Fig 6A ) Immuno - fluorescence images of LC3 - II and P . gingivalis within MoDCs pre - treated with GP120 ( DC - SIGN blocker ) after 12 hours of infection . LC3 - II was detected in red - fluorescent dye and the bacterial strains were pre - labeled with green CFSE . B ) Fluorescent intensity of LC3 - II and P . gingivalis strains signal ( in triplicates ) were statistically analyzed ( * and # p < 0 . 001 ) . C ) Flow cytometry of MoDCs treated with siRNA for DC - SIGN ( si - DC - SIGN ) after 12 hours . Left panels show the decrease of DC - SIGN in Cont . ( un - infected ) , Pg381 and Mfa1 + Pg - infected MoDCs . D ) Epifluorescence microscopy images of MoDCs ( si - DC - SIGN ) infected with Pg381 and Mfa1 + Pg . LC3 - II was detected in red - fluorescent dye and the bacterial strains were pre - labeled with green CFSE . E ) The figure shows the CFU counts of the P . gingivalis strains with MoDCs that lack DC - SIGN ( si - DC - SIGN ) . The analysis of readings used One - way ANOVA analysis of different groups and Tukey ' s test for multiple comparisons ( * p < 0 . 001 ) .

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Fig 6A) Immuno-fluorescence images of LC3-II and P. gingivalis within MoDCs pre-treated with GP120 (DC-SIGN blocker) after 12 hours of infection. LC3-II was detected in red-fluorescent dye and the bacterial strains were pre-labeled with green CFSE.B) Fluorescent intensity of LC3-II and P. gingivalis strains signal (in triplicates) were statistically analyzed (* and # p<0.001).C) Flow cytometry of MoDCs treated with siRNA for DC-SIGN (si-DC-SIGN) after 12 hours. Left panels show the decrease of DC-SIGN in Cont. (un-infected), Pg381 and Mfa1+Pg-infected MoDCs.D) Epifluorescence microscopy images of MoDCs (si-DC-SIGN) infected with Pg381 and Mfa1+Pg. LC3-II was detected in red-fluorescent dye and the bacterial strains were pre-labeled with green CFSE.E) The figure shows the CFU counts of the P. gingivalis strains with MoDCs that lack DC-SIGN (si-DC-SIGN). The analysis of readings used One-way ANOVA analysis of different groups and Tukey's test for multiple comparisons (* p<0.001).


Fig 7A ) Western blot detection of LC3 in MoDCs infected with Pg381 and mutant strains for 12 hours . B ) The mean ± standard deviation of LC3 - II intensity represents three different experiments and the values were analyzed using Kruskal - Wallis test analysis of different groups and Dunn ' s test for multiple comparisons . C ) Blot shows the autophagy flux test after blocking the lysosomal fusion with Bafilomycin in MoDCs . Cells were treated with Bafilomycin for 4 hours at 4nM . D ) LC3 - II intensity in MoDCs infected with Pg381 strains with and without Bafilomycin . The analysis of the intensity of three different experiments used Kruskal - Wallis test analysis of different groups and Dunn ' s test for multiple comparisons .

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Fig 7A) Western blot detection of LC3 in MoDCs infected with Pg381 and mutant strains for 12 hours. B) The mean ±standard deviation of LC3-II intensity represents three different experiments and the values were analyzed using Kruskal-Wallis test analysis of different groups and Dunn's test for multiple comparisons.C) Blot shows the autophagy flux test after blocking the lysosomal fusion with Bafilomycin in MoDCs. Cells were treated with Bafilomycin for 4 hours at 4nM. D) LC3-II intensity in MoDCs infected with Pg381 strains with and without Bafilomycin. The analysis of the intensity of three different experiments used Kruskal-Wallis test analysis of different groups and Dunn's test for multiple comparisons.


Fig 8A ) Flow cytometry of CD83 on MoDCs after incubation of TLR4 ligand ( E . coli LPS ) and TLR1 and 2 ligand ( Pam3csk4 ) for 4 hour . B ) Immuno - fluorescence images of LC3 - II ( red ) within MoDCs after incubation with TLR4 and TLR1 & amp ; amp ; 2 ligands ( E . coli LPS and Pam3csk4 ) C ) The plot represents the means ± standard deviation of CFU within MoDCs harvested from three healthy individuals after 24 hours ( * * p < 0 . 001 ) .

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Fig 8A) Flow cytometry of CD83 on MoDCs after incubation of TLR4 ligand (E. coli LPS) and TLR1 and 2 ligand (Pam3csk4) for 4 hour.B) Immuno-fluorescence images of LC3-II (red) within MoDCs after incubation with TLR4 and TLR1&amp;2 ligands (E. coli LPS and Pam3csk4)C) The plot represents the means ±standard deviation of CFU within MoDCs harvested from three healthy individuals after 24 hours (** p<0.001).


Fig 9A ) Scanning electron microscopy ( SEM ) of MoDCs of the early interaction of MoDCs with Pg381 and Mfa1 + Pg ( upper panel 10000x and lower panel 25000x ) . B ) SEM for MoDCs interacting with Pg381 ( green stains for bacteria are computer generated ) . C ) Transmission electron microscopy ( TEM ) of autophagosome like structures within MoDCs infected with Pg381 , Mfa1 + Pg and FimA + Pg for 12 hours ( upper , middle and lower panels , respectively ) . The right and left sections show the different magnifications of randomly selected sections . Pg381 and FimA + Pg strains are mostly enclosed in the characteristics double - membrane intracellular vesicles ( Orange arrows ) . Contrary , Mfa1 + Pg escaped these autophagic ( double - membrane ) vesicles and enclosed within single membrane structures or freely occupy the cytoplasm ( Green arrows ) . D ) The ratio of bacteria included in the double membrane were compared to total number of bacteria within MoDCs and plotted as percentage . Counting of the bacteria included in single or doubled membrane vesicles after 12 hours of infection . Each strain was counted in three randomly selected grids for each sample . The analysis of the bacterial counts used Kruskal - Wallis test of different groups and Dunn ' s test for multiple comparisons ( * p < 0 . 01 ) .

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Fig 9A) Scanning electron microscopy (SEM) of MoDCs of the early interaction of MoDCs with Pg381 and Mfa1+Pg (upper panel 10000x and lower panel 25000x). B) SEM for MoDCs interacting with Pg381 (green stains for bacteria are computer generated).C) Transmission electron microscopy (TEM) of autophagosome like structures within MoDCs infected with Pg381, Mfa1+Pg and FimA+Pg for 12 hours (upper, middle and lower panels, respectively). The right and left sections show the different magnifications of randomly selected sections. Pg381 and FimA+Pg strains are mostly enclosed in the characteristics double-membrane intracellular vesicles (Orange arrows). Contrary, Mfa1+Pg escaped these autophagic (double-membrane) vesicles and enclosed within single membrane structures or freely occupy the cytoplasm (Green arrows).D) The ratio of bacteria included in the double membrane were compared to total number of bacteria within MoDCs and plotted as percentage. Counting of the bacteria included in single or doubled membrane vesicles after 12 hours of infection. Each strain was counted in three randomly selected grids for each sample. The analysis of the bacterial counts used Kruskal-Wallis test of different groups and Dunn's test for multiple comparisons (*p<0.01).


Fig 10A ) Epifluorescence microscopy images of MoDCs infected with pre - labeled bacteria ( green - CFSE ) . LAMP1 detected by transduction of red fluorescent protein ( RFP ) chimera using baculovirus transgenes to MoDCs . B ) The mean of fluorescent intensities ± standard deviation of LAMP1 were quantified in three different experiments ( * p < 0 . 001 ) . C ) The upper scattered plot shows the co - localization of red and green signals in sections of MoDCs infected with Pg381 and mutants strains . The lower plot shows the average of Pearson ' s correlation measures of three randomly selected region of interest ( ROI ) within each field within 3 different experiments ( * p < 0 . 0001 ) ( Table 4 and 5 ) . All analysis of fluorescence intensity used One - way ANOVA analysis of different groups and Tukey ' s test for multiple comparisons .

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Fig 10A) Epifluorescence microscopy images of MoDCs infected with pre-labeled bacteria (green-CFSE). LAMP1 detected by transduction of red fluorescent protein (RFP) chimera using baculovirus transgenes to MoDCs.B) The mean of fluorescent intensities ±standard deviation of LAMP1 were quantified in three different experiments (* p<0.001).C) The upper scattered plot shows the co-localization of red and green signals in sections of MoDCs infected with Pg381 and mutants strains. The lower plot shows the average of Pearson's correlation measures of three randomly selected region of interest (ROI) within each field within 3 different experiments (* p<0.0001) (Table 4 and 5). All analysis of fluorescence intensity used One-way ANOVA analysis of different groups and Tukey's test for multiple comparisons.


Figure 1C , D . Immunolabeling of HH25 chick embryo sections 50 hours post - engraftment of A375P : : GFP cells , using an anti - GFP antibody ( in green in C , in brown in D , A375P : GFP cells ) , an anti - HNK1 antibody ( in white , migrating and early post - migrating NCCs ) , an anti - Ki67 antibody ( in red in C , cycling cells ) , an anti - SOX10 antibody ( in red in D , also stains chick endogenous NCCs ) and an anti - MITF antibody ( in yellow in D ) . Nuclei were stained with Hoechst ( in blue ) . In C , the upper left photo shows a grafted embryo prior to cryosection . In D , right panels are enlargements of the lower left panel . Scale bar : 200 µm . F . 3D views ( light - sheet imaging ) of HH25 chick embryos engrafted with different melanoma patient samples , showing different patterns of tumor cell localization 50 hours post - engraftment . G . Immunofluorescent labeling of HH25 chick embryo sections 50 hours post - engraftment of OF - MEL - 020 patient sample , labeled with CFSE ( in green ) prior to the graft . An anti - SOX10 antibody ( in red ) and an anti - MITF antibody ( in yellow ) were used . Nuclei were stained with Hoechst ( in blue ) . Right panels are enlargements of the dotted area in the left panel .

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Figure 1C,D. Immunolabeling of HH25 chick embryo sections 50 hours post-engraftment of A375P::GFP cells, using an anti-GFP antibody (in green in C, in brown in D, A375P:GFP cells), an anti-HNK1 antibody (in white, migrating and early post-migrating NCCs), an anti-Ki67 antibody (in red in C, cycling cells), an anti-SOX10 antibody (in red in D, also stains chick endogenous NCCs) and an anti-MITF antibody (in yellow in D). Nuclei were stained with Hoechst (in blue). In C, the upper left photo shows a grafted embryo prior to cryosection. In D, right panels are enlargements of the lower left panel. Scale bar: 200 µm.F. 3D views (light-sheet imaging) of HH25 chick embryos engrafted with different melanoma patient samples, showing different patterns of tumor cell localization 50 hours post-engraftment.G. Immunofluorescent labeling of HH25 chick embryo sections 50 hours post-engraftment of OF-MEL-020 patient sample, labeled with CFSE (in green) prior to the graft. An anti-SOX10 antibody (in red) and an anti-MITF antibody (in yellow) were used. Nuclei were stained with Hoechst (in blue). Right panels are enlargements of the dotted area in the left panel.


Figure 2A , B . Immunofluorescent labeling ( A ) of HH25 chick embryo sections 50 hours post - engraftment of A375P : : GFP cells , using an anti - GFP antibody , an anti - MITF antibody ( pink ) and an anti - SOX10 ( white ) antibody . Nuclei were stained with Hoechst . Right panels are enlargements of the dotted area in left panel . In B , the mean fraction of MITF - negative ( MITFneg ) , - low ( MITFlow ) and - positive ( MITFpos ) A375P : : GFP cells in the tumor mass and in migrating cells was quantified ( n = 16 slices from 3 embryos ) . Error bars show SEM . Mann - Whitney test comparing tumors vs migrating cells for each class of MITF expression was performed , p - values are indicated on the graph . C , D . Immunofluorescent labeling ( C ) of HH25 chick embryo sections 50 hours post - engraftment of A375P : : GFP cells , using an anti - GFP antibody and an anti - Ki67 antibody . Nuclei were stained with Hoechst . Right panels are enlargements of the dotted area in the left panel . In D , the mean fraction of Ki67 - positive ( Ki67pos ) A375P : : GFP cells in the tumor mass and in migrating cells was quantified ( n = 25 slices from 3 embryos ) . Arrows point at cells having a mesenchymal morphology . Error bars show SEM . Student t - test , * * p = 0 . 0070 . H - K . 3D views ( H , J ) and quantification of tumor volumes ( I , K ) of HH25 chick embryos engrafted with GLO ( H , I ) or GLO - R ( J , K ) cells and treated with a combination of Vemurafenib and Cobimetinib , or Vorinostat alone , or a combination of the three molecules . The number of embryos analyzed for each experimental condition are indicated on the graphs . Bars and error bars indicate mean + SEM . Student t - tests , * * : p < 0 . 01 , ns : not significant . Exact p - values are indicated on the graphs .

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Figure 2A,B. Immunofluorescent labeling (A) of HH25 chick embryo sections 50 hours post-engraftment of A375P::GFP cells, using an anti-GFP antibody, an anti-MITF antibody (pink) and an anti-SOX10 (white) antibody. Nuclei were stained with Hoechst. Right panels are enlargements of the dotted area in left panel. In B, the mean fraction of MITF-negative (MITFneg), -low (MITFlow) and -positive (MITFpos) A375P::GFP cells in the tumor mass and in migrating cells was quantified (n = 16 slices from 3 embryos). Error bars show SEM. Mann-Whitney test comparing tumors vs migrating cells for each class of MITF expression was performed, p-values are indicated on the graph.C,D. Immunofluorescent labeling (C) of HH25 chick embryo sections 50 hours post-engraftment of A375P::GFP cells, using an anti-GFP antibody and an anti-Ki67 antibody. Nuclei were stained with Hoechst. Right panels are enlargements of the dotted area in the left panel. In D, the mean fraction of Ki67-positive (Ki67pos) A375P::GFP cells in the tumor mass and in migrating cells was quantified (n = 25 slices from 3 embryos). Arrows point at cells having a mesenchymal morphology. Error bars show SEM. Student t-test, **p = 0.0070.H-K. 3D views (H, J) and quantification of tumor volumes (I, K) of HH25 chick embryos engrafted with GLO (H, I) or GLO-R (J, K) cells and treated with a combination of Vemurafenib and Cobimetinib, or Vorinostat alone, or a combination of the three molecules. The number of embryos analyzed for each experimental condition are indicated on the graphs. Bars and error bars indicate mean + SEM. Student t-tests, **: p < 0.01, ns: not significant. Exact p-values are indicated on the graphs.


Figure 3A , B . 3D views ( A ) and quantification of variations in the mean tumor volume ( B ) of HH25 chick embryos engrafted with BRAFwt ( OF - MEL - 028 ) or BRAFV600E ( OF - MEL - 020 , OF - MEL - 027 ) patient samples and treated with excipient or a combination of Vemurafenib and Cobimetinib . The number of embryos analyzed for each patient sample is indicated on the graphs . Student t - test for OF - MEL - 020 ; Mann - Whintey test for OF - MEL - 028 , * : p < 0 . 05 , ns : not significant . Exact p - values are indicated on the graphs . C , D . Survival rate ( left axis ) and mean body surface area ( BSA , right axis ) of chick embryos injected with increasing doses of Dabrafenib ( C ) and Trametinib ( D ) . The maximum tolerated dose ( MTD , in red on the X - axis ) was defined as the higher dose of drug associated with a survival rate higher than 75 % and a mean BSA similar ( i . e . , non - statistically different ) from embryos treated with excipient ( 17 . 1 and 0 . 0228 mg / kg respectively ) . N = 10 replicates per experimental group . Error bars indicate SEM . ns , non - significant using Student ' s t - test compared with excipient . ( F - H ) of HH25 chick embryos engrafted with 3 different patient samples ( OF - MEL - 033 ( F ) , OF - MEL - 034 ( G ) , OF - MEL - 035 ( H ) ) and treated with excipient or a combination of Dabrafenib and Trametinib . The clinical response of each patient after a 3 months treatment with Dabrafenib / Trametinib is indicated above the graphs . Error bars indicate SEM . Mann - Whitney tests , exact p - values are indicated on the graphs .

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Figure 3A,B. 3D views (A) and quantification of variations in the mean tumor volume (B) of HH25 chick embryos engrafted with BRAFwt (OF-MEL-028) or BRAFV600E (OF-MEL-020, OF-MEL-027) patient samples and treated with excipient or a combination of Vemurafenib and Cobimetinib. The number of embryos analyzed for each patient sample is indicated on the graphs. Student t-test for OF-MEL-020; Mann-Whintey test for OF-MEL-028, *: p < 0.05, ns: not significant. Exact p-values are indicated on the graphs.C,D. Survival rate (left axis) and mean body surface area (BSA, right axis) of chick embryos injected with increasing doses of Dabrafenib (C) and Trametinib (D). The maximum tolerated dose (MTD, in red on the X-axis) was defined as the higher dose of drug associated with a survival rate higher than 75% and a mean BSA similar (i.e., non-statistically different) from embryos treated with excipient (17.1 and 0.0228 mg/kg respectively). N=10 replicates per experimental group. Error bars indicate SEM. ns, non-significant using Student's t-test compared with excipient.(F-H) of HH25 chick embryos engrafted with 3 different patient samples (OF-MEL-033 (F), OF-MEL-034 (G), OF-MEL-035 (H)) and treated with excipient or a combination of Dabrafenib and Trametinib. The clinical response of each patient after a 3 months treatment with Dabrafenib/Trametinib is indicated above the graphs. Error bars indicate SEM. Mann-Whitney tests, exact p-values are indicated on the graphs.


f1a , Fluorescence microscopyHeLa cells infected with Shigella and Listeria visualized with 4 ′ , 6 - diamidino - 2 - phenylindole ( DAPI ) ( pseudo - coloured red ) , and Salmonella carrying mCherry - expressing vector ( red ) . The cis - Golgi ( green ) was detected by α - GM130 antibodies . Scale bar , 10 µm . b , Fluorescence microscopy of HeLa cells transfected with either IpaJ or VirA . The cis - Golgi ( GM130 , green ) and F - actin ( red ) are shown . Scale bar , 10 µm . c , Fluorescence microscopy of HeLa cells infected with indicated Shigella mutant strains . Scale bar , 10 µm . d , Percentage of HeLa cells with disrupted Golgi morphology in 100 cells infected with either wild - type Shigella ( M90T ) or the indicated mutants . Error bars , means ± s . e . m . calculated from three independent experiments . e , Number of recoverable c . f . u . per mouselung24 h after intranasal inoculation with 1 × 106 of wild - type M90T or each mutant Shigella strain as indicated . Limit of detection ( LOD ) for this assay is indicated by the dotted line . Error bars , geometrical means ± s . e . m . * * P < 0 . 001 .

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f1a, Fluorescence microscopyHeLa cells infected with Shigella and Listeria visualized with 4′,6-diamidino-2-phenylindole (DAPI) (pseudo-coloured red), and Salmonella carrying mCherry-expressing vector (red). The cis-Golgi (green) was detected by α-GM130 antibodies. Scale bar, 10 µm.b, Fluorescence microscopy of HeLa cells transfected with either IpaJ or VirA. The cis-Golgi (GM130, green) and F-actin (red) are shown. Scale bar, 10 µm.c, Fluorescence microscopy of HeLa cells infected with indicated Shigella mutant strains. Scale bar, 10 µm.d, Percentage of HeLa cells with disrupted Golgi morphology in 100 cells infected with either wild-type Shigella (M90T) or the indicated mutants. Error bars, means ± s.e.m. calculated from three independent experiments.e, Number of recoverable c.f.u. per mouselung24 h after intranasal inoculation with 1 × 106 of wild-type M90T or each mutant Shigella strain as indicated. Limit of detection (LOD) for this assay is indicated by the dotted line. Error bars, geometrical means ± s.e.m. **P<0.001.


f2b , Fluorescence microscopy of HeLa cells transfected with the indicated IpaJ mutants . The cis - Golgi ( GM130 , green ) and F - actin ( red ) are shown . Scale bar , 10 µm . c , IpaJ or its catalytic mutants were expressed from a galactose - inducible promoter ( pGal413 vector ) and assayed for a growth arrest phenotype on galactose or glucose ( control ) carbon source . e , Yeast strain harbouring a galactose - inducible IpaJ gene were transformed with a multi - copy vector containing the indicated genes . Yeast were assayed for a growth arrest phenotype on galactose or glucose ( control ) carbon source .

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f2b, Fluorescence microscopy of HeLa cells transfected with the indicated IpaJ mutants. The cis-Golgi (GM130, green) and F-actin (red) are shown. Scale bar, 10 µm.c, IpaJ or its catalytic mutants were expressed from a galactose-inducible promoter (pGal413 vector) and assayed for a growth arrest phenotype on galactose or glucose (control) carbon source.e, Yeast strain harbouring a galactose-inducible IpaJ gene were transformed with a multi-copy vector containing the indicated genes. Yeast were assayed for a growth arrest phenotype on galactose or glucose (control) carbon source.


f3a - c , Mass spectra of purified ARF1 - strep in untreated ( a ) , IpaJ - treated ( b ) or IpaJ C64A - treated ( c ) cells , with the observed monoisotopic mass reported in daltons . * Observed 1 Da shift in molecular mass is accounted for in MS / MS data ( Supplementary Fig . 7 ) . f , In - gel fluorescence assay ( top panel ) visualizing Alexa Fluor 647 - labelled myristoylated ARF1 - His isolated from bacteria either not expressing ( lane 1 ) or expressing ( lane 2 ) NMTp . Bacterial cell lysates treated with recombinant IpaJ or IpaJ C64A as indicated . The expression levels of ARF1 - His were determined by Coomassie blue stain . g , In - gel fluorescence assay ( top panel ) visualizing Alexa Fluor 647 - labelled myristoylated ARF1 - strep purified from HeLa cell lysates left untreated or incubated with IpaJ or its catalytic mutant as indicated . The expressed amounts of ARF1 - strep are shown ( bottom panel ) . h , In - gel fluorescence assay visualizing protein extracts isolated from HeLa cells incubated with azide myristic acid , azide palmitic acid or geranylgeranyl alcohol azide and subsequently labelled with Alexa Fluor 647 alkyne by click chemistry . Arrows indicate proteins that are proteolytically demyristoylated by IpaJ .

B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

f3a-c, Mass spectra of purified ARF1-strep in untreated (a), IpaJ-treated (b) or IpaJ C64A-treated (c) cells, with the observed monoisotopic mass reported in daltons. *Observed 1 Da shift in molecular mass is accounted for in MS/MS data (Supplementary Fig. 7).f, In-gel fluorescence assay (top panel) visualizing Alexa Fluor 647-labelled myristoylated ARF1-His isolated from bacteria either not expressing (lane 1) or expressing (lane 2) NMTp. Bacterial cell lysates treated with recombinant IpaJ or IpaJ C64A as indicated. The expression levels of ARF1-His were determined by Coomassie blue stain.g, In-gel fluorescence assay (top panel) visualizing Alexa Fluor 647-labelled myristoylated ARF1-strep purified from HeLa cell lysates left untreated or incubated with IpaJ or its catalytic mutant as indicated. The expressed amounts of ARF1-strep are shown (bottom panel).h, In-gel fluorescence assay visualizing protein extracts isolated from HeLa cells incubated with azide myristic acid, azide palmitic acid or geranylgeranyl alcohol azide and subsequently labelled with Alexa Fluor 647 alkyne by click chemistry. Arrows indicate proteins that are proteolytically demyristoylated by IpaJ.


f4b , In - gel fluorescence assay showing myristoylated peptides of the indicated proteins in cells expressing IpaJ or catalytic mutant . The peptide - eGFP and IpaJ inputs are indicated . * Demyristoylation of GRASP65 and hVPS15 resulted in slower mobility of the resulting peptide , potentially owing to reduced mobility in SDS - polyacrylamide gel electrophoresis caused by proteolytic reaction . c , In - gel fluorescence assay showing myristoylated ARF1 T31N mutant ( a GDP - locked mutant ) or ARF1 Q71L ( a GTP - locked mutant ) after in cells co - expressing IpaJ or catalytic mutants as indicated . Equal loading of ARF1 ( middle panel ) and IpaJ ( lower panel ) was determined by western blot analysis . d , Fluorescence microscopy showing ARF1 - mCherry and the Golgi apparatus ( GM130 ) after cellular microinjection of recombinant IpaJ or IpaJ C64A , over the indicated period . Scale bar , 10 µm . e , Fluorescence microscopy showing ARF1 - eGFP ( green ) and Golgi membranes ( blue , GM130 ) after infection with Shigella M90T and the indicated mutants ( red ; mCherry - expressing bacteria ) . Scale bar , 10µm .

B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

f4b, In-gel fluorescence assay showing myristoylated peptides of the indicated proteins in cells expressing IpaJ or catalytic mutant. The peptide-eGFP and IpaJ inputs are indicated. *Demyristoylation of GRASP65 and hVPS15 resulted in slower mobility of the resulting peptide, potentially owing to reduced mobility in SDS-polyacrylamide gel electrophoresis caused by proteolytic reaction.c, In-gel fluorescence assay showing myristoylated ARF1 T31N mutant (a GDP-locked mutant) or ARF1 Q71L (a GTP-locked mutant) after in cells co-expressing IpaJ or catalytic mutants as indicated. Equal loading of ARF1 (middle panel) and IpaJ (lower panel) was determined by western blot analysis.d, Fluorescence microscopy showing ARF1-mCherry and the Golgi apparatus (GM130) after cellular microinjection of recombinant IpaJ or IpaJ C64A, over the indicated period. Scale bar, 10 µm.e, Fluorescence microscopy showing ARF1-eGFP (green) and Golgi membranes (blue, GM130) after infection with Shigella M90T and the indicated mutants (red; mCherry-expressing bacteria). Scale bar, 10µm.


Figure 1 ( A ) Mice bearing established A375 were treated with vehicle ( n = 7 ) , PLX4720 ( n = 5 ) bevacizumab ( n = 7 ) or COMBO ( n = 5 ) . Tumor growth is expressed as % change of the initial tumor , * * P < 0 . 01 , * * * P < 0 . 001 versus vehicle ( PLX4720 P = 0 . 0049 ; bevacizumab P = 0 . 0003 ; P = 4 . 86E - 10 ) ŦP < 0 . 05 compared to PLX4720 ( P = 0 . 015 ) . ( B ) Mice bearing established COLO205 were treated with vehicle ( n = 7 ) , PLX4720 ( n = 8 ) bevacizumab ( n = 7 ) or COMBO ( n = 8 ) . Tumor growth is expressed as % change of the initial tumor , * * * P < 0 . 001 versus vehicle ( PLX4720 P = 2 . 75E - 05 ; bevacizumab P = 2 . 96E - 05 ; COMBO P = 2 . 13E - 07 ) Ŧp < 0 . 05 compared to PLX4720 ( P = 0 . 039 ) . ( C ) Melanoma MC - 1 cells ( 5 x 105 ) were injected into the tail vein of CD1 mice . Lung colonization was assayed by HE staining and calculating the number of nodules and their total area normalized per the total area of the lungs . The mice analyzed were : start point , n = 6 ; vehicle , n = 7 ; PLX4720 , n = 6 ; bevacizumab , n = 6 ; COMBO , n = 7 , * P < 0 . 001 versus vehicle ( P = 0 . 031 ) . ( D ) Representative images of tumor cell proliferation determined by immunofluorescence Ki67 staining in A375 xenografts treated as indicated . Bar graphs indicate the Ki67 + area / tumor area ( n = 4 tumors ) , * * * P < 0 . 001 versus vehicle ( PLX4720 P = 2 . 83E - 08 ; bevacizumab P = 4 . 25E - 13 ; COMBO P = 6 . 16E - 08 ) . ( E ) Representative images of tumor cell apoptosis determined by immunofluorescence staining with TUNEL in A375 xenografts treated as indicated . Bar graphs indicate the TUNEL + area / tumor area ( n = 3 tumors ) . * * * P < 0 . 001 versus vehicle ( P = 6 . 2 . 7E - 10 ) .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1(A) Mice bearing established A375 were treated with vehicle (n=7), PLX4720 (n=5) bevacizumab (n=7) or COMBO (n=5). Tumor growth is expressed as % change of the initial tumor, **P < 0.01, ***P < 0.001 versus vehicle (PLX4720 P = 0.0049; bevacizumab P = 0.0003; P = 4.86E-10) ŦP < 0.05 compared to PLX4720 (P = 0.015).(B) Mice bearing established COLO205 were treated with vehicle (n=7), PLX4720 (n=8) bevacizumab (n=7) or COMBO (n=8). Tumor growth is expressed as % change of the initial tumor, ***P < 0.001 versus vehicle (PLX4720 P = 2.75E-05; bevacizumab P = 2.96E-05; COMBO P = 2.13E-07) Ŧp < 0.05 compared to PLX4720 (P = 0.039).(C) Melanoma MC-1 cells (5 x 105) were injected into the tail vein of CD1 mice. Lung colonization was assayed by HE staining and calculating the number of nodules and their total area normalized per the total area of the lungs. The mice analyzed were: start point, n=6; vehicle, n=7; PLX4720, n=6; bevacizumab, n=6; COMBO, n=7, *P < 0.001 versus vehicle (P = 0.031).(D) Representative images of tumor cell proliferation determined by immunofluorescence Ki67 staining in A375 xenografts treated as indicated. Bar graphs indicate the Ki67+ area/tumor area (n=4 tumors), ***P < 0.001 versus vehicle (PLX4720 P = 2.83E-08; bevacizumab P = 4.25E-13; COMBO P = 6.16E-08).(E) Representative images of tumor cell apoptosis determined by immunofluorescence staining with TUNEL in A375 xenografts treated as indicated. Bar graphs indicate the TUNEL+ area/tumor area (n=3 tumors). ***P < 0.001 versus vehicle (P = 6.2.7E-10).


Figure 2 ( A ) Representative images of vasculature stained by an antibody anti - CD31 in A375 xenografts treated as indicated . Bar graphs indicate quantitative micro vessel density ( MVD ) and micro vessel area ( MVA ) analysis ( n = 5 tumors ) , * * * P < 0 . 001 versus vehicle ( MVD : bevacizumab P = 5 . 94E - 25 ; COMBO P = 1 . 84E - 04 ) ( MVA : PLX4720 P = 4 . 98E - 09 ; bevacizumab P = 1 . 16E - 19 ; COMBO P = 3 . 00E - 08 ) . ( B ) Representative images of vessel lumen in A375 xenografts treated as indicated . Bar graphs indicate the quantitative analysis of lumen diameters in A375 and COLO205 xenografts ( n = 3 tumors ) , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 versus vehicle ( A375 : PLX4720 P = 0 . 0078 ; COMBO P = 0 . 0012 ) ( COLO205 : PLX4720 P = 2 . 01E - 06 ; COMBO P = 1 . 33E - 07 ) . ( C ) Representative images of perfusing fluorescent beads and their relationship with microvessels in A375 xenografts treated as indicated . Bar graphs indicate the % of CD31 + vessels co - stained with fluorescent beads ( n = 4 tumors ) , * * P < 0 . 01 versus vehicle ( PLX4720 P = 0 . 0031 ; COMBO P = 0 . 0033 ) . ( D ) Representative images of hypoxia marker pimonidazole in A375 xenografts treated as indicated . Bar graphs show the % of tumor hypoxic area in A375 and COLO205 xenografts ( n = 5 tumors ) , * * P < 0 . 01 , * * * P < 0 . 001 versus vehicle ( A375 : PLX4720 P = 6 . 94E - 12 ; COMBO P = 3 . 34E - 12 ) ( COLO205 : PLX4720 P = 0 . 0015 ; bevacizumab P = 0 . 0004 ; COMBO P = 0 . 0003 ) .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2(A) Representative images of vasculature stained by an antibody anti-CD31 in A375 xenografts treated as indicated. Bar graphs indicate quantitative micro vessel density (MVD) and micro vessel area (MVA) analysis (n=5 tumors), ***P < 0.001 versus vehicle (MVD: bevacizumab P = 5.94E-25; COMBO P = 1.84E-04) (MVA: PLX4720 P = 4.98E-09; bevacizumab P = 1.16E-19; COMBO P = 3.00E-08).(B) Representative images of vessel lumen in A375 xenografts treated as indicated. Bar graphs indicate the quantitative analysis of lumen diameters in A375 and COLO205 xenografts (n=3 tumors), *P < 0.05, **P < 0.01, ***P < 0.001 versus vehicle (A375: PLX4720 P = 0.0078; COMBO P = 0.0012) (COLO205: PLX4720 P = 2.01E-06; COMBO P = 1.33E-07).(C) Representative images of perfusing fluorescent beads and their relationship with microvessels in A375 xenografts treated as indicated. Bar graphs indicate the % of CD31+ vessels co-stained with fluorescent beads (n=4 tumors), **P < 0.01 versus vehicle (PLX4720 P = 0.0031; COMBO P = 0.0033).(D) Representative images of hypoxia marker pimonidazole in A375 xenografts treated as indicated. Bar graphs show the % of tumor hypoxic area in A375 and COLO205 xenografts (n=5 tumors), **P < 0.01, ***P < 0.001 versus vehicle (A375: PLX4720 P = 6.94E-12; COMBO P = 3.34E-12) (COLO205: PLX4720 P = 0.0015; bevacizumab P = 0.0004; COMBO P = 0.0003).


Figure 3 ( D ) GSEA enrichment plots for " Reactome Cytokine Signaling in Immune System " and " Reactome Innate Immune System " ( upper panels ) and " Reactome Extracellular Matrix Organization " and " Reactome Collagen Formation " ( lower panels ) highlight significant enrichment of the pathways relative to the immune response and a decreased expression of the pathways relative to extracellular matrix remodeling in COMBO treated tumors as compared to the other treatments ( vehicle , PLX4720 , bevacizumab ) . ( E ) Heatmap representation of gene expression changes within the " Reactome Innate Immune System " ( left panel ) and " Reactome Extracellular matrix Organization " ( right panel ) gene set . Genes in heatmaps are shown in rows and samples are shown in columns . Expression level is represented as a gradient from high ( red ) to low ( blue ) . V , P , B and C respectively indicate vehicle , PLX4720 , bevacizumab and COMBO .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3(D) GSEA enrichment plots for "Reactome Cytokine Signaling in Immune System" and "Reactome Innate Immune System" (upper panels) and "Reactome Extracellular Matrix Organization" and "Reactome Collagen Formation" (lower panels) highlight significant enrichment of the pathways relative to the immune response and a decreased expression of the pathways relative to extracellular matrix remodeling in COMBO treated tumors as compared to the other treatments (vehicle, PLX4720, bevacizumab).(E) Heatmap representation of gene expression changes within the "Reactome Innate Immune System" (left panel) and "Reactome Extracellular matrix Organization" (right panel) gene set. Genes in heatmaps are shown in rows and samples are shown in columns. Expression level is represented as a gradient from high (red) to low (blue). V, P, B and C respectively indicate vehicle, PLX4720, bevacizumab and COMBO.


Figure 4 ( A ) Representative images of leukocytes infiltration determined by immunofluorescence CD45 staining in of A375 xenografts treated as indicated . Bar graphs indicate the CD45 + area / tumor area in A375 and COLO205 xenografts ( n = 5 tumors ) , * * * P < 0 . 001 versus vehicle ( A375 : P = 2 . 19E - 20 ) ( COLO205 : P = 1 . 81E - 06 ) . ( B ) Representative images of macrophage infiltration determined by F4 / 80 immunofluorescence staining in A375 xenografts treated as indicated . Bar graphs indicate the F4 / 80 + area / tumor area in A375 and COLO205 xenografts ( n = 5 tumors ) * * * P < 0 . 001 versus vehicle ( A375 : COMBO P = 3 . 42E - 13 ) ( COLO205 : COMBO P = 1 . 94E - 06 ) . ( C ) A375 xenograft tumors after 14 days of treatment were subjected to FACS and tumor infiltrate was analyzed . Gating strategy for CD45 + cells and graph showing the quantification of FACS analysis of infiltrating CD45 + leukocytes in vehicle ( n = 4 tumors ) compared to COMBO ( n = 6 tumors ) treated tumors , * * P < 0 . 01 versus vehicle ( P = 0 . 0064 ) . ( D ) Graph showing the quantification of FACS analysis of infiltrating F4 / 80 + macrophages and CD11c + MHCII + dendritic cells in vehicle ( n = 4 tumors ) compared to COMBO ( n = 6 tumors ) treated tumors , * * P < 0 . 01 versus vehicle ( TAM P = 0 . 0061 ; DC P = 0 . 0091 ) .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4(A) Representative images of leukocytes infiltration determined by immunofluorescence CD45 staining in of A375 xenografts treated as indicated. Bar graphs indicate the CD45+ area/tumor area in A375 and COLO205 xenografts (n=5 tumors), ***P < 0.001 versus vehicle (A375: P = 2.19E-20) (COLO205: P = 1.81E-06).(B) Representative images of macrophage infiltration determined by F4/80 immunofluorescence staining in A375 xenografts treated as indicated. Bar graphs indicate the F4/80+ area/tumor area in A375 and COLO205 xenografts (n=5 tumors) ***P < 0.001 versus vehicle (A375: COMBO P = 3.42E-13) (COLO205: COMBO P = 1.94E-06).(C) A375 xenograft tumors after 14 days of treatment were subjected to FACS and tumor infiltrate was analyzed. Gating strategy for CD45+ cells and graph showing the quantification of FACS analysis of infiltrating CD45+ leukocytes in vehicle (n=4 tumors) compared to COMBO (n=6 tumors) treated tumors, **P < 0.01 versus vehicle (P = 0.0064).(D) Graph showing the quantification of FACS analysis of infiltrating F4/80+ macrophages and CD11c+MHCII+ dendritic cells in vehicle (n=4 tumors) compared to COMBO (n=6 tumors) treated tumors, **P < 0.01 versus vehicle (TAM P = 0.0061; DC P = 0.0091).


Figure 5 ( A ) Real - time quantitative PCR of the indicated genes ( M1 - like and M2 - like macrophages markers ) of A375 xenograft treated with PLX4720 , bevacizumab or COMBO . Data are presented as expression fold change ( log2 ) compared with vehicle after normalization for housekeeping gene TBP ( n = 3 tumors ) , ( * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 versus vehicle ( PLX4720 : m - Arg1 P = 5 . 65E - 06 ) ( bevacizumab : m - Ccl5 P = 0 . 025 ) ( COMBO : m - Ccl5 P = 0 . 0070 ; m - Cd40 P = 0 . 00099 ; m - Cxcl10 P = 0 . 043 ; m - Cxcl9 P = 0 . 046 ; m - Il1b P = 0 . 0367 ; m - Stat1 P = 0 . 049 ; m - Tlr2 = 0 . 00182 ; m - Arg - 1 P = 0 . 0013 ) . ( B ) Representative images of COMBO - treated tumor sections co - stained with an antibody against CD68 and an antibody anti - CCR7 ( M1 - like polarization ) or anti - CD206 ( M2 - like polarization ) . ( C ) Graphs showing the quantification of FACS analysis of infiltrating F4 / 80 + tumor macrophages and average percentage of median fluorescence intensity ( MFI ) of TNFα + or INOS + on F4 / 80 + cells after ex vivo stimulation with PMA and ionomycin in vehicle ( n = 4 tumors ) and COMBO ( n = 6 tumors ) , * P < 0 . 05 versus vehicle ( TNFα P = 0 . 037 ; INOS = 0 . 038 ) . ( D ) Graphs showing the quantification of FACS analysis of infiltrating CD11b + Ly6C + tumor monocytes and average percentage of MFI of TNFα + or INOS + on CD11b + Ly6C + cells after ex vivo stimulation with PMA and ionomycin in vehicle ( n = 4 tumors ) and COMBO ( n = 6 tumors ) , * P < 0 . 05 versus vehicle ( TNFα P = 0 . 048 ; INOS = 0 . 038 ) . ( E ) Graph showing the quantification of circulating CD11b + Ly6C + monocytes in peripheral blood analyzed by FACS in vehicle - ( n = 4 tumors ) and COMBO - ( n = 6 tumors ) treated tumors . ( F ) Cell viability measured as fluorescence intensity of ZsGreen - A375 cells co - cultured with cells isolated from xenograft treated with vehicle or COMBO with after 48h . Data are representative of one experiment out of two done in triplicate * P < 0 . 05 versus vehicle ( P = 0 . 046 ) .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5(A) Real-time quantitative PCR of the indicated genes (M1-like and M2-like macrophages markers) of A375 xenograft treated with PLX4720, bevacizumab or COMBO. Data are presented as expression fold change (log2) compared with vehicle after normalization for housekeeping gene TBP (n=3 tumors), (*P < 0.05, **P < 0.01, ***P < 0.001 versus vehicle (PLX4720: m-Arg1 P = 5.65E-06) (bevacizumab: m-Ccl5 P = 0.025) (COMBO: m-Ccl5 P = 0.0070; m-Cd40 P = 0.00099; m-Cxcl10 P = 0.043; m-Cxcl9 P = 0.046; m-Il1b P = 0.0367; m-Stat1 P = 0.049; m-Tlr2 = 0.00182; m-Arg-1 P = 0.0013).(B) Representative images of COMBO-treated tumor sections co-stained with an antibody against CD68 and an antibody anti-CCR7 (M1-like polarization) or anti-CD206 (M2-like polarization).(C) Graphs showing the quantification of FACS analysis of infiltrating F4/80+ tumor macrophages and average percentage of median fluorescence intensity (MFI) of TNFα+ or INOS+ on F4/80+ cells after ex vivo stimulation with PMA and ionomycin in vehicle (n=4 tumors) and COMBO (n=6 tumors), *P < 0.05 versus vehicle (TNFα P = 0.037; INOS = 0.038).(D) Graphs showing the quantification of FACS analysis of infiltrating CD11b+Ly6C+ tumor monocytes and average percentage of MFI of TNFα+ or INOS+ on CD11b+Ly6C+ cells after ex vivo stimulation with PMA and ionomycin in vehicle (n=4 tumors) and COMBO (n=6 tumors), *P < 0.05 versus vehicle (TNFα P = 0.048; INOS = 0.038).(E) Graph showing the quantification of circulating CD11b+Ly6C+ monocytes in peripheral blood analyzed by FACS in vehicle- (n=4 tumors) and COMBO- (n=6 tumors) treated tumors.(F) Cell viability measured as fluorescence intensity of ZsGreen-A375 cells co-cultured with cells isolated from xenograft treated with vehicle or COMBO with after 48h. Data are representative of one experiment out of two done in triplicate *P < 0.05 versus vehicle (P = 0.046).


Figure 6 ( A ) Representative images of collagen deposition determined by collagen I immunofluorescent staining in A375 xenografts treated as indicated . Bar graphs indicate the collagen I + area / tumor area ( n = 5 tumors ) , * * * P < 0 . 001 versus vehicle ( P = 2 . 93E - 06 ) . ( B ) Representative images of CAF density determined by α - SMA immunofluorescence staining in A375 xenografts treated as indicated . Bar graphs indicate α - SMA + area / tumor area ( n = 5 tumors ) , * * * P < 0 . 001 versus vehicle ( P = 0 . 0006 ) . ( C ) Representative images of Lysyl Oxidase enzyme determined by LOX immunofluorescence staining in A375 xenografts treated as indicated . Bar graphs indicate the LOX + area / tumor area ( n = 3 tumors ) , * P < 0 . 05 versus vehicle ( bevacizumab P = 0 . 033 ; COMBO P = 0 . 040 ) . ( E ) Quantitative Real - time PCR of h - TGFB1 and m - Tgfb1 of A375 xenograft treated with PLX4720 , bevacizumab or COMBO . Data are presented as expression fold change ( log2 ) compared with vehicle after normalization for the housekeeping gene TBP ( n = 3 tumors ) , * P < 0 . 05 versus vehicle ( P = 0 . 043 ) . ( F ) Western blot for human TGFβ in total protein extract from A375 tumors treated as indicate . Vinculin was used as an internal control .

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Figure 6(A) Representative images of collagen deposition determined by collagen I immunofluorescent staining in A375 xenografts treated as indicated. Bar graphs indicate the collagen I+ area/tumor area (n=5 tumors), ***P < 0.001 versus vehicle (P = 2.93E-06).(B) Representative images of CAF density determined by α-SMA immunofluorescence staining in A375 xenografts treated as indicated. Bar graphs indicate α-SMA+ area/tumor area (n=5 tumors), ***P < 0.001 versus vehicle (P = 0.0006).(C) Representative images of Lysyl Oxidase enzyme determined by LOX immunofluorescence staining in A375 xenografts treated as indicated. Bar graphs indicate the LOX+ area/tumor area (n=3 tumors), *P < 0.05 versus vehicle (bevacizumab P = 0.033; COMBO P = 0.040).(E) Quantitative Real-time PCR of h-TGFB1 and m-Tgfb1 of A375 xenograft treated with PLX4720, bevacizumab or COMBO. Data are presented as expression fold change (log2) compared with vehicle after normalization for the housekeeping gene TBP (n=3 tumors), *P < 0.05 versus vehicle (P = 0.043).(F) Western blot for human TGFβ in total protein extract from A375 tumors treated as indicate. Vinculin was used as an internal control.


Figure 7 ( A ) Effect of clodronate liposomes on tumor growth inhibition induced by COMBO treatment in in mice bearing A375 tumors . Tumor growth is expressed as % change of the initial tumor . Macrophage depletion by clodronate liposome enhanced the tumor growth of tumors treated with the combination of PLX4720 and bevacizumab ( n = 5 mice / group ) , * P < 0 . 05 , * * * P < 0 . 001 versus vehicle ( vehicle Clodronate P = 0 . 049 , COMBO P = 5 . 72E - 06 ) ŦP < 0 . 05 compared to COMBO ( P = 0 . 033 ) . ( B ) Representative images of macrophage infiltration determined by F4 / 80 immunofluorescence staining in A375 xenografts treated as indicated . Bar graphs indicate the F4 / 80 + area / tumor ( n = 3 tumors ) , * * * P < 0 . 001 versus vehicle ( P = 1 . 53E - 25 ) . ( C ) Representative images of collagen deposition determined by collagen I immunofluorescence staining in A375 xenografts treated as indicated . Bar graphs indicate the collagen I + area / tumor ( n = 3 tumors ) . ( D ) Waterfall plots showing the percent change in volume ( relative to the initial tumor volume ) for the individual A375 xenografts in each treatment group ( vehicle , PLX4720 , bevacizumab and COMBO ) from week 1 to week 6 . ( E ) Representative images of macrophage infiltration determined by F4 / 80 immunofluorescence staining in a responder and a relapsing A375 xenografts . Bar graphs indicate the F4 / 80 + area / tumor ( n = 3 mice ) , * * * P < 0 . 001 versus vehicle ( P = 1 . 25E - 06 ) . ( F ) Representative images of collagen deposition determined by collagen I immunofluorescence staining in a responder and a relapsing A375 xenografts . Bar graphs indicate the collagen I + area / tumor ( n = 3 mice ) , * * * P < 0 . 001 versus vehicle ( P = 4 . 03E - 05 ) . ( G ) Real - time quantitative PCR of h - TGFB1 of A375 xenograft treated with PLX4720 , bevacizumab or COMBO . Data are presented as expression fold change ( log2 ) of relapsing tumors compared to responder tumors after normalization for housekeeping gene TBP ( n = 3 tumors ) * * * P < 0 . 001 versus vehicle ( P = 0 . 0006 ) .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 7(A) Effect of clodronate liposomes on tumor growth inhibition induced by COMBO treatment in in mice bearing A375 tumors. Tumor growth is expressed as % change of the initial tumor. Macrophage depletion by clodronate liposome enhanced the tumor growth of tumors treated with the combination of PLX4720 and bevacizumab (n=5 mice/group), *P < 0.05, ***P < 0.001 versus vehicle (vehicle Clodronate P = 0.049, COMBO P = 5.72E-06) ŦP < 0.05 compared to COMBO (P = 0.033).(B) Representative images of macrophage infiltration determined by F4/80 immunofluorescence staining in A375 xenografts treated as indicated. Bar graphs indicate the F4/80+ area/tumor (n=3 tumors), ***P < 0.001 versus vehicle (P = 1.53E-25).(C) Representative images of collagen deposition determined by collagen I immunofluorescence staining in A375 xenografts treated as indicated. Bar graphs indicate the collagen I+ area/tumor (n=3 tumors).(D) Waterfall plots showing the percent change in volume (relative to the initial tumor volume) for the individual A375 xenografts in each treatment group (vehicle, PLX4720, bevacizumab and COMBO) from week 1 to week 6.(E) Representative images of macrophage infiltration determined by F4/80 immunofluorescence staining in a responder and a relapsing A375 xenografts. Bar graphs indicate the F4/80+ area/tumor (n=3 mice), ***P < 0.001 versus vehicle (P = 1.25E-06).(F) Representative images of collagen deposition determined by collagen I immunofluorescence staining in a responder and a relapsing A375 xenografts. Bar graphs indicate the collagen I+ area/tumor (n=3 mice), ***P < 0.001 versus vehicle (P = 4.03E-05).(G) Real-time quantitative PCR of h-TGFB1 of A375 xenograft treated with PLX4720, bevacizumab or COMBO. Data are presented as expression fold change (log2) of relapsing tumors compared to responder tumors after normalization for housekeeping gene TBP (n=3 tumors) ***P < 0.001 versus vehicle (P = 0.0006).


Figure 2A Isothermal calorimetry ( ITC ) assays using ADP - ribose ligand and purified macrodomains of human macroH2A1 . 1 and macroH2A2 . B In vitro PARP1 activity assay in the presence of increasing concentrations ( 10 , 25 and 50 μM ) of purified macrodomains of human macroH2A1 . 1 , macroH2A1 . 1 G224E mutant and macroH2A2 . PARP1 activity is assessed by its auto - PARylation detected by an anti - PAR immunoblot . The naphtol blue staining shows the increasing amounts of purified macrodomain added to each reaction . A representative blot of one of three independent experiments is shown .

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Figure 2A Isothermal calorimetry (ITC) assays using ADP-ribose ligand and purified macrodomains of human macroH2A1.1 and macroH2A2.B In vitro PARP1 activity assay in the presence of increasing concentrations (10, 25 and 50 μM) of purified macrodomains of human macroH2A1.1, macroH2A1.1 G224E mutant and macroH2A2. PARP1 activity is assessed by its auto-PARylation detected by an anti-PAR immunoblot. The naphtol blue staining shows the increasing amounts of purified macrodomain added to each reaction. A representative blot of one of three independent experiments is shown.


Figure 3A The diameter ( d ) of the fluorescence signaling of H2B tagged with photo - activatable GFP ( PAGFP - H2B ) is used to quantify chromatin expansion across 120 seconds after induction of DNA damage using intercalating DNA dye and local irradiation with a laser . B Chromatin expansion in absence and presence of intercalating DNA dye ( - and + DNA damage ) and pretreatment of 1h with 1μM of Olaparib PARP inhibitor . Boxplots represent single cell measurement of chromatin expansion at 120 sec . post DNA damage from 3 biological replicates with n > 30 cells each . The box limits correspond to 25th and 75th percentiles , the bold line indicates median and cross indicates the average values ( * , p < 0 . 05 using unpaired , two - tailed Student ' s T - test ) . C Quantified chromatin expansion in cells not transfected ( control ) and cells transfected with each mCherry - tagged histone ( as indicated ) . Data is represented as in B ( n > 30 cells , 3 bio . repl . ; * , p < 0 . 05 compared to mCherry - H2A - expressing cells using unpaired , two - tailed Student ' s T - test ) . D Chromatin expansion in Hela cells containing a doxocyclin ( doxo . ) - inducible macroH2A1 . 1 transgene . Chromatin expansion assay was performed 48 hours after induction and expression was controlled by anti - macroH2A1 . 1 - immunoblotting . Data is represented as in B ( n > 30 cells , 3 bio . repl . ; * , p < 0 . 05 using unpaired , two - tailed Student ' s T - test ) .

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Figure 3A The diameter (d) of the fluorescence signaling of H2B tagged with photo-activatable GFP (PAGFP-H2B) is used to quantify chromatin expansion across 120 seconds after induction of DNA damage using intercalating DNA dye and local irradiation with a laser.B Chromatin expansion in absence and presence of intercalating DNA dye (- and + DNA damage) and pretreatment of 1h with 1μM of Olaparib PARP inhibitor. Boxplots represent single cell measurement of chromatin expansion at 120 sec. post DNA damage from 3 biological replicates with n > 30 cells each. The box limits correspond to 25th and 75th percentiles, the bold line indicates median and cross indicates the average values (*, p < 0.05 using unpaired, two-tailed Student's T-test).C Quantified chromatin expansion in cells not transfected (control) and cells transfected with each mCherry-tagged histone (as indicated). Data is represented as in B (n > 30 cells, 3 bio. repl.; *, p < 0.05 compared to mCherry-H2A-expressing cells using unpaired, two-tailed Student's T-test).D Chromatin expansion in Hela cells containing a doxocyclin (doxo.)-inducible macroH2A1.1 transgene. Chromatin expansion assay was performed 48 hours after induction and expression was controlled by anti-macroH2A1.1-immunoblotting. Data is represented as in B (n > 30 cells, 3 bio. repl.; *, p < 0.05 using unpaired, two-tailed Student's T-test).


Figure 4Quantified chromatin expansion in cells transfected with indicated histone constructs . Boxplots represent single cell measurement of chromatin expansion at 120 sec . post DNA damage ( n > 30 cells , 3 bio . repl . ) . The box limits correspond to 25th and 75th percentiles , the bold line indicates median and cross indicates the average values . ( * , p < 0 . 05 using unpaired two - tailed Student ' s T - test comparing to H2A ; n . s . , not significant ) . Quantified chromatin expansion in cells transfected with indicated histone constructs . Boxplots represent single cell measurement of chromatin expansion at 120 sec . post DNA damage ( n > 30 cells , 3 bio . repl . ) . The box limits correspond to 25th and 75th percentiles , the bold line indicates median and cross indicates the average values . ( * , p < 0 . 05 using unpaired two - tailed Student ' s T - test comparing to H2A ; n . s . , not significant ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4Quantified chromatin expansion in cells transfected with indicated histone constructs. Boxplots represent single cell measurement of chromatin expansion at 120 sec. post DNA damage (n > 30 cells, 3 bio. repl.). The box limits correspond to 25th and 75th percentiles, the bold line indicates median and cross indicates the average values. (*, p<0.05 using unpaired two-tailed Student's T-test comparing to H2A; n.s., not significant).Quantified chromatin expansion in cells transfected with indicated histone constructs. Boxplots represent single cell measurement of chromatin expansion at 120 sec. post DNA damage (n > 30 cells, 3 bio. repl.). The box limits correspond to 25th and 75th percentiles, the bold line indicates median and cross indicates the average values. (*, p<0.05 using unpaired two-tailed Student's T-test comparing to H2A; n.s., not significant).


Figure 5A Western blot showing the expression of macroH2A proteins in control HepG2 cells , knock - down for macroH2A1 and macroH2A2 ( DKD ) and DKD with ectopic expression of the indicated YFP - tagged constructs . H3 is used as a loading control . B H3K9me3 immunostaining in the indicated HepG2 cell lines with nuclear counterstaining in DAPI . The indicated areas are zoomed - in for a better observation of the H3K9me3 signal profile . All images are maximum intensity Z - projections of confocal stacks . Quantification of the nuclear area The box limits correspond to 25th and 75th percentiles , the bold line indicates median and cross indicates the average values . ( * , p < 0 . 05 using unpaired two - tailed Student ' s T - test ; n . s . , not significant ) . Quantification of H3K9me3 foci per nucleus , The box limits correspond to 25th and 75th percentiles , the bold line indicates median and cross indicates the average values . ( * , p < 0 . 05 using unpaired two - tailed Student ' s T - test ; n . s . , not significant ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5A Western blot showing the expression of macroH2A proteins in control HepG2 cells, knock-down for macroH2A1 and macroH2A2 (DKD) and DKD with ectopic expression of the indicated YFP-tagged constructs. H3 is used as a loading control.B H3K9me3 immunostaining in the indicated HepG2 cell lines with nuclear counterstaining in DAPI. The indicated areas are zoomed-in for a better observation of the H3K9me3 signal profile. All images are maximum intensity Z-projections of confocal stacks.Quantification of the nuclear area The box limits correspond to 25th and 75th percentiles, the bold line indicates median and cross indicates the average values. (*, p<0.05 using unpaired two-tailed Student's T-test; n.s., not significant).Quantification of H3K9me3 foci per nucleus, The box limits correspond to 25th and 75th percentiles, the bold line indicates median and cross indicates the average values. (*, p<0.05 using unpaired two-tailed Student's T-test; n.s., not significant).


Figure 1 ( c - f ) The indicated marker genes were used to select clusters for subsetting into stromal cells ( c ) , epithelial cells ( d ) , endothelial cells ( e ) , and leukocytes ( f ) . ( g - j ) The heatmaps show the relative gene expression levels for the indicated marker genes for the indicated stromal ( g ) , epithelial ( h ) , endothelial ( i ) , and leukocyte ( j ) cell types .

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Figure 1(c-f) The indicated marker genes were used to select clusters for subsetting into stromal cells (c), epithelial cells (d), endothelial cells (e), and leukocytes (f).(g-j) The heatmaps show the relative gene expression levels for the indicated marker genes for the indicated stromal (g), epithelial (h), endothelial (i), and leukocyte (j) cell types.


Figure 2 ( d ) Differential gene expression between end stage lung disease patients and controls across cohorts was compared for the indicated cell type identities . The color code demonstrates similarities of gene expression changes calculated by Pearson correlation of the t - value coefficient , which represents differences in the likelihood of detection for any gene between health and disease . ( e ) The heatmap shows the top 79 genes differentially expressed in the indicated cell type identities . ( f - h ) The box plots illustrate differences in mRNA detection for the indicated genes between tissues from end stage lung fibrosis patients in ( f ) alveolar epithelial cells , ( g ) fibroblasts , and ( h ) macrophages when compared to control tissue . The boxes represent the interquartile range , the horizontal line in the box is the median , and the whiskers represent 1 . 5 times the interquartile range ( Chicago cohort : ILD n = 9 , controls n = 8 ; Nashville cohort : ILD n = 20 , controls n = 10 ; Munich cohort : ILD n = 3 , controls n = 11 ) .

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Figure 2(d) Differential gene expression between end stage lung disease patients and controls across cohorts was compared for the indicated cell type identities. The color code demonstrates similarities of gene expression changes calculated by Pearson correlation of the t-value coefficient, which represents differences in the likelihood of detection for any gene between health and disease.(e) The heatmap shows the top 79 genes differentially expressed in the indicated cell type identities.(f-h) The box plots illustrate differences in mRNA detection for the indicated genes between tissues from end stage lung fibrosis patients in (f) alveolar epithelial cells, (g) fibroblasts, and (h) macrophages when compared to control tissue. The boxes represent the interquartile range, the horizontal line in the box is the median, and the whiskers represent 1.5 times the interquartile range (Chicago cohort: ILD n = 9, controls n = 8; Nashville cohort: ILD n = 20, controls n = 10; Munich cohort: ILD n = 3, controls n = 11).


Figure 3 ( e ) The heatmap shows changes of the indicated cell type signatures in published bulk RNA - seq data ( GEO GSE124685 ) across different histopathological stages that represent increasing extent of fibrosis from stage 1 - 3 , as determined by quantitative micro - CT imaging and tissue histology Samples used in this study were 10 IPF and 6 control patients . ( f ) The heatmaps show z - scores for the individual marker genes of the indicated cell types across IPF stages and controls .

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Figure 3(e) The heatmap shows changes of the indicated cell type signatures in published bulk RNA-seq data (GEO GSE124685) across different histopathological stages that represent increasing extent of fibrosis from stage 1-3, as determined by quantitative micro-CT imaging and tissue histology Samples used in this study were 10 IPF and 6 control patients.(f) The heatmaps show z-scores for the individual marker genes of the indicated cell types across IPF stages and controls.


Figure 4 ( b ) The boxplots show the number of proteins quantified ( y - axis ) across various diagnoses ( x - axis ) . The mean and 10 - 90 percentiles are shown ; the dotted line marks 1000 proteins ( IPF n = 16 , HP / EAA n = 8 , COP n = 11 , NSIP n = 10 , RB - IL , n = 3 , Sarcoidosis n = 22 , other ILDs n = 25 , non - IL , n = 29 ) .

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Figure 4(b) The boxplots show the number of proteins quantified (y-axis) across various diagnoses (x-axis). The mean and 10-90 percentiles are shown; the dotted line marks 1000 proteins (IPF n=16, HP/EAA n=8, COP n=11, NSIP n=10, RB-IL, n=3, Sarcoidosis n=22, other ILDs n=25, non-IL, n=29).


Figure 5 ( c Empirical cumulative density plots depict the distribution of correlation coefficients for ( c ) Myofibroblast markers ( red points ) with DLCO in comparison to all background proteins ( black line ) . d ) Empirical cumulative density plots depict the distribution of correlation coefficients for ( d ) Plasma cell markers ( red points ) with % alveolar macrophages in BAL in comparison to all background proteins ( black line ) . ( e ) Barplot displays coefficients derived from correlating lung function associations at the protein level with cell type specific ILD associations at the RNA level . ( f ) The heatmap illustrates gene expression changes associated with lung fibrosis across indicated cell types ( columns ) for selected BALF protein biomarkers ( rows ) . The dotplot visualizes the frequency changes of the indicated cell types inferred from the deconvolution of bulk mRNA data of ILD samples compared to controls ( GSE47460 ) . Samples used in this study from the LTRC n = 254 ILD patients and n = 108 controls .

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Figure 5(c Empirical cumulative density plots depict the distribution of correlation coefficients for (c) Myofibroblast markers (red points) with DLCO in comparison to all background proteins (black line).d) Empirical cumulative density plots depict the distribution of correlation coefficients for (d) Plasma cell markers (red points) with % alveolar macrophages in BAL in comparison to all background proteins (black line).(e) Barplot displays coefficients derived from correlating lung function associations at the protein level with cell type specific ILD associations at the RNA level.(f) The heatmap illustrates gene expression changes associated with lung fibrosis across indicated cell types (columns) for selected BALF protein biomarkers (rows). The dotplot visualizes the frequency changes of the indicated cell types inferred from the deconvolution of bulk mRNA data of ILD samples compared to controls (GSE47460). Samples used in this study from the LTRC n = 254 ILD patients and n = 108 controls.


Figure 6 ( a , b ) Scatter plots stratify genes based on the protein lung function associations ( y - axis ) and cell type specific ILD associations ( x - axis ) . The size of the dots represents the detection level of each gene in the corresponding cell type . Colors highlight genes with marginal associations at the protein and RNA levels . ( c , d ) The box plots illustrate differences in mRNA detection for the indicated genes between tissues from end stage lung fibrosis patients in ( c ) basal cells , and ( d ) alveolar epithelial cells when compared to controls . The boxes represent the interquartile range , the horizontal line in the box is the median , and the whiskers represent 1 . 5 times the interquartile range ( Chicago cohort : ILD n = 9 , controls n = 8 ; Nashville cohort : ILD n = 20 , controls n = 10 ; Munich cohort : ILD n = 3 , controls n = 11 ) . ( e , f ) The scatter plots show the positive correlation of the indicated proteins in BALF ( MS - intensity , x - axis ) with meta lung function ( y - axis ) . ( g - j ) Immunofluorescence analysis of the indicated proteins and cell type markers in IPF ( n = 3 ) and control samples ( n = 3 ) .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6(a, b) Scatter plots stratify genes based on the protein lung function associations (y-axis) and cell type specific ILD associations (x-axis). The size of the dots represents the detection level of each gene in the corresponding cell type. Colors highlight genes with marginal associations at the protein and RNA levels.(c, d) The box plots illustrate differences in mRNA detection for the indicated genes between tissues from end stage lung fibrosis patients in (c) basal cells, and (d) alveolar epithelial cells when compared to controls. The boxes represent the interquartile range, the horizontal line in the box is the median, and the whiskers represent 1.5 times the interquartile range (Chicago cohort: ILD n = 9, controls n = 8; Nashville cohort: ILD n = 20, controls n = 10; Munich cohort: ILD n = 3, controls n = 11).(e, f) The scatter plots show the positive correlation of the indicated proteins in BALF (MS-intensity, x-axis) with meta lung function (y-axis).(g-j) Immunofluorescence analysis of the indicated proteins and cell type markers in IPF (n=3) and control samples (n=3).


Figure 7 ( a ) The scatter plots show the positive correlation of CFHR1 in BALF ( MS - intensity , x - axis ) with meta lung function ( y - axis ) . ( c ) The volcano plot depicts the fold changes ( x - axis ) and the - log10 p - values ( y - axis ) derived from the differential gene expression analysis using diffxpy between ILD and controls within the subset of PDGFRB + pericytes ( outlier values truncated ) . ( e ) The heatmap shows the expression of genes most strongly associated with SSTR2 expression within the pericytes ( left color bar indicates significant correlation or anti - correlation ) . ( f ) Immunofluorescence analysis of SSTR2 , the pericyte cell type marker PDGFRB and a marker for smooth muscle cells DESMIN in IPF ( n = 3 ) and control samples ( n = 3 ) . ( g ) Representative images of immunohistochemistry analysis of SSTR2 protein expression in tissue regions ( n = 474 ) from ILD patients ( n = 53 ) and control patients ( n = 26 ) . ( h ) Correlation of immunohistochemistry signal of SSTR2 with Ashcroft scores . ( i ) The bar graph shows the genes most strongly correlated with SSTR2 belonging to the GO category " transcription regulators " . The dotted line marks a correlation coefficient of zero .

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Figure 7(a) The scatter plots show the positive correlation of CFHR1 in BALF (MS-intensity, x-axis) with meta lung function (y-axis).(c) The volcano plot depicts the fold changes (x-axis) and the -log10 p-values (y-axis) derived from the differential gene expression analysis using diffxpy between ILD and controls within the subset of PDGFRB+ pericytes (outlier values truncated).(e) The heatmap shows the expression of genes most strongly associated with SSTR2 expression within the pericytes (left color bar indicates significant correlation or anti-correlation).(f) Immunofluorescence analysis of SSTR2, the pericyte cell type marker PDGFRB and a marker for smooth muscle cells DESMIN in IPF (n=3) and control samples (n=3).(g) Representative images of immunohistochemistry analysis of SSTR2 protein expression in tissue regions (n = 474) from ILD patients (n = 53) and control patients (n = 26).(h) Correlation of immunohistochemistry signal of SSTR2 with Ashcroft scores.(i) The bar graph shows the genes most strongly correlated with SSTR2 belonging to the GO category "transcription regulators". The dotted line marks a correlation coefficient of zero.


Figure 8 ( a ) The scatter plots show the positive correlation of CFHR1 in BALF ( MS - intensity , x - axis ) with meta lung function ( y - axis ) . ( c ) Relative expression level of CRTAC1 across human organs . ( d ) The box plots illustrate differences in mRNA detection for CRTAC1 in alveolar epithelial cells from fibrosis patients compared to control samples across the three indicated patient cohorts ( Chicago cohort : ILD n = 9 , controls n = 8 ; Nashville cohort : ILD n = 20 , controls n = 10 ; Munich cohort : ILD n = 3 , controls n = 11 ) . ( e ) Relative gene expression levels of CRTAC1 in GSE47460 . Dots represent average expression in the tissue of individual patients . The line represents the mean , error bars show SD . CRTAC1 is significantly downregulated in ILD but not COPD patients ( f ) For each single cell cohort , the gene - gene correlations with CRTAC1 within the SFTPC + AT - 2 cells were calculated . The indicated genes were selected based on their common direction of correlation across cohorts . ( h ) The bar graph shows the gene categories most strongly correlated with CRTAC1 belonging to the GO category of " transcription regulators " . The dotted line marks a correlation coefficient of zero . ( n ) Immunofluorescence analysis of SPRR1A , KRT8 as well as SFTPC in IPF ( n = 3 ) and control samples ( n = 2 ) . ( o ) A high throughput experimental workflow for plasma proteomics ( Niu allowed for profiling of two independent cohorts of ILD patients ( Munich , n = 30 and Hannover , n = 81 ; healthy age matched controls , n = 30 ) . All proteins quantified in plasma are shown , ranked by their abundance measured by mass spectrometry ( MS - intensity ) . ( p ) The indicated proteins from the plasma analysis were selected based on their common direction of correlation with patient lung function in two independent patient cohorts with distinct clinical characteristics . ( q ) The heatmap shows the predicted relative contribution of lung cell types to the association of protein biomarker signatures in plasma with lung function ( forced vital capacity - FVC ) . Patients were split in two groups , one with a mild decline in lung function [ FVC 60 - 100 % ] and one with severe loss of lung function [ FVC 20 - 60 % ] and compared to healthy age matched controls .

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Figure 8(a) The scatter plots show the positive correlation of CFHR1 in BALF (MS-intensity, x-axis) with meta lung function (y-axis).(c) Relative expression level of CRTAC1 across human organs.(d) The box plots illustrate differences in mRNA detection for CRTAC1 in alveolar epithelial cells from fibrosis patients compared to control samples across the three indicated patient cohorts (Chicago cohort: ILD n = 9, controls n = 8; Nashville cohort: ILD n = 20, controls n = 10; Munich cohort: ILD n = 3, controls n = 11).(e) Relative gene expression levels of CRTAC1 in GSE47460. Dots represent average expression in the tissue of individual patients. The line represents the mean, error bars show SD. CRTAC1 is significantly downregulated in ILD but not COPD patients(f) For each single cell cohort, the gene-gene correlations with CRTAC1 within the SFTPC+ AT-2 cells were calculated. The indicated genes were selected based on their common direction of correlation across cohorts.(h) The bar graph shows the gene categories most strongly correlated with CRTAC1 belonging to the GO category of "transcription regulators". The dotted line marks a correlation coefficient of zero.(n) Immunofluorescence analysis of SPRR1A, KRT8 as well as SFTPC in IPF (n=3) and control samples (n=2).(o) A high throughput experimental workflow for plasma proteomics(Niu allowed for profiling of two independent cohorts of ILD patients (Munich, n=30 and Hannover, n=81; healthy age matched controls, n=30). All proteins quantified in plasma are shown, ranked by their abundance measured by mass spectrometry (MS-intensity).(p) The indicated proteins from the plasma analysis were selected based on their common direction of correlation with patient lung function in two independent patient cohorts with distinct clinical characteristics.(q) The heatmap shows the predicted relative contribution of lung cell types to the association of protein biomarker signatures in plasma with lung function (forced vital capacity - FVC). Patients were split in two groups, one with a mild decline in lung function [FVC 60-100%] and one with severe loss of lung function [FVC 20-60%] and compared to healthy age matched controls.


Figure 1CSTB gene expression analysis in hCOs , starting from day ( d ) 16 until d140 . For every time point , at least 3 different samples were analyzed ; each sample was made by a pool of 3 to 4 hCOs . Data are represented as mean ± SEM . Unpaired t - test ( * P < 0 . 05 , * * P < 0 . 01 ) . Micrographs of sections of control hCOs immunostained with CSTB antibody at different time points in culture . The dashed lines represent the apical surface of the cavities / ventricles ( V - like ) . The cortical plate like side is indicated ( CP - like ) . Nuclei ( blue ) are stained with DAPI . Real time qPCR results of PAX6 , NEUN , NESTIN , CSTB gene expression levels in PAX6 - and NEUN - FACS sorted nuclei from f - CTRL d135 hCOs . Data are represented as mean ± SEM . Statistical significance was based on T - student test ( * P < 0 . 05 , * * P < 0 . 01 ) . For PAX6 , 10 samples were analyzed as biological replicates : 5 for Pax6 + sorted Nuclei and 5 for Neun + sorted Nuclei ; for NEUN , NESTIN and CSTB , 9 samples were analyzed as biological replicates : 5 for Pax6 + sorted Nuclei and 4 for Neun + sorted Nuclei . Western blot analysis and relative quantifications ( arbitrary units , a . u . ) on protein extracts from cortical ( Cx ) and ganglionic eminence ( GE ) tissues for Cstb using Actb as a loading control . Cx and GE tissues from mouse embryos at different time of development as indicated in figure . Micrograph of coronal sections of E16 mouse cerebral cortices immunostained for Cstb and acetylated tubulin ( acTub ) as marker for stabilized microtubules . Ventricle ( V ) and cortical plate ( CP ) are indicated . The dashed lines represent the apical surface of the ventricles . Nuclei ( blue ) are stained with DAPI .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1CSTB gene expression analysis in hCOs, starting from day (d) 16 until d140. For every time point, at least 3 different samples were analyzed; each sample was made by a pool of 3 to 4 hCOs. Data are represented as mean ± SEM. Unpaired t-test (*P < 0.05, **P < 0.01).Micrographs of sections of control hCOs immunostained with CSTB antibody at different time points in culture. The dashed lines represent the apical surface of the cavities/ventricles (V-like). The cortical plate like side is indicated (CP-like). Nuclei (blue) are stained with DAPI.Real time qPCR results of PAX6, NEUN, NESTIN, CSTB gene expression levels in PAX6- and NEUN- FACS sorted nuclei from f-CTRL d135 hCOs. Data are represented as mean ± SEM. Statistical significance was based on T-student test (*P < 0.05, **P < 0.01). For PAX6, 10 samples were analyzed as biological replicates: 5 for Pax6+sorted Nuclei and 5 for Neun+sorted Nuclei; for NEUN, NESTIN and CSTB, 9 samples were analyzed as biological replicates: 5 for Pax6+sorted Nuclei and 4 for Neun+sorted Nuclei.Western blot analysis and relative quantifications (arbitrary units, a.u.) on protein extracts from cortical (Cx) and ganglionic eminence (GE) tissues for Cstb using Actb as a loading control. Cx and GE tissues from mouse embryos at different time of development as indicated in figure.Micrograph of coronal sections of E16 mouse cerebral cortices immunostained for Cstb and acetylated tubulin (acTub) as marker for stabilized microtubules. Ventricle (V) and cortical plate (CP) are indicated. The dashed lines represent the apical surface of the ventricles. Nuclei (blue) are stained with DAPI.


Figure 2Micrograph sections of d40 hCOs electroporated with GFP - empty vector control or GFP - CSTB and analyzed 5 dpe . Sections were then immunostained for GFP and KI67 . The dashed lines represent the apical surface of the cavities / ventricles ( V - like ) . The cortical plate like side is indicated ( CP - like ) . Quantification of the total number of proliferating KI67 + cells / area ( µm2 ) of V - like structures transfected with GFP - empty vector control or GFP - CSTB in ( A ) . Micrograph of coronal sections of E16 mouse cerebral cortices electroporated at E14 with GFP - empty vector or GFP - Cstb and analyzed 2 dpe and immunostained with GFP , Ki67 and BrdU 30 minutes after BrdU injection . Ventricle ( V ) and cortical plate ( CP ) are indicated . The dashed lines represent the apical surface of the ventricles . Quantification of the total number of proliferating Ki67 + cells / area ( µm2 ) ( D ) and Ki67 + Brdu + cells / area ( µm2 ) ( E ) of ventricles transfected with GFP - empty vector or GFP - Cstb in ( C ) . Micrograph of coronal sections of E16 mouse cerebral cortices electroporated at E14 with GFP - empty vector or GFP - Cstb , analyzed 2 dpe and immunostained with GFP and Phospho - Histone H3 ( PH3 ) ( F ) and with GFP , Pax6 and Tbr2 ( G ) . Ventricle ( V ) and cortical plate ( CP ) are indicated . The dashed lines represent the apical surface of the ventricles . Distribution of proliferating Ki67 + / BrdU + cells ( H ) and mitotic progenitor PH3 + cells ( I ) in the mouse cortex . The cortex was subdivided in 5 equal Bins - Bin1 corresponded to apical side and Bin5 to pial side of the cortex and PH3 + cells or Ki67 + / BrdU + cells calculated in each Bin . Distribution of Tbr2 + intermediate progenitors ( J ) and Pax6 + Tbr2 + cells ( K ) in the mouse cortex . The cortex was subdivided in 5 equal Bins - Bin1 corresponded to apical side and Bin5 to pial side of the cortex and Tbr2 + cells or Pax6 + Tbr2 + cells calculated in each Bin .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2Micrograph sections of d40 hCOs electroporated with GFP-empty vector control or GFP-CSTB and analyzed 5 dpe. Sections were then immunostained for GFP and KI67. The dashed lines represent the apical surface of the cavities/ventricles (V-like). The cortical plate like side is indicated (CP-like). Quantification of the total number of proliferating KI67+ cells/area (µm2) of V-like structures transfected with GFP-empty vector control or GFP-CSTB in (A).Micrograph of coronal sections of E16 mouse cerebral cortices electroporated at E14 with GFP-empty vector or GFP-Cstb and analyzed 2 dpe and immunostained with GFP,Ki67 and BrdU 30 minutes after BrdU injection. Ventricle (V) and cortical plate (CP) are indicated. The dashed lines represent the apical surface of the ventricles. Quantification of the total number of proliferating Ki67+ cells/area(µm2)(D) and Ki67+Brdu+ cells/area(µm2)(E) of ventricles transfected with GFP-empty vector or GFP-Cstb in (C).Micrograph of coronal sections of E16 mouse cerebral cortices electroporated at E14 with GFP-empty vector or GFP-Cstb, analyzed 2 dpe and immunostained with GFP and Phospho-Histone H3 (PH3)(F) and with GFP, Pax6 and Tbr2 (G). Ventricle (V) and cortical plate (CP) are indicated. The dashed lines represent the apical surface of the ventricles.Distribution of proliferating Ki67+/BrdU+cells (H) and mitotic progenitor PH3+ cells (I) in the mouse cortex. The cortex was subdivided in 5 equal Bins -Bin1 corresponded to apical side and Bin5 to pial side of the cortex and PH3+ cells or Ki67+/BrdU+ cells calculated in each Bin.Distribution of Tbr2+ intermediate progenitors (J) and Pax6+Tbr2+ cells (K) in the mouse cortex. The cortex was subdivided in 5 equal Bins -Bin1 corresponded to apical side and Bin5 to pial side of the cortex and Tbr2+ cells or Pax6+Tbr2+ cells calculated in each Bin.