C ) Dopamine fibres in striatum of 6 - OHDA - lesionned rats , ten after treatment . Representative of coronal sections of striatum showing TH ( left panel ) , intact side on left 6 - OHDA lesioned side on right . ( Scale bar , 2000 µm ) . Optical density measurement of TH - positive fibres in lesioned side of striatum ( bar graph , right panel ) . values mean ± SD are shown , N > 10 in each group . Tukey post hoc after one - way ANOVA , * P < 0 . 05 , * * P < 0 . 01 compared to vehicle group . are oriented with dorsal part of tissue at top , ventral part at

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(C) Dopamine fibres in the striatum of 6-OHDA-lesionned rats, ten weeks after treatment. Representative images of coronal sections of the striatum showing TH IHC staining (left panel), intact side on the left and 6-OHDA lesioned side on the right. (Scale bar, 2000 µm). Optical density measurement of TH-positive fibres in the lesioned side of the striatum (bar graph, right panel). Individual values and mean ± SD are shown, N > 10 animals in each group. Tukey post hoc analysis after one-way ANOVA, *P < 0.05, **P < 0.01 compared to vehicle group. Images are oriented with the dorsal part of the tissue at the top, and ventral part at the bottom


( D ) Dopamine bodies in SN of 6 - OHDA - lesionned rats , ten after treatment . Representative of coronal sections of SN ( lesioned side ) showing TH - positive bodies stained by ( left panel ) ( Scale bar , 2000 µm ) . TH - positive in SN ( right panel ) . values mean + SD are shown , N > 10 in each group . Tukey post hoc after one - way ANOVA , * * P < 0 . 01 compared to vehicle group . are oriented with dorsal part of tissue at top , ventral part at bottom .

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(D) Dopamine cell bodies in the SN of 6-OHDA-lesionned rats, ten weeks after treatment. Representative images of coronal sections of the SN (lesioned side) showing TH-positive cell bodies stained by IHC (left panel) (Scale bar, 2000 µm). TH-positive cell count in the SN (right panel). Individual values and mean + SD are shown, N > 10 animals in each group. Tukey post hoc analysis after one-way ANOVA, **P < 0.01 compared to vehicle group. Images are oriented with the dorsal part of the tissue at the top, and ventral part at the bottom.


( A ) Netrin - 1 gene expression profiling by array of substantiae nigrae from PD non - PD ( Control ) using GEO dataset GSE7621 that has a total of 25 samples ( n = 9 control n = 16 PD cases ) . Unpaired t - , * * * P < 0 . 0005 , Mean ± SD are shown .

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(A) Netrin-1 gene expression profiling by array of substantiae nigrae from PD and non-PD (Control) patients using the GEO dataset GSE7621 that has a total of 25 samples (n=9 control and n=16 PD cases). Unpaired t-test, ***P < 0.0005, Mean ± SD are shown.


( B ) Immunoblot of netrin - 1 , DCC , UNC5B , active caspase - 3 , alpha synuclein FL , alpha synuclein pS129 , Tyrosine Hydroxylase ( TH ) in - matched controls ( n = 3 ) vs Parkinson ' s disease brain samples ( n = 3 ) ( left panel ) . Band bar graph ( right panel ) . Bars error bars represent mean ± . Statistical significance was determined using a one - way ANOVA followed by post hoc Tukey for multiple group comparison . P * * < 0 . 01 . N = 3

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(B) Immunoblot of netrin-1, DCC, UNC5B, active caspase-3, alpha synuclein FL, alpha synuclein pS129, and Tyrosine Hydroxylase (TH) levels in age-matched controls (n=3) vs Parkinson's disease patients brain samples (n=3) (left panel). Band quantification bar graph (right panel). Bars and error bars represent the mean ± SEM. Statistical significance was determined using a one-way ANOVA followed by post hoc Tukey test for multiple group comparison. P**< 0.01. N=3 replicates


F Representative BLI histology of organotypic cultures with H2030 - BrM cancer treated with DMSO or decreasing concentrations of DEBIO - 0932 . Scale bar : 100 µm ; high magnification : 50 µm . G of bioluminescence signal emitted by each condition shown in ( F ) at day 3 normalized by initial value obtained at day 0 normalized to organotypic cultures treated with DMSO . Day 0 is considered 12 - 16h after addition of cancer treatment or DMSO . Values are shown in box - - whisker plots where each dot is an organotypic culture in box corresponds to median . boxes go from upper to lower quartiles whiskers go from minimum to maximum value ( n = 8 DMSO , n = 8 BKM120 n = 7 per of DEBIO - 0932 - treated organotypic cultures , 2 independent experiments ) . P value was calculated using two - t - .

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F Representative BLI and histology of organotypic cultures with H2030-BrM cancer cells treated with DMSO or decreasing concentrations of DEBIO-0932. Scale bar: 100 µm; high magnification: 50 µm. G Quantification of the bioluminescence signal emitted by each condition shown in (F) at day 3 normalized by the initial value obtained at day 0 and normalized to the organotypic cultures treated with DMSO. Day 0 is considered 12-16h after the addition of cancer cells and treatment or DMSO. Values are shown in box-and-whisker plots where each dot is an organotypic culture and the line in the box corresponds to the median. The boxes go from the upper to the lower quartiles and the whiskers go from the minimum to the maximum value (n=8 DMSO, n=8 BKM120 and n=7 per concentration of DEBIO-0932-treated organotypic cultures, 2 independent experiments). P value was calculated using two-tailed t-test.


B Representative of organotypic cultures with established metastases with various glial components of microenvironment labelled . Scale bar : 75 µm . Each condition was evaluated in several organotypic cultures ( 3 - 6 slices ) .

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B Representative images of organotypic cultures with established metastases with various glial components of the microenvironment labelled. Scale bar: 75 µm. Each individual condition was evaluated in several organotypic cultures (3-6 slices).


D of LDH in conditioned media of organotypic slices cultured during 3 days relative to a lysate of same preparation . Values are shown as mean + s . e . m . ( n = 3 organotypic cultures per experimental condition , 1 independent experiment ) .

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D Quantification of LDH levels in the conditioned media of organotypic slices cultured during 3 days relative to a lysate of the same preparation. Values are shown as mean + s.e.m. (n=3 organotypic cultures per experimental condition, 1 independent experiment).


A - C Immunofluorescence against HSP90 in mouse brains with established metastases . ( A ) HSP90 positive structures in areas not affected by metastasis includes medial habenula , where neurons co - localize with chaperone . Scale bars : 100 µm ( low magnification ) , 50 µm ( medial habenula nucleus ) , 12 µm ( high magnification neurons ) . ( B ) Established metastases from different origins oncogenomic profiles stained with HSP90 . Dotted lines delineate metastasis ( cc : cancer ) . Scale bars : 75 µm . ( C ) Iba1 colocalizes with HSP90 within areas affected by metastases . BB : bisbenzamide . Scale bar : 75 µm ( low magnification ) , 12 µm ( high magnification ) .

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A-C Immunofluorescence against HSP90 in mouse brains with established metastases. (A) HSP90 positive structures in areas not affected by the metastasis includes the medial habenula, where neurons co-localize with the chaperone. Scale bars: 100 µm (low magnification), 50 µm (medial habenula nucleus), 12 µm (high magnification neurons). (B) Established metastases from different primary origins and oncogenomic profiles stained with HSP90. Dotted lines delineate the metastasis (cc: cancer cells). Scale bars: 75 µm. (C) Iba1 colocalizes with HSP90 within areas affected by metastases. BB: bisbenzamide. Scale bar: 75 µm (low magnification), 12 µm (high magnification).


E Representative human brain metastases showing different intensities or scores for HSP90 . Scale bar : 50 µm . F of HSP90 in human brain metastases . 59 out of 60 ( 98 % ) showed positive of HSP90 in , 15 ( 25 % ) scored with 3 ( strong ) , 36 ( 60 % ) with 2 ( moderate ) 8 ( 13 % ) with 1 ( weak ) according to signal of HSP90 in cytoplasm of cancer .

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E Representative human brain metastases showing different intensities or scores for HSP90. Scale bar: 50 µm. F Quantification of HSP90 in human brain metastases. 59 out of 60 (98%) showed positive staining of HSP90 in the tumor, 15 (25%) scored with 3 (strong), 36 (60%) with 2 (moderate) and 8 (13%) with 1 (weak) according to the signal intensity of HSP90 in the cytoplasm of cancer cells.


Representative human brain metastases showing different percentages of HSP90 . Scale bars : ( J ) 50 µm Black arrows point to cancer positive for HSP90 in nucleus .

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Representative human brain metastases showing different percentages of nuclear HSP90. Scale bars: (J) 50 µm Black arrows point to cancer cells positive for HSP90 in the nucleus.


Representative human brain metastases showing different percentages of HSP90 . Scale bars : ( K ) low magnification : 100 µm ; high magnification : 10 µm .

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Representative human brain metastases showing different percentages of nuclear HSP90. Scale bars: (K) low magnification: 100 µm; high magnification: 10 µm.


L of HSP90 in human brain metastases . 54 out of 60 samples ( 90 % ) showed positive HSP90 in . 27 ( 45 % ) showed 1 - 5 % ( moderate ) 27 ( 45 % ) showed > 5 % ( high ) of HSP90 . M of HSP90 in human tumors . 19 out of 30 ( 63 % ) showed positive HSP90 in . 9 ( 30 % ) showed 1 - 5 % ( moderate ) 10 ( 33 % ) showed > 5 % ( high ) of HSP90 .

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L Quantification of nuclear HSP90 in human brain metastases. 54 out of 60 samples (90%) showed positive nuclear HSP90 in the tumor. 27 (45%) showed 1-5% (moderate) and 27 (45%) showed >5% (high) of nuclear HSP90. M Quantification of nuclear HSP90 in human primary tumors. 19 out of 30 (63%) showed positive nuclear HSP90 in the tumor. 9 (30%) showed 1-5% (moderate) and 10 (33%) showed >5% (high) of nuclear HSP90.


B Representative in vivo ex vivo of vehicle DEBIO - 0932 treated mice 5 ( experimental endpoint ) after intracardiac inoculation of H2030 - BrM .

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B Representative in vivo and ex vivo images of vehicle and DEBIO-0932 treated mice 5 weeks (experimental endpoint) after intracardiac inoculation of H2030-BrM cells.


E Representative sections of brains from vehicle DEBIO - 0932 treated mice in ( B - D ) . dotted lines surround metastases ( GFP + ) . Representative field of view of metastasis stained with GFP caspase 3 . Scale bars : slices , 1mm ; caspase 3 , 50 µm . F of established metastases found in vehicle DEBIO - 0932 treated brains from panel ( E ) . Values are shown in box - - whisker plots where every dot represents a different brain in box corresponds to median . boxes go from upper to lower quartiles whiskers go from minimum to maximum value ( vehicle : n = 10 brains ; DEBIO - 0932 : n = 14 brains ) . P value was calculated using two - t - . G of number of caspase 3 ( CC3 + ) in cancer found in vehicle DEBIO - 0932 treated brains from panel ( E ) . Values are shown in box - - whisker plots where every dot is a metastatic lesion in box corresponds to median . boxes go from upper to lower quartiles whiskers go from minimum to maximum value ( n = 8 metastatic lesions from 4 brains per condition ) . P value was calculated using two - t - .

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E Representative sections of brains from vehicle and DEBIO-0932 treated mice in (B-D). The dotted lines surround the metastases (GFP+). Representative field of view of metastasis stained with GFP and cleaved caspase 3. Scale bars: slices,1mm; cleaved caspase 3, 50 µm. F Quantification of established metastases found in vehicle and DEBIO-0932 treated brains from panel (E). Values are shown in box-and-whisker plots where every dot represents a different brain and the line in the box corresponds to the median. The boxes go from the upper to the lower quartiles and the whiskers go from the minimum to the maximum value (vehicle: n=10 brains; DEBIO-0932: n=14 brains). P value was calculated using two-tailed t-test. G Quantification of number of cleaved caspase 3 (CC3+) in cancer cells found in vehicle and DEBIO-0932 treated brains from panel (E). Values are shown in box-and-whisker plots where every dot is a metastatic lesion and the line in the box corresponds to the median. The boxes go from the upper to the lower quartiles and the whiskers go from the minimum to the maximum value (n=8 metastatic lesions from 4 brains per condition). P value was calculated using two-tailed t-test.


I Representative BrM - PDOC stained with proliferation markers ( BrdU ) markers of microenvironment ( GFAP for astrocytes , Iba1 for microglia / macrophages ) . Scale bar : 50 µm .

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I Representative BrM-PDOC stained with proliferation markers (BrdU) and markers of the microenvironment (GFAP for astrocytes, Iba1 for microglia/ macrophages). Scale bar: 50 µm.


F qRT - PCR of H2030 - BrM brain metastases obtained from during neurosurgery compared to relapsed metastases from corresponding . A panel of five genes related to HSP90 pathway is evaluated . Values are shown in box - - whisker plots where every dot represents a different in box corresponds to median . boxes go from upper to lower quartiles whiskers go from minimum to maximum value ( n = 5 mice per experimental condition ) . P value was calculated using two - t - .

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F qRT-PCR of H2030-BrM brain metastases obtained from animals during neurosurgery compared to relapsed metastases from the corresponding animals. A panel of five genes related to HSP90 pathway is evaluated. Values are shown in box-and-whisker plots where every dot represents a different animal and the line in the box corresponds to the median. The boxes go from the upper to the lower quartiles and the whiskers go from the minimum to the maximum value (n=5 mice per experimental condition). P value was calculated using two-tailed t-test.


H Representative of vehicle DEBIO - 0932 treated mice after neurosurgery until experimental endpoint at 6 8 for vehicle DEBIO - 0932 treated mice , respectively .

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H Representative images of vehicle and DEBIO-0932 treated mice after neurosurgery until experimental endpoint at 6 and 8 weeks for vehicle and DEBIO-0932 treated mice, respectively.


C Volcano plot with deregulated ( red : upregulated ; green : downregulated ) found in brain metastases treated with DEBIO - 0932 compared to DMSO ( n = 3 biological replicates ( mice ) per condition , n ≥ 12 brain metastases per mouse were pooled together ) . with a P value < 0 . 05 a log2 > 1 or < - 1 were defined as deregulated . Gray dotted lines indicate P value log2 cut offs . names of top deregulated are shown .

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C Volcano plot with deregulated proteins (red: upregulated; green: downregulated) found in brain metastases treated with DEBIO-0932 compared to DMSO (n=3 biological replicates (mice) per condition, n≥12 brain metastases per mouse were pooled together). Proteins with a P value <0.05 and a log2 ratio >1 or <-1 were defined as deregulated. Gray dotted lines indicate P value and log2 ratio cut offs. The names of the top deregulated proteins are shown.


G Kaplan - Meier curves showing significant correlation between worse survival post - brain metastasis high expression of HSP90 signature ( AHR , DDA1 , UBE4B , GPATCH8 ) in a cohort of 45 breast cancer brain metastasis .

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G Kaplan-Meier curves showing significant correlation between worse survival post-brain metastasis and high expression levels of the HSP90 signature (AHR, DDA1, UBE4B, GPATCH8) in a cohort of 45 breast cancer brain metastasis patients.


H Representative ( selected cases obtained from Fig EV6M ) histological score of AHR , DDA1 UBE4B in human brain metastases ( n = 16 ) according to signal of corresponding in cancer .

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H Representative images (selected cases obtained from Fig EV6M) and histological score of AHR, DDA1 and UBE4B in human brain metastases (n=16) according to the signal intensity of the corresponding protein in cancer cells.


K of ex vivo BLI of brains thoracic regions at endpoint of experiment . Values are shown in box - - whisker plots where every dot represents a different in box corresponds to median . boxes go from upper to lower quartiles whiskers go from minimum to maximum value ( n = 9 shControl mice n = 10 shAHR # 1 mice ) . P value was calculated using two - t - .

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K Quantification of ex vivo BLI of brains and thoracic regions at the endpoint of the experiment. Values are shown in box-and-whisker plots where every dot represents a different animal and the line in the box corresponds to the median. The boxes go from the upper to the lower quartiles and the whiskers go from the minimum to the maximum value (n=9 shControl mice and n=10 shAHR#1 mice). P value was calculated using two-tailed t-test.


N Representative of tumors with high ( red dot ) or low ( grey dot ) UBE4B . A few cases of matched - metastases allowed to evaluate HSP90 - dependent . Scale bar : : 100 µm .

N Representative images of primary tumors with high (red dot) or low (grey dot) UBE4B levels. A few cases of matched primary-metastases allowed to evaluate the HSP90-dependent protein. Scale bar: : 100 µm.


P Kaplan - Meier curve comparing relapse - free survival of tumors with high low values of UBE4B . P value was calculated using log - rank ( Mantel - Cox ) .

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P Kaplan-Meier curve comparing relapse-free survival of primary tumors with high and low values of UBE4B. P value was calculated using log-rank (Mantel-Cox) test.


D Representative organotypic cultures from experiment in panel ( C ) . RFP is an internal control probe labelling cancer independent of autophagy flux GFP indicate GFP - LC3 + . dotted in upper panel delimits a high magnification shown in lower panel respect to GFP signal derived from GFP - LC3 accumulation . Dotted lines in lower panel surround cancer . Asterisk labels in magnified in high magnification panel showing GFP - LC3 + . Scale bar : low magnificantion , 25 µm ; high magnification ( ) , 10 µm ; high magnification ( ) , 2 . 5 µm . E of GFP - LC3 + vesicles per of experiment in panel ( C ) . Values are shown in box - - whisker plots where every dot represents a field of view of an organotypic culture in box corresponds to median . boxes go from upper to lower quartiles whiskers go from minimum to maximum value ( DMSO : n = 15 fields of view , 2232 cancer from 3 organotypic cultures ; DEBIO - 0932 : n = 20 fields of view , 3260 cancer from 4 organotypic cultures ) . P value was calculated using two - t - .

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D Representative organotypic cultures from the experiment in panel (C). RFP is an internal control probe labelling cancer cells independent of autophagy flux and GFP indicate GFP-LC3+ puncta. The dotted line in the upper panel delimits a high magnification area shown in the lower panel respect to the GFP signal derived from GFP-LC3 accumulation. Dotted lines in lower panel surround individual cancer cells. Asterisk labels the area in the cell magnified in the high magnification panel showing the GFP-LC3+ puncta. Scale bar: low magnificantion, 25 µm; high magnification (cells), 10 µm; high magnification (puncta), 2.5 µm. E Quantification of GFP-LC3+ vesicles per cell of the experiment in panel (C). Values are shown in box-and-whisker plots where every dot represents a field of view of an organotypic culture and the line in the box corresponds to the median. The boxes go from the upper to the lower quartiles and the whiskers go from the minimum to the maximum value (DMSO: n=15 fields of view, 2232 cancer cells from 3 organotypic cultures; DEBIO-0932: n=20 fields of view, 3260 cancer cells from 4 organotypic cultures). P value was calculated using two-tailed t-test.


Growth of wild type Xcc8004 , EPS deficient mutants ( B5 B12 ) , ndvB ndvB ( pHMJ ) grown in PS medium ( A ) ; PS medium containing 50 µm extracellularion chelator , diethylenetriaminepentaacetic acid ( DETAPAC ) ( B ) ; or supplemented with either with ( C ) secreted glucan ( 0 . 5 mg / ml ) ; ( D ) xanthan ( 0 . 5 mg / ml ) ; ( E ) FeSO4 ( 40µM ) ; ( F ) MnCl2 ( 40µM ) . Data shown are mean ± SD ( n = 3 ) . From here onward , until not mentioned , ' n ' indicates three biological replicates .

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Growth analysis of wild type Xcc8004, EPS deficient mutants (B5 and B12), ndvB and ndvB (pHMJ) grown in PS medium (A); PS medium containing 50 µm extracellularion chelator, diethylenetriaminepentaacetic acid (DETAPAC) (B); or supplemented with either with (C) secreted glucan (0.5 mg/ml); (D) xanthan (0.5 mg/ml); (E) FeSO4 (40µM); and (F) MnCl2 (40µM). Data shown are mean ± SD (n=3). From here onward, until not mentioned, the 'n' indicates three biological replicates.


( A ) MALDI - MS on 2 . 1 RT eluted fraction from wild type Xcc8004 EPS . Shown one quasimolecular ion at m / z ( mass - to - charge ) 2632 . 8 which matched with calculated mass for an [ M + Na ] + ion on an unsubstituted 16 - member cyclic glucan ( inset ) of Xanthomonas campestris . Peaks with m / z of below 1100 were matrix derived peaks after comparing MALDI - MS of matrix only control ( See also Fig . EV3A - B ; Fig . EV4D ) .

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(A) MALDI-MS analysis on the 2.1 RT eluted fraction from the wild type Xcc8004 EPS. Shown one quasimolecular ion at m/z (mass-to-charge ratio) 2632.8 which matched with the calculated mass for an [M+Na] + ion based on an unsubstituted 16-member cyclic glucan (inset) of Xanthomonas campestris. Peaks with m/z of below 1100 were matrix derived peaks after comparing the MALDI-MS of matrix only control (See also Fig. EV3A-B; Fig. EV4D).


( B ) Top : GC - MS of acid hydrolyzed BSTFA - derivatized , 2 . 1 RT eluted fraction from EPS of wild type Xcc . Bottom : matching spectrum from NIST database , corresponding to glucopyranose pentakis - O - trimethyl silyl ( See also Fig . EV3C ) .

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(B) Top: GC-MS analysis of acid hydrolyzed and BSTFA-derivatized, 2.1 RT eluted fraction from the EPS of wild type Xcc strain. Bottom: matching spectrum from NIST database, corresponding to glucopyranose pentakis-O-trimethyl silyl (See also Fig. EV3C).


( C ) of total EPS , secreted ( S ) periplasmic ( P ) glucan by phenol - sulfuric acid colorimetric from 2 - day - old cultures . Secreted xanthan glucan was isolated from - free culture supernatant by addition of KCl [ 1 % ( w / v ) ] 2 volumes of ethanol , to precipitate xanthan . Supernatant containing cyclic - β - ( 1 , 2 ) was purified by BioGel - P4 - exclusion chromatography . To isolate periplasmic glucan , pellet was treated with 1 % trichloroacetic acid ( TCA ) to release periplasmic glucans , purified by BioGel - P4 - exclusion chromatography . Data shown are mean ± SD ( n = 3 ) .

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(C) Quantification of total EPS, secreted (S) and periplasmic (P) glucan by phenol-sulfuric acid colorimetric assay from 2-day-old cultures. Secreted xanthan and glucan was isolated from the cell-free culture supernatant by addition of KCl [1% (w/v)] and 2 volumes of ethanol, to precipitate xanthan. Supernatant containing cyclic-β-(1, 2) was purified by BioGel-P4 size-exclusion chromatography. To isolate periplasmic glucan, cell pellet was treated with 1% trichloroacetic acid (TCA) to release periplasmic glucans, purified by BioGel-P4 size-exclusion chromatography. Data shown are mean ± SD (n=3).


( A ) Iron ( Fe2 + ) - binding potential of different polysaccharide [ used at 5 . 56 micromole ( glucose equivalent ) ] using Fe2 + - ferrozine colorimetric . Lipopolysacchrides ( LPS ; E . coli 0111 : B4 ) , mannan ( Saccharomyces cerevisiae ) , isolated commercial ( purchased from Sigma Aldrich ) xanthan , extracellular glucan from Xcc . Data shown are mean ± SD ( n = 3 ) .

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(A) Iron (Fe2+)-binding potential of different polysaccharide [used at 5.56 micromole (glucose equivalent)] using the Fe2+-ferrozine colorimetric assay. Lipopolysacchrides (LPS; E. coli 0111:B4), mannan (Saccharomyces cerevisiae), isolated and commercial (purchased from Sigma Aldrich) xanthan, extracellular glucan from Xcc. Data shown are mean ± SD (n=3).


( B ) Colorimetric iron - binding . Glucan from diverse source binds Fe2 + iron . Extracellular ( ECG ) or periplasmic ( PPG ) glucan isolated from wild type Xcc8004 or ndvB ( pHM1J ) ; commercial glucan from yeast , barley Euglena purchased from Sigma Aldrich . Glucans from various sources were incubated with FeSO4 at room for 15 min , amount of free iron was determined using standard of ferrozine - Fe2 + .

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(B) Colorimetric iron-binding assay. Glucan from diverse source binds Fe2+ iron. Extracellular (ECG) or periplasmic (PPG) glucan isolated from the wild type Xcc8004 or the ndvB (pHM1J); commercial glucan from yeast, barley and Euglena purchased from Sigma Aldrich. Glucans from various sources were incubated with FeSO4 at room temperature for 15 min, amount of free iron was determined using the standard concentration of ferrozine-Fe2+ complex.


( C ) Iron - binding measured by Inductively Coupled Plasma - Optical Emission Spectrometry ( ICP - OES ) . Xanthan various glucans were mixed with 2 mM FeSO4 , dialysed against deionized water before determination of Fe2 + content by ICP - OES . Data shown are mean ± SD ( n ≥ 3 ) . * * indicates p - value < 0 . 01 * * * indicates p - value < 0 . 001 by paired student t - .

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(C) Iron-binding measured by Inductively Coupled Plasma-Optical Emission Spectrometry (ICP-OES). Xanthan and various glucans were mixed with 2 mM FeSO4, dialysed against deionized water before determination of Fe2+ content by ICP-OES. Data shown are mean ± SD (n≥3). ** indicates p-value < 0.01 and *** indicates p-value < 0.001 by paired student t-test.