Representative Western blotting and quantification of pluripotency factors during EB formation .

Representative Western blotting and quantification of pluripotency factors during EB formation.


Representative Western blotting and quantification of neural lineage markers during mESCs neural differentiation .

Representative Western blotting and quantification of neural lineage markers during mESCs neural differentiation.


Western blotting in the WT and the Trim71 ( R783H ) mESCs with dox - inducible expression of Lsd1 - GFP .

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

Western blotting in the WT and the Trim71(R783H) mESCs with dox-inducible expression of Lsd1-GFP.


Exit pluripotency assay for mESCs.


Representative Western blotting and quantification of pluripotency factors during the monolayer differentiation of mESCs .

Representative Western blotting and quantification of pluripotency factors during the monolayer differentiation of mESCs.


Representative Western blotting and quantification in the WT and the R783H / + mESCs .

Representative Western blotting and quantification in the WT and the R783H/+ mESCs.


Exit pluripotency assay for the WT and the R783H / + mESCs .

Exit pluripotency assay for the WT and the R783H/+ mESCs.


Representative Western blotting and quantification of pluripotency factors in differentiating mESCs .

Representative Western blotting and quantification of pluripotency factors in differentiating mESCs.


Western blotting in the WT and the R783H / + mESCs with dox - inducible expression of Lsd1 - GFP .

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

Western blotting in the WT and the R783H/+ mESCs with dox-inducible expression of Lsd1-GFP.


Exit pluripotency assay for the WT and the R783H / + mESCs with dox - inducible expression of Lsd1 - GFP .

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

Exit pluripotency assay for the WT and the R783H/+ mESCs with dox-inducible expression of Lsd1-GFP.


Representative Western blotting and quantification of pluripotency factors during the differentiation of the WT and the R783H / + mESCs with dox - inducible expression of Lsd1 - GFP .

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

Representative Western blotting and quantification of pluripotency factors during the differentiation of the WT and the R783H/+ mESCs with dox-inducible expression of Lsd1-GFP.


Representative Western blotting and quantification of neural lineage markers during the neural differentiation of the WT and the R783H / + mESCs with dox - inducible expression of Lsd1 - GFP .

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

Representative Western blotting and quantification of neural lineage markers during the neural differentiation of the WT and the R783H/+ mESCs with dox-inducible expression of Lsd1-GFP.


( C ) Electrophoretic mobility - shift assay ( EMSA ) with biotin - labeled RORA consensus , rs2978980T or rs2978980G probes and HGC - 27 or MGC80 - 3 nuclear extracts . HGC - 27 or MGC80 - 3 left panel : EMSA with RORA consensus or rs2978980T probes . Lane 1 - 8 : from the first lane at the left side to the right side . Lanes 4 and 8 , probe only ; lanes 2 and 6 , probe and nuclear extracts ; lanes 1 , 3 , 5 and 7 , probe and nuclear extracts plus 100 × unlabeled rs2978980T ( lanes 1 and 5 ) or RORA consensus probes ( lanes 3 and 7 ) . HGC - 27 or MGC80 - 3 right panel : EMSA with rs2978980T or rs2978980G probes . Lanes 1 and 6 , probe only ; lanes 2 and 7 , probe and nuclear extracts ; lanes 3 - 5 and 8 - 10 , probe and nuclear extracts plus 100 × unlabeled rs2978980T ( lanes 5 and 8 ) , rs2978980G ( lanes 3 and 10 ) or RORA consensus probes ( lanes 4 and 9 ) .

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

(C) Electrophoretic mobility-shift assay (EMSA) with biotin-labeled RORA consensus, rs2978980T or rs2978980G probes and HGC-27 or MGC80-3 nuclear extracts. HGC-27 or MGC80-3 left panel: EMSA with RORA consensus or rs2978980T probes. Lane 1-8: from the first lane at the left side to the right side. Lanes 4 and 8, probe only; lanes 2 and 6, probe and nuclear extracts; lanes 1, 3, 5 and 7, probe and nuclear extracts plus 100× unlabeled rs2978980T (lanes 1 and 5) or RORA consensus probes (lanes 3 and 7). HGC-27 or MGC80-3 right panel: EMSA with rs2978980T or rs2978980G probes. Lanes 1 and 6, probe only; lanes 2 and 7, probe and nuclear extracts; lanes 3-5 and 8-10, probe and nuclear extracts plus 100× unlabeled rs2978980T (lanes 5 and 8), rs2978980G (lanes 3 and 10) or RORA consensus probes (lanes 4 and 9).


( F ) Suppression of RORA using siRNAs ( siRORA - 1 and siRORA - 2 ) could markedly down - regulate lncPSCA expression in MGC80 - 3 cells but not in HGC - 27 cells . In detail , MGC80 - 3 and HGC - 27 cells were transfected with siRORA - 1 and siRORA - 2 , lncPSCA and RORA expression levels were measured by RT - qPCR and Western Blot .

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

(F) Suppression of RORA using siRNAs (siRORA-1 and siRORA-2) could markedly down-regulate lncPSCA expression in MGC80-3 cells but not in HGC-27 cells. In detail, MGC80-3 and HGC-27 cells were transfected with siRORA-1 and siRORA-2, lncPSCA and RORA expression levels were measured by RT-qPCR and Western Blot.


( E ) lncPSCA - knockout significantly promoted growth of MGC80 - 3 ( n = 8 ) or HGC - 27 ( n = 7 ) xenografts compared with control xenografts after 27 or 18 days .

O O O O O O O O O O O O O O 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) lncPSCA-knockout significantly promoted growth of MGC80-3 (n = 8) or HGC-27 (n = 7) xenografts compared with control xenografts after 27 or 18 days.


( F ) HE and Ki67 staining of MGC80 - 3 or HGC - 27 xenografts . Scale bar = 100 μm .

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

(F) HE and Ki67 staining of MGC80-3 or HGC-27 xenografts. Scale bar = 100 μm.


( B ) lncPSCA inhibits invasion abilities of MGC80 - 3 and HGC - 27 cells . Cells on the lower surface of the chamber were stained by crystal violet . Scale bar = 100 μm .

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

(B) lncPSCA inhibits invasion abilities of MGC80-3 and HGC-27 cells. Cells on the lower surface of the chamber were stained by crystal violet. Scale bar = 100 μm.


( C ) In MGC80 - 3 and HGC - 27 cells , expression changes of different markers ( β - catenin , ZO - 1 , SNAIL and VIMENTIN ) of epithelial - to - mesenchymal transition were examined after overexpression or knockout of lncPSCA .

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

(C) In MGC80-3 and HGC-27 cells, expression changes of different markers (β-catenin, ZO-1, SNAIL and VIMENTIN) of epithelial-to-mesenchymal transition were examined after overexpression or knockout of lncPSCA.


( F ) Gastric cancer cells after lncPSCA - knockout or stably overexpressing lncPSCA and control cells were treated with cycloheximide ( CHX ) or vehicle for the indicated periods of time . DDX5 levels were analyzed by Western blot .

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

(F) Gastric cancer cells after lncPSCA-knockout or stably overexpressing lncPSCA and control cells were treated with cycloheximide (CHX) or vehicle for the indicated periods of time. DDX5 levels were analyzed by Western blot.


( I ) Knockdown of DDX5 with siRNA ( siD - 1 and siD - 2 ) substantially reduced the proliferation of gastric cancer cells . Data show one representative example of three biological replicates . ( J ) Silencing of DDX5 with shRNA ( shD ) significantly reduced the proliferation of gastric cancer cells . Data show one representative example of three biological replicates . (

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

(I) Knockdown of DDX5 with siRNA (siD-1 and siD-2) substantially reduced the proliferation of gastric cancer cells. Data show one representative example of three biological replicates. (J) Silencing of DDX5 with shRNA (shD) significantly reduced the proliferation of gastric cancer cells. Data show one representative example of three biological replicates. (


( I ) Knockdown of DDX5 with siRNA ( siD - 1 and siD - 2 ) substantially reduced the proliferation of gastric cancer cells . Data show one representative example of three biological replicates .

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

(I) Knockdown of DDX5 with siRNA (siD-1 and siD-2) substantially reduced the proliferation of gastric cancer cells. Data show one representative example of three biological replicates.


( E ) RNA Pol II ChIPseq and RNAseq signals of PMAIP1 , CDKN1A , SESN2 and SESN3 in HGC - 27 cells . Red bars represent the RNA Pol II ChIPseq signal . Orange or blue bars represent the RNAseq signals of HGC - 27 cells overexpressing lncPSCA ( lncP ) or with silenced DDX5 ( shD ) , respectively .

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

(E) RNA Pol II ChIPseq and RNAseq signals of PMAIP1, CDKN1A, SESN2 and SESN3 in HGC-27 cells. Red bars represent the RNA Pol II ChIPseq signal. Orange or blue bars represent the RNAseq signals of HGC-27 cells overexpressing lncPSCA (lncP) or with silenced DDX5 (shD), respectively.


( F ) Validation of candidate downstream genes of the lncPSCA - DDX5 axis identified through the integrated analyses . qRT - PCR was performed for PMAIP1 , CDKN1A and SESN2 genes upon overexpression of lncPSCA ( lncP ) , silencing of DDX5 ( shD ) or lncPSCA konckout . Error bars indicate the SEM . Data show one representative example of three biological replicates .

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

(F) Validation of candidate downstream genes of the lncPSCA-DDX5 axis identified through the integrated analyses. qRT-PCR was performed for PMAIP1, CDKN1A and SESN2 genes upon overexpression of lncPSCA (lncP), silencing of DDX5 (shD) or lncPSCA konckout. Error bars indicate the SEM. Data show one representative example of three biological replicates.


( A ) Swelling of neurons in the vestibular nucleus due to fine vesiculation ( arrows ) and clear vacuolization ( arrowhead ) of the cytoplasm . HE , scale bar 100 μm .

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

(A) Swelling of neurons in the vestibular nucleus due to fine vesiculation (arrows) and clear vacuolization (arrowhead) of the cytoplasm. HE, scale bar 100 μm.


( B ) Clear cytoplasmic vacuolization ( arrows ) in cerebellar corticalPurkinje cells . HE , scale bar 100 μm .

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

(B) Clear cytoplasmic vacuolization (arrows) in cerebellar corticalPurkinje cells. HE, scale bar 100 μm.


( C ) Normal cerebellar cortex of an unaffected dog shows viable Purkinje cells ( arrows ) and a dense granular cell layer . HE , scale bar 100 μm . ( D ) Marked neuronal loss is present the cerebellar cortex of an affected dog . The number of neurons in the granular cell layer is reduced and only scattered Purkinje cells remain ( arrow ) . HE , scale bar 100 μm .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Normal cerebellar cortex of an unaffected dog shows viable Purkinje cells (arrows) and a dense granular cell layer. HE, scale bar 100 μm. (D) Marked neuronal loss is present the cerebellar cortex of an affected dog. The number of neurons in the granular cell layer is reduced and only scattered Purkinje cells remain (arrow). HE, scale bar 100 μm.


( E ) Axonal spheroids of varying quality were seen in the white matter ( arrows ) of cerebellum and brainstem . HE , scale bar 100 μm .

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

(E) Axonal spheroids of varying quality were seen in the white matter (arrows) of cerebellum and brainstem. HE, scale bar 100 μm.


( F ) Diffuse cytoplasmic vacuolization of the exocrine pancreatic acinar cells . HE , scale bar 100 μm .

(F) Diffuse cytoplasmic vacuolization of the exocrine pancreatic acinar cells. HE, scale bar 100 μm.


( G ) Purkinje cell with numerous single - membrane bound , cytoplasmic vacuoles tethering to each other ( arrows , inset ) . Electronmicrograph , scale bar 2 μm . Inset : scale bar 1 μm .

(G) Purkinje cell with numerous single-membrane bound, cytoplasmic vacuoles tethering to each other (arrows, inset). Electronmicrograph, scale bar 2 μm. Inset: scale bar 1 μm.


( H ) Axonal spheroid containing aggregated degenerated mitochondria , occasional double - membrane - bound autophagosomes ( arrows ) and free electron dense material , compressed by a peripheral clear vacuolar space . Electronmicrograph , scale bar 0 . 5 μm . Abbreviations : n , nucleus ; ml , molecular layer ; pl , Purkinje cell layer ; gl , granular cell layer .

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

(H) Axonal spheroid containing aggregated degenerated mitochondria, occasional double-membrane-bound autophagosomes (arrows) and free electron dense material, compressed by a peripheral clear vacuolar space. Electronmicrograph, scale bar 0.5 μm. Abbreviations: n, nucleus; ml, molecular layer; pl, Purkinje cell layer; gl, granular cell layer.


( A ) Axonal spheroids stain diffusely positive for LC3B . IHCLC3B , scale bar 100 μm . ( B , C ) The granular cores of the spheroids are positive for ( B ) ubiquitin ( arrow ) and ( C ) p62 . IHCubiquitin and p62 , scale bars 20 and 100 μm , respectively . ( D ) Smooth axonal swellings in the cerebellar cortex contain ATG4D . Inset : control . IHCATG4D , scale bar 100 μm . ( E ) Affected neurons show increased perinuclear granular LC3B positivity . Inset : control . IHCLC3B , scale bar 100 μm . ( F ) Neuronalvacuoles are partially LAMP2 positive ( arrow ) and partially negative ( arrow head ) . Inset : control . IHCLAMP2 , scale bar 20 μm .

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

(A) Axonal spheroids stain diffusely positive for LC3B. IHCLC3B, scale bar 100 μm. (B,C) The granular cores of the spheroids are positive for (B) ubiquitin (arrow) and (C) p62. IHCubiquitin and p62, scale bars 20 and 100 μm, respectively. (D) Smooth axonal swellings in the cerebellar cortex contain ATG4D. Inset: control. IHCATG4D, scale bar 100 μm. (E) Affected neurons show increased perinuclear granular LC3B positivity. Inset: control. IHCLC3B, scale bar 100 μm. (F) Neuronalvacuoles are partially LAMP2 positive (arrow) and partially negative (arrow head). Inset: control. IHCLAMP2, scale bar 20 μm.


( A - F ) Lateral views of control and morphant zebrafishembryos . ( A , B ) Control embryos appear normal at 1 dpf . ( C , D ) Morphant embryos show severe abnormalities in different regions of the brain at 1 dpf . Black arrowheads denote the developing cerebellum . Purple arrows indicate the hindbrain irregularities . ( E ) Control embryo at 2 dpf . ( F ) A morphant embryo at 2 dpf displaying hydrocephalus ( yellow arrow ) and small head and eye .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-F) Lateral views of control and morphant zebrafishembryos. (A,B) Control embryos appear normal at 1 dpf. (C,D) Morphant embryos show severe abnormalities in different regions of the brain at 1 dpf. Black arrowheads denote the developing cerebellum. Purple arrows indicate the hindbrain irregularities. (E) Control embryo at 2 dpf. (F) A morphant embryo at 2 dpf displaying hydrocephalus (yellow arrow) and small head and eye.


( G ) RT - PCR assay showing the efficiency of the atg4daSMO .

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

(G) RT-PCR assay showing the efficiency of the atg4daSMO.


Immunostaining with ( A - C ) anti - Pvalb7 and ( D - F ) anti - zebrinII antibody show loss of cerebellarPurkinje cells in atg4da morphants . ( A , D ) Control embryos at 4 . 5 dpf . ( B , E ) Morphants with mild phenotype show partial loss of cerebellarPurkinje cells . ( C , F ) Morphants with strong phenotype show either total loss of Purkinje cells or presence of few differentiated neurons , which are laterally located in the cerebellum . ( G - I ) Labeling of cerebellar granule cells with anti - Vglut1 antibody in control and morphant embryos . ( H ) Mildly affected morphants show reduced expression of Vglut1 in the cerebellum . ( I ) Vglut1 expression is strongly reduced in embryos showing severe phenotype . White arrowheads indicate the region of cerebellum . Abbreviations : hb , hindbrain .

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

Immunostaining with (A-C) anti-Pvalb7 and (D-F) anti-zebrinII antibody show loss of cerebellarPurkinje cells in atg4da morphants. (A,D) Control embryos at 4.5 dpf. (B,E) Morphants with mild phenotype show partial loss of cerebellarPurkinje cells. (C,F) Morphants with strong phenotype show either total loss of Purkinje cells or presence of few differentiated neurons, which are laterally located in the cerebellum. (G-I) Labeling of cerebellar granule cells with anti-Vglut1 antibody in control and morphant embryos. (H) Mildly affected morphants show reduced expression of Vglut1 in the cerebellum. (I) Vglut1 expression is strongly reduced in embryos showing severe phenotype. White arrowheads indicate the region of cerebellum. Abbreviations: hb, hindbrain.


A ) Transmission electron microscopy ( TEM ) of MoDCs infected with P . gingivalis for 2 , 12 and 24 hours ( left , middle and right panels ) . The sections show the intra - and extra - cellular contents of Cont . ( un - infected ) , Pg381 , Mfa1 + Pg and FimA + Pg infected MoDCs for the different time

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

A) Transmission electron microscopy (TEM) of MoDCs infected with P. gingivalis for 2, 12 and 24 hours (left, middle and right panels). The sections show the intra-and extra-cellular contents of Cont. (un-infected), Pg381, Mfa1+Pg and FimA+Pg infected MoDCs for the different time points


B ) The figure shows the intracellular content of P . gingivalis strains after 24 hours of infection . MoDCs infected with Pg381 or isogenic mutants strains were lysed and the survived intracellular bacteria were re - suspended and maintained in anaerobic broth for 5 days . The data represents CFU within MoDCs harvested from three healthy individuals . The means ± standard deviation ( in triplicates ) were analyzed by One - way ANOVA of different groups and Tukey ' s test for multiple group comparisons within 3 different experiments ( * statistical significance at the p < 0 . 001 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) The figure shows the intracellular content of P. gingivalis strains after 24 hours of infection. MoDCs infected with Pg381 or isogenic mutants strains were lysed and the survived intracellular bacteria were re-suspended and maintained in anaerobic broth for 5 days. The data represents CFU within MoDCs harvested from three healthy individuals. The means ±standard deviation (in triplicates) were analyzed by One-way ANOVA of different groups and Tukey's test for multiple group comparisons within 3 different experiments (* statistical significance at the p<0.001).


A ) DC - SIGN mRNA expression in P . gingivalis - infected MoDCs at 0 . 1 , 1 and 10 MOIs . The figure shows the gene expression after 12 hours of Pg381 and mutant strains infections . The target gene ( DC - SIGN ) was normalized using the endogenous control GAPDH ( ΔCt ) and fold regulations were calculated using 2 - ( ΔΔCt ) method . The statistical analysis was performed using the t - test , which accounts for the clustering of infected and un - infected controls within 3 different experiments ( * p < 0 . 001 ) .

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

A) DC-SIGN mRNA expression in P. gingivalis-infected MoDCs at 0.1, 1 and 10 MOIs. The figure shows the gene expression after 12 hours of Pg381 and mutant strains infections. The target gene (DC-SIGN) was normalized using the endogenous control GAPDH (ΔCt) and fold regulations were calculated using 2-(ΔΔCt) method. The statistical analysis was performed using the t-test, which accounts for the clustering of infected and un-infected controls within 3 different experiments (* p<0.001).


B ) Immuno - electron microscopy of un - infected MoDCs ( Cont . ) ( upper panel ) , MoDCs infected with Pg381 ( middle panel ) and Mfa1 + Pg mutants ( lower panel ) . Gold particles ( marked with red rings ) for positive DC - SIGN were detected in the cell membrane and cytoplasm of cells infected with Mfa1 + Pg strains . Minimal positive staining for DC - SIGN was detected in the membranes of MoDCs infected with Pg381 , while no cytoplasmic gold labeling was detected in these cells .

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

B) Immuno-electron microscopy of un-infected MoDCs (Cont.) (upper panel), MoDCs infected with Pg381 (middle panel) and Mfa1+Pg mutants (lower panel). Gold particles (marked with red rings) for positive DC-SIGN were detected in the cell membrane and cytoplasm of cells infected with Mfa1+Pg strains. Minimal positive staining for DC-SIGN was detected in the membranes of MoDCs infected with Pg381, while no cytoplasmic gold labeling was detected in these cells.


C ) Flow cytometry analysis of surface DC - SIGN in human MoDCs after infection with Pg381 , Mfa1 + Pg and FimA + Pg . The analysis of the intensity used Kruskal - Wallis test analysis of different groups and Dunn ' s test for multiple comparisons 3 different experiments ( * p < 0 . 01 ) .

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

C) Flow cytometry analysis of surface DC-SIGN in human MoDCs after infection with Pg381, Mfa1+Pg and FimA+Pg. The analysis of the intensity used Kruskal-Wallis test analysis of different groups and Dunn's test for multiple comparisons 3 different experiments (* p<0.01).


A ) Pg381 and mutant strains count after 24 hr incubation with human MoDCs with / without Rapamycin treatment . The survived bacteria were measured after maintaining the lysed MoDCs suspension in anaerobic broth for 5 days . The plot represents the means ± standard deviation of CFU within MoDCs harvested from three healthy individuals ( * P < 0 . 001 ) . The analysis of readings used One - way AVOVA analysis of different groups and Tukey ' s test for multiple comparisons .

O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Pg381 and mutant strains count after 24 hr incubation with human MoDCs with/without Rapamycin treatment. The survived bacteria were measured after maintaining the lysed MoDCs suspension in anaerobic broth for 5 days. The plot represents the means ±standard deviation of CFU within MoDCs harvested from three healthy individuals (* P<0.001). The analysis of readings used One-way AVOVA analysis of different groups and Tukey's test for multiple comparisons.


B ) Epifluorescence microscopy images of MoDCs treated with Rapamycin 1 hour after P . gingivalis infections . LC3 - II ( red - fluorescent dye ) and the bacterial strains ( green CFSE ) were studied in MoDCs 11 hours after Rapamycin treatment ( 12 hours after infections ) .

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

B) Epifluorescence microscopy images of MoDCs treated with Rapamycin 1 hour after P. gingivalis infections. LC3-II (red-fluorescent dye) and the bacterial strains (green CFSE) were studied in MoDCs 11 hours after Rapamycin treatment (12 hours after infections).


C ) Quantifications of the fluorescent intensity of CFSE - labeled P . gingivalis and LC3 - II signals within infected MoDCs using NIS - Elements BR software . One - way ANOVA analysis was used to compare the means of intensity of different groups and Tukey ' s test for multiple comparisons ( * P < 0 . 001 ) .

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 O O O O O O O O O O O O O O O O O O O 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) Quantifications of the fluorescent intensity of CFSE-labeled P. gingivalis and LC3-II signals within infected MoDCs using NIS-Elements BR software. One-way ANOVA analysis was used to compare the means of intensity of different groups and Tukey's test for multiple comparisons (* P<0.001).


A ) Survival of P . gingivalis strains within MoDCs after 6 , 12 , 24 and 48 hours . Blue lines show P . gingivalis strains survival within MoDCs and their survival in anaerobic condition in the absence of DCs are showed in black lines .

O O O O O O O O O O O O O O O O O O O O O O O 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) Survival of P. gingivalis strains within MoDCs after 6, 12, 24 and 48 hours. Blue lines show P. gingivalis strains survival within MoDCs and their survival in anaerobic condition in the absence of DCs are showed in black lines.


The effect of rapamycin on Mfa1 + Pg , Pg381 and FimA + Pg survival within MoDCs are shown in figures B , C and D respectively . A three - factor repeated measures ANOVA using mixed models was used to test the effect of strain and rapamycin treatment over time on OD reading . The survival curves for the strains are showed in blue , while the effect of rapamycin treatments are in red . Bacterial survivals in the absence of MoDCs with and without rapamycin are plotted in grey and black , respectively . Statistical analysis showed that the strain by rapamycin treatment overtime interaction indicates the pattern of means in each strain ( Mfa1 + Pg , Pg381 and FimA + Pg ) between treated ( rapamycin ) and untreated were significantly different overtime ( p - value < 0 . = 001 ) .

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

The effect of rapamycin on Mfa1+Pg, Pg381 and FimA+Pg survival within MoDCs are shown in figures B, C and D respectively. A three-factor repeated measures ANOVA using mixed models was used to test the effect of strain and rapamycin treatment over time on OD reading. The survival curves for the strains are showed in blue, while the effect of rapamycin treatments are in red. Bacterial survivals in the absence of MoDCs with and without rapamycin are plotted in grey and black, respectively. Statistical analysis showed that the strain by rapamycin treatment overtime interaction indicates the pattern of means in each strain (Mfa1+Pg, Pg381 and FimA+Pg) between treated (rapamycin) and untreated were significantly different overtime (p-value <0.=001).


A ) Epifluorescence microscopy images of MoDCs infected with Pg381 , Mfa1 + Pg , FimA + Pg and MFB strains after 12 hours . LC3 - II was detected in red - fluorescent ( Texas red ) dye and the bacterial strains were pre - labeled with CFSE ( green ) . Co - localization of P . gingivalis and LC3 - II showed in the right panels .

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

A) Epifluorescence microscopy images of MoDCs infected with Pg381, Mfa1+Pg, FimA+Pg and MFB strains after 12 hours. LC3-II was detected in red-fluorescent (Texas red) dye and the bacterial strains were pre-labeled with CFSE (green). Co-localization of P. gingivalis and LC3-II showed in the right panels.


B ) Quantifications of the fluorescent intensity of LC3 - II within infected MoDCs using NIS - Elements BR software . One - way ANOVA analysis was used to compare the means of intensity of different groups and Tukey ' s test for multiple comparisons of three different experiments ( * p < 0 . 001 , # p < 0 . 01 ) .

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

B) Quantifications of the fluorescent intensity of LC3-II within infected MoDCs using NIS-Elements BR software. One-way ANOVA analysis was used to compare the means of intensity of different groups and Tukey's test for multiple comparisons of three different experiments (* p<0.001, # p<0.01).


C ) Bacterial uptake by MoDCs was determined by CFSE fluorescent intensity ( * p < 0 . 001 , # p < 0 . 01 ) .

O O O O 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) Bacterial uptake by MoDCs was determined by CFSE fluorescent intensity (* p<0.001, # p<0.01).


A ) 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 .

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

A) 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 ) .

O O O O 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

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 .

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

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 .

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

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 ) .

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

E) The 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).


A ) 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 .

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 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

A) 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 .

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

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.


A ) Flow cytometry of CD83 on MoDCs after incubation of TLR4 ligand ( E . coli LPS ) and TLR1 and 2 ligand ( Pam3csk4 ) for 4 hour .

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

A) 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 )

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

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 ) .

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

C) The plot represents the means ±standard deviation of CFU within MoDCs harvested from three healthy individuals after 24 hours (** p<0.001).


A ) 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 ) .

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

A) 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 ) .

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

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 ) .

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

D) The 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).


A ) 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 .

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

A) 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 ) .

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

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 .

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

C) The 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.


C , 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 .

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

C,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 .

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

F. 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 .

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

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.


A , 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 .

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

A,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 .

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

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 .

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

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.


A , 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 .

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

A,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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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,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 .

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

(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.


a , 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 .

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

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 .

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

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 .

O O O O O O O O O O O O O O O O O O O O O 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, 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, 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.


b , 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 .

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

b, 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 .

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

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 .

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

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.


a - 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 ) .

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

a-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 .

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

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 ) .

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

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 .

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

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 , 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 .

O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O B-CONTROLLED_VAR O O O O O O O B-MEASURED_VAR 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, 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 .

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

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 .

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

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 .

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

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.


( 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 ) .

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

(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 ) .

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

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

(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).


( 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 ) .

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

(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 ) .

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

(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 ) .

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

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

(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).


( 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 ) .

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

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

(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.


( 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 ) .

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

(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 ) .

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 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

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

(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).


( 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 ) .

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

(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 ) .

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

(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 ) .

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

(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 ) .

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

(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 .

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

(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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(F) 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).


( 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 ) .

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

(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 ) .

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

(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 .

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

(F) Western blot for human TGFβ in total protein extract from A375 tumors treated as indicate. Vinculin was used as an internal control.


( 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 ) .

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

(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 ) .

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

(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 .

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

(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 ) .

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

(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).


A Isothermal calorimetry ( ITC ) assays using ADP - ribose ligand and purified macrodomains of human macroH2A1 . 1 and macroH2A2 .

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

A 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 .

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

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.


A 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 .

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

A 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 ) .

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

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 ) .

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

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 ) .

O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O 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 O O O O O O O O O O O O O O O O O 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 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).


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

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).


A 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 .

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

A 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.