A . Immunoblots of subcellular fractions from HeLa cells exposed to normoxia . P : post - nuclear supernatant ; C : cytosol ; ER : endoplasmic reticulum ; MT : mitochondria ; MAM : mitochondrial - associated membranes . B . Immunoblots of subcellular fractions from HeLa cells exposed to hypoxia ( 1 % O2 ) for 5 h . P : post - nuclear supernatant ; C : cytosol ; ER : endoplasmic reticulum ; MT : mitochondria ; MAM : mitochondrial - associated membranes . C . Quantification of the MAM : PNS ratio of FUNDC1 or DRP1 under normoxic or hypoxic conditions . All quantitative data are presented as mean ± s . e . m . from 3 independent experiments ; * * P < 0 . 01 .

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A. Immunoblots of subcellular fractions from HeLa cells exposed to normoxia. P: post-nuclear supernatant; C: cytosol; ER: endoplasmic reticulum; MT: mitochondria; MAM: mitochondrial-associated membranes.B. Immunoblots of subcellular fractions from HeLa cells exposed to hypoxia (1% O2) for 5 h. P: post-nuclear supernatant; C: cytosol; ER: endoplasmic reticulum; MT: mitochondria; MAM: mitochondrial-associated membranes.C. Quantification of the MAM:PNS ratio of FUNDC1 or DRP1 under normoxic or hypoxic conditions. All quantitative data are presented as mean ± s.e.m. from 3 independent experiments; ** P < 0.01.


D . Control HeLa cells or hypoxia - treated HeLa cells ( 1 % O2 for 5 h ) were processed for immunogold electron microscopy with anti - FUNDC1 antibody ( 12 nm gold particles ) . Bar = 500 nm . The boxed areas in the left panels are magnified in the right panels . Arrows indicate gold particles .

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D. Control HeLa cells or hypoxia-treated HeLa cells (1% O2 for 5 h) were processed for immunogold electron microscopy with anti-FUNDC1 antibody (12 nm gold particles). Bar = 500 nm. The boxed areas in the left panels are magnified in the right panels. Arrows indicate gold particles.


E . Control HeLa cells or hypoxia - treated HeLa cells ( 1 % O2 for 5 h ) were processed for immunogold electron microscopy with anti - TOM20 antibody ( 12 nm gold particles ) . Bar = 500 nm .

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E. Control HeLa cells or hypoxia-treated HeLa cells (1% O2 for 5 h) were processed for immunogold electron microscopy with anti-TOM20 antibody (12 nm gold particles). Bar = 500 nm.


F . Quantification of the MAM : mitochondrion ratio of FUNDC1 immunogold particles from D under normoxic and hypoxic ( 1 % O2 ) conditions ; Data ( mean ± s . e . m . ) are from 3 independent experiments . n = 60 MAMs ( one MAM means one mitochondrial ER tethering site in the corresponding mitochondria ) , * * P < 0 . 01 .

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F. Quantification of the MAM:mitochondrion ratio of FUNDC1 immunogold particles from D under normoxic and hypoxic (1% O2) conditions; Data (mean ± s.e.m.) are from 3 independent experiments. n = 60 MAMs (one MAM means one mitochondrial ER tethering site in the corresponding mitochondria), ** P < 0.01.


G . Quantification of the MAM : mitochondrion ratio of TOM20 immunogold particles from E under normoxic and hypoxic ( 1 % O2 ) conditions . Data ( mean ± s . e . m . ) are from 3 independent experiments . n = 50 MAMs ( one MAM means one mitochondrial ER tethering site in the corresponding mitochondria ) . NS means not significant .

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G. Quantification of the MAM:mitochondrion ratio of TOM20 immunogold particles from E under normoxic and hypoxic (1% O2) conditions. Data (mean ± s.e.m.) are from 3 independent experiments. n = 50 MAMs (one MAM means one mitochondrial ER tethering site in the corresponding mitochondria). NS means not significant.


H . The quantification of MAM counts from 50 cells , * * * P < 0 . 001 .

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H. The quantification of MAM counts from 50 cells, ***P < 0.001.


I . The quantification of FACL4 immunogold particles from 50 cells , * * * P < 0 . 001 .

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I. The quantification of FACL4 immunogold particles from 50 cells, *** P < 0.001.


J . Hypoxia - treated HeLa cells ( 1 % O2 for 5 h ) were processed for immunogold electron microscopy with anti - FUNDC1 antibody ( 12 nm gold particles ) and anti - FACL4 ( 6 nm gold particles ) . Bar = 500 nm . The boxed areas in the left panels are magnified in the right panels . Yellow arrows indicate FUNDC1 - gold particles and green arrows indicate FACL4 - gold particles .

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J. Hypoxia-treated HeLa cells (1% O2 for 5 h) were processed for immunogold electron microscopy with anti-FUNDC1 antibody (12 nm gold particles) and anti-FACL4 (6 nm gold particles). Bar = 500 nm. The boxed areas in the left panels are magnified in the right panels. Yellow arrows indicate FUNDC1-gold particles and green arrows indicate FACL4-gold particles.


K . The ratio of FACL4 immunogold particles on the MAM and mitochondrion . n = 80 MAMs ( one MAM means one mitochondrial ER tethering site in the corresponding mitochondria ) .

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K. The ratio of FACL4 immunogold particles on the MAM and mitochondrion. n = 80 MAMs (one MAM means one mitochondrial ER tethering site in the corresponding mitochondria).


L . Hypoxia - treated HeLa cells ( 1 % O2 for 5 h ) were processed for immunogold electron microscopy with anti - FUNDC1 antibody ( 12 nm gold particles ) and anti - DRP1 ( 6 nm gold particles ) . Bar = 500 nm . The boxed areas in the left panels are magnified in the right panels . Yellow arrows indicate FUNDC1 - gold particles and green arrows indicate DRP1 - gold particles .

L. Hypoxia-treated HeLa cells (1% O2 for 5 h) were processed for immunogold electron microscopy with anti-FUNDC1 antibody (12 nm gold particles) and anti-DRP1 (6 nm gold particles). Bar = 500 nm. The boxed areas in the left panels are magnified in the right panels. Yellow arrows indicate FUNDC1-gold particles and green arrows indicate DRP1-gold particles.


B . HeLa cells were transfected with FUNDC1 - MYC or empty vector for 24 h . Cell lysates were immunoprecipitated by anti - MYC antibody and immunoblotted with indicated antibodies .

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B. HeLa cells were transfected with FUNDC1-MYC or empty vector for 24 h. Cell lysates were immunoprecipitated by anti-MYC antibody and immunoblotted with indicated antibodies.


C . HeLa cells were transfected with FLAG - FUNDC1 . About 24 h post - transfection , cell lysates were immunoprecipitated by anti - Calnexin and immunoblotted with anti - FLAG and anti - Calnexin antibodies .

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C. HeLa cells were transfected with FLAG-FUNDC1. About 24 h post-transfection, cell lysates were immunoprecipitated by anti-Calnexin and immunoblotted with anti-FLAG and anti-Calnexin antibodies.


D and E . HeLa cells were transfected with the indicated FUNDC1 - MYC constructs for 24 h . Cell lysates were immunoprecipitated using anti - Calnexin and immunoblotted using anti - MYC and anti - Calnexin antibodies .

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D and E. HeLa cells were transfected with the indicated FUNDC1-MYC constructs for 24 h. Cell lysates were immunoprecipitated using anti-Calnexin and immunoblotted using anti-MYC and anti-Calnexin antibodies.


F . Immunoblots of subcellular fractions from HeLa cells transfected with scramble siRNA and exposed to hypoxia ( 1 % O2 ) for 5 h . PNS : post - nuclear supernatant ; CYTO : cytosol ; ER : endoplasmic reticulum ; MITO : mitochondria ; MAM : mitochondrial - associated membrane . DRP1 ( S ) and DRP1 ( L ) indicate short and long exposures , respectively . G . Immunoblots of subcellular fractions from HeLa cells transfected with Calnexin siRNA ( si - CNX ) and exposed to hypoxia ( 1 % O2 ) for 5 h . PNS : post nuclear supernatant ; CYTO : cytosol ; ER : endoplasmic reticulum ; MITO : mitochondria ; MAM : mitochondrial - associated membrane . DRP1 ( S ) and DRP1 ( L ) indicate short and long exposures , respectively . H . Quantification of the MAM : PNS ratio of FUNDC1 or DRP1 in HeLa cells treated with scramble siRNA or si - CNX and exposed to hypoxia ( 1 % O2 ) for 5 h . Data are presented as mean ± s . e . m . from 3 independent experiments ; * * * P < 0 . 001 .

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F. Immunoblots of subcellular fractions from HeLa cells transfected with scramble siRNA and exposed to hypoxia (1% O2) for 5 h. PNS: post-nuclear supernatant; CYTO: cytosol; ER: endoplasmic reticulum; MITO: mitochondria; MAM: mitochondrial-associated membrane. DRP1 (S) and DRP1 (L) indicate short and long exposures, respectively.G. Immunoblots of subcellular fractions from HeLa cells transfected with Calnexin siRNA (si-CNX) and exposed to hypoxia (1% O2) for 5 h. PNS: post nuclear supernatant; CYTO: cytosol; ER: endoplasmic reticulum; MITO: mitochondria; MAM: mitochondrial-associated membrane. DRP1 (S) and DRP1 (L) indicate short and long exposures, respectively.H. Quantification of the MAM:PNS ratio of FUNDC1 or DRP1 in HeLa cells treated with scramble siRNA or si-CNX and exposed to hypoxia (1% O2) for 5 h. Data are presented as mean ± s.e.m. from 3 independent experiments; *** P < 0.001.


A . HeLa cells were transfected with the indicated si - RNAs . 24 h post - transfection , cells were exposed to hypoxia ( 1 % O2 ) for 12 h and then immunostained by anti - TOM20 . Bar = 50 µm . Boxed regions in the left panels are enlarged in the right panels . C . Quantification of mitochondria morphology ( fragmented , intermediate and elongated mitochondria ) in 100 cells from ( A ) . Data represent mean ± s . e . m . ( * * * P < 0 . 001 ) .

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A. HeLa cells were transfected with the indicated si-RNAs. 24 h post-transfection, cells were exposed to hypoxia (1% O2) for 12 h and then immunostained by anti-TOM20. Bar = 50 µm. Boxed regions in the left panels are enlarged in the right panels.C. Quantification of mitochondria morphology (fragmented, intermediate and elongated mitochondria) in 100 cells from (A). Data represent mean ± s.e.m. (*** P<0.001).


B . Electron microscopy analysis of HeLa cells treated with the indicated si - RNAs for 24 h and then exposed to hypoxia ( 1 % O2 ) for 12 h . Bar = 1 μm . Boxed regions in the left panels are enlarged in the right panels .

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B. Electron microscopy analysis of HeLa cells treated with the indicated si-RNAs for 24 h and then exposed to hypoxia (1% O2) for 12 h. Bar = 1 μm. Boxed regions in the left panels are enlarged in the right panels.


A . HeLa cells were treated with the indicated si - RNAs and exposed to hypoxia for 12 h . Cells were fixed and immunostained by anti - TOM20 and anti - DRP1 antibodies . Bar = 40 µm .

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A. HeLa cells were treated with the indicated si-RNAs and exposed to hypoxia for 12 h. Cells were fixed and immunostained by anti-TOM20 and anti-DRP1 antibodies. Bar = 40 µm.


B . Immunogold electron microscopy of control HeLa cells ( top ) or HeLa cells transfected with si - FUNDC1 ( bottom ) and then exposed to hypoxia ( 1 % O2 ) for 12 h . DRP1 was detected with anti - DRP1 ( 12 nm gold particles ) . Blue stars indicate mitochondria . Yellow arrows indicate gold particles . Red arrows indicate double - membraned autophagosomes .

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B. Immunogold electron microscopy of control HeLa cells (top) or HeLa cells transfected with si-FUNDC1 (bottom) and then exposed to hypoxia (1% O2) for 12 h. DRP1 was detected with anti-DRP1 (12 nm gold particles). Blue stars indicate mitochondria. Yellow arrows indicate gold particles. Red arrows indicate double-membraned autophagosomes.


C - H . Immunoblots of subcellular fractions from control HeLa cells or HeLa cells treated with the indicated si - RNA and then exposed to hypoxia for 5 h . PNS : post nuclear supernatant ; Cyto : cytosol ; Mito ( crude ) : crude mitochondrial fraction containing mitochondria and MAMs .

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C-H. Immunoblots of subcellular fractions from control HeLa cells or HeLa cells treated with the indicated si-RNA and then exposed to hypoxia for 5 h. PNS: post nuclear supernatant; Cyto: cytosol; Mito (crude): crude mitochondrial fraction containing mitochondria and MAMs.


A . HeLa cells were co - transfected with FLAG - DRP1 and FUNDC1 - MYC for 24 h . Cells were lysed and immunoprecipitated using anti - FLAG antibody . FLAG - DRP1 and FUNDC1 - MYC were analyzed with the corresponding antibodies .

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A. HeLa cells were co-transfected with FLAG-DRP1 and FUNDC1-MYC for 24 h. Cells were lysed and immunoprecipitated using anti-FLAG antibody. FLAG-DRP1 and FUNDC1-MYC were analyzed with the corresponding antibodies.


B . HeLa cells were co - transfected with empty vector , MYC - DRP1 and FLAG - FUNDC1 . 24 h after transfection , cell lysates were immunoprecipitated using anti - FLAG antibody and immunoblotted with anti - MYC and anti - FLAG antibodies .

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B. HeLa cells were co-transfected with empty vector, MYC-DRP1 and FLAG-FUNDC1. 24 h after transfection, cell lysates were immunoprecipitated using anti-FLAG antibody and immunoblotted with anti-MYC and anti-FLAG antibodies.


C . MEFs were transfected with FUNDC1 - MYC . 24 h post - transfection , cells were fixed and immunostained by anti - MYC and anti - DRP1 . Bar = 10 µm .

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C. MEFs were transfected with FUNDC1-MYC. 24 h post-transfection, cells were fixed and immunostained by anti-MYC and anti-DRP1. Bar = 10 µm.


D and E . HeLa cells were co - transfected with the indicated FUNDC1 - MYC and FLAG - DRP1 constructs for 24 h . Cell lysates were analyzed by immunoprecipitation using anti - FLAG and immunoblotted with anti - MYC and anti - FLAG antibodies .

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D and E. HeLa cells were co-transfected with the indicated FUNDC1-MYC and FLAG-DRP1 constructs for 24 h. Cell lysates were analyzed by immunoprecipitation using anti-FLAG and immunoblotted with anti-MYC and anti-FLAG antibodies.


F . HeLa cells were exposed to hypoxia ( 1 % O2 ) for the indicated times and cell lysates were immunoprecipitated using anti - FUNDC1 antibody . Endogenous DRP1 , Calnexin , LC3 and FUNDC1 were detected by anti - DRP1 , anti - Calnexin , anti - LC3 , and anti - FUNDC1 antibodies .

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F. HeLa cells were exposed to hypoxia (1% O2) for the indicated times and cell lysates were immunoprecipitated using anti-FUNDC1 antibody. Endogenous DRP1, Calnexin, LC3 and FUNDC1 were detected by anti-DRP1, anti-Calnexin, anti-LC3, and anti-FUNDC1 antibodies.


G . 10 - 20 g GST - FUNDC1 or GST was immobilized on glutathione sepharose resin ( GE Healthcare ) . 20 - 50 g DRP1 was incubated with the GST - FUNDC1 or GST bound resin overnight at 4 ° C . The resin was then extensively rinsed and eluted . Samples were subjected to SDS - PAGE , and then visualized by coomassie blue staining and western blot , respectively .

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G. 10-20 g GST-FUNDC1 or GST was immobilized on glutathione sepharose resin (GE Healthcare). 20-50 g DRP1 was incubated with the GST-FUNDC1 or GST bound resin overnight at 4 °C. The resin was then extensively rinsed and eluted. Samples were subjected to SDS-PAGE, and then visualized by coomassie blue staining and western blot, respectively.


H . Full - length FUNDC1 - MYC , FUNDC1 - MYC ( AA 96 - 155 ) , FL ( Δ129 - 138 ) or vector control were transfected with Mito - DsRed ( red ) into FUNDC1 KD MEFs . 12 h post - transfection , cells were exposed to hypoxia for 12 h and then stained with anti - MYC ( blue ) and anti - DRP1 ( green ) . Bar = 10 µm . Numbers at the right show the average length of mitochondria in Mito - DsRed - positive cells .

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H. Full-length FUNDC1-MYC, FUNDC1-MYC (AA 96-155), FL (Δ129-138) or vector control were transfected with Mito-DsRed (red) into FUNDC1 KD MEFs. 12 h post- transfection, cells were exposed to hypoxia for 12 h and then stained with anti-MYC (blue) and anti-DRP1 (green). Bar = 10 µm. Numbers at the right show the average length of mitochondria in Mito-DsRed-positive cells.


A . HeLa cells were transfected with the indicated si - RNAs for 24 h and then exposed to hypoxia for 24 h or not . Cells were fixed and immunostained by anti - TOM20 and anti - LC3 antibodies . The colocalization of LC3 and mitochondria was quantified and listed on the right side . Bar = 5 µm .

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A. HeLa cells were transfected with the indicated si-RNAs for 24 h and then exposed to hypoxia for 24 h or not. Cells were fixed and immunostained by anti-TOM20 and anti-LC3 antibodies. The colocalization of LC3 and mitochondria was quantified and listed on the right side. Bar = 5 µm.


B and C . HeLa cells were transfected with the indicated si - RNAs for 24 h and then exposed to hypoxia for 24 h . Cells were then treated with or without 50 nM BAF A1 ( Bafilomycin A1 ) for 6 h before harvesting . Cell lysates were immunoblotted with the indicated antibodies .

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B and C. HeLa cells were transfected with the indicated si-RNAs for 24 h and then exposed to hypoxia for 24 h. Cells were then treated with or without 50 nM BAF A1 (Bafilomycin A1) for 6 h before harvesting. Cell lysates were immunoblotted with the indicated antibodies.


D and E . Quantification of the TIM23 : Actin and TOM20 : Actin ratios from ( B and C ) . All data are from 3 independent experiments .

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D and E. Quantification of the TIM23:Actin and TOM20:Actin ratios from (B and C). All data are from 3 independent experiments.


F . HeLa cells were transfected by different truncated FUNDC1 - MYC . About 24h post - transfection , cell lysates were immunoblotted using indicated anti - bodies .

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F. HeLa cells were transfected by different truncated FUNDC1-MYC. About 24h post-transfection, cell lysates were immunoblotted using indicated anti-bodies.


( A ) Relative mRNA levels of FX in CD45 + leukocytes in the peripheral blood of E0771 ( abbreviated to E ) or LLC tumour - bearing mice . The mean sizes of E0771 and LLC tumours were 9 . 8 mm and 9 . 5 mm , respectively . Shown are averages ( N = 8 mice / group ) with SEM and one - way ANOVA

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(A) Relative mRNA levels of FX in CD45+ leukocytes in the peripheral blood of E0771 (abbreviated to E) or LLC tumour-bearing mice. The mean sizes of E0771 and LLC tumours were 9.8 mm and 9.5 mm, respectively. Shown are averages (N = 8 mice/group) with SEM and one-way ANOVA


( B ) Relative mRNA levels of FX in CD45 + leukocytes in various organs such as lung ( Lu ) , Liver ( Li ) , spleen ( Sp ) , bone marrow ( BM ) and lymph node ( Lymph ) derived from no tumour - bearing or E0771 - bearing mice . " Tumour " stands for mRNA levels of FX in E0771 tumours . Shown are averages ( N = 6 : organs from no tumour - bearing mice , N = 6 : organs from E0771 - bearing mice ) with SEM and one - way

O O O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O B-GENEPROD O B-CELL_TYPE O O O O O B-TISSUE O B-TISSUE O O B-TISSUE O B-TISSUE O O B-TISSUE O B-TISSUE O O B-TISSUE I-TISSUE O B-TISSUE O O B-TISSUE I-TISSUE O B-TISSUE O O O O B-DISEASE O O O O O O B-ORGANISM O O B-DISEASE O O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O B-DISEASE O O O O O O O O O O O O B-DISEASE O O B-ORGANISM O O O O O O O O O O B-ORGANISM O O O O O O O

(B) Relative mRNA levels of FX in CD45+ leukocytes in various organs such as lung (Lu), Liver (Li), spleen (Sp), bone marrow (BM) and lymph node (Lymph) derived from no tumour-bearing or E0771-bearing mice. "Tumour" stands for mRNA levels of FX in E0771 tumours. Shown are averages (N = 6: organs from no tumour-bearing mice, N = 6: organs from E0771-bearing mice) with SEM and one-way ANOVA


( C ) Immunohistochemical quantifications of FX expression in CD45 + leukocytes in the liver and lungs of no tumour - bearing or E0771 - bearing mice . Shown are averages ( N = 6 ) with SEM and Welch " s t - test

O O O B-EXP_ASSAY O O B-GENEPROD B-EXP_ASSAY O B-GENEPROD O B-CELL_TYPE O O B-TISSUE O B-TISSUE O O B-DISEASE O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O

(C) Immunohistochemical quantifications of FX expression in CD45+ leukocytes in the liver and lungs of no tumour-bearing or E0771-bearing mice. Shown are averages (N = 6) with SEM and Welch"s t-test


( D ) Immunohistochemical quantifications of fibrinogen deposition in no tumour - bearing - and tumour - bearing - mouse lungs . Shown are averages ( N = 7 / group , 14 random fields ) with SEM . Welch " s t - test

O O O B-EXP_ASSAY O O B-SUBCELLULAR O O O B-DISEASE O O O O B-DISEASE O O O B-ORGANISM B-TISSUE 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) Immunohistochemical quantifications of fibrinogen deposition in no tumour-bearing- and tumour-bearing- mouse lungs. Shown are averages (N = 7/ group, 14 random fields) with SEM. Welch"s t-test


( E ) Representative photos of immunohistochemical co - localisation of FX + CD45 + cells and fibrinogen deposition in tumour - bearing mouse lungs . Circles show high fibrinogen deposition areas ( scale bar , 50 μm )

O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O B-GENEPROD O O O B-SUBCELLULAR O O B-DISEASE O O B-ORGANISM B-TISSUE O O O O B-SUBCELLULAR O O O O O O O O O

(E) Representative photos of immunohistochemical co-localisation of FX+CD45+ cells and fibrinogen deposition in tumour-bearing mouse lungs. Circles show high fibrinogen deposition areas (scale bar, 50 μm)


( B ) KikGR red cells were detected in the TCM - stimulated - liver and lungs after liver exposure to violet light ( arrow ) . Images taken from animals with no light exposure were also shown ( scale bar , 100 μm )

O O O B-GENEPROD O O O O O O O O O O B-TISSUE O B-TISSUE O B-TISSUE O O O O O O O O B-EXP_ASSAY O O B-ORGANISM O O O O O O O O O O O O O O

(B) KikGR red cells were detected in the TCM-stimulated- liver and lungs after liver exposure to violet light (arrow). Images taken from animals with no light exposure were also shown (scale bar, 100 μm)


( C ) Flow cytometric quantifications of photoconverted HepELs in TCM ( 3 times ) - stimulated KikGR mouseliver and lungs . Cells were isolated 72 h after photoconversion . Ratio was calculated as the number of photoconverted cells ( KikGR redin comparison with the number of liver or lung cells pre - sorted with CD45 - beads . Shown are averages ( N = 5 ) with SEM and Welch " s t -

O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O B-GENEPROD B-ORGANISM O B-TISSUE O O O O O O O O O O O O O O B-EXP_ASSAY O O O O B-GENEPROD O O O O B-EXP_ASSAY O B-TISSUE O B-TISSUE O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O

(C) Flow cytometric quantifications of photoconverted HepELs in TCM (3 times)-stimulated KikGR mouseliver and lungs. Cells were isolated 72 h after photoconversion. Ratio was calculated as the number of photoconverted cells (KikGR redin comparison with the number of liver or lung cells pre-sorted with CD45-beads. Shown are averages (N = 5) with SEM and Welch"s t-test


( D ) Surface marker analyses of photoconverted KikGR cells . Vertical axes represent ratio of each marker + cell / photoconverted KikGR cell . Shown are averages ( conCM , control : N = 4 , TCM : N = 9 ) with SEM and Welch " s t - test

O O O O O O O O B-GENEPROD O O O O O O O O O O O O O B-GENEPROD O O O O O 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) Surface marker analyses of photoconverted KikGR cells. Vertical axes represent ratio of each marker+ cell/photoconverted KikGR cell. Shown are averages (conCM, control: N = 4, TCM: N = 9) with SEM and Welch"s t-test


( E ) Representative immunostaining images of FX expression in NK1 . 1 + , CD11c + , B220 + and CD4 + cells in tumour - bearing lungs ( scale bar , 10 μm )

O O O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD B-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD O O B-GENEPROD O O B-GENEPROD O O B-GENEPROD O O O B-DISEASE O O B-TISSUE O O O O O O O

(E) Representative immunostaining images of FX expression in NK1.1+, CD11c+, B220+ and CD4+ cells in tumour-bearing lungs (scale bar, 10 μm)


( A ) Flow cytometric analysis of B220 + CD11c + NK1 . 1 + cells in the lung of TCM - stimulated mice . Values in the dot plots present ratios for total cells gated KikGR red and KikGR green cell

O O O B-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-TISSUE O O O O B-ORGANISM O O O O O O O O O O O O B-GENEPROD O O B-GENEPROD O O

(A) Flow cytometric analysis of B220+CD11c+NK1.1+ cells in the lung of TCM-stimulated mice. Values in the dot plots present ratios for total cells gated KikGR red and KikGR green cell


( B ) Representative flow cytometric analyses of the relocation of B220 + CD11c + NK1 . 1 + cells in lungs that were photoconverted in TCM - stimulated KikGR mouse liver ( gated in red polygonal region ) . Percent is shown for total cells ( upper panel in B ) , and for gated KikGR red or KikGR green cells ( lower panels in B ) . Region where the photoconverted cells locate is magnified in the right . Yellow arrow shows the photoconversion direction . Three independent experiments . LTCM stands for TCM derived from LLC

O O O O B-EXP_ASSAY I-EXP_ASSAY O O O B-EXP_ASSAY O B-GENEPROD O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-TISSUE O O O O O O O B-GENEPROD B-ORGANISM B-TISSUE 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-GENEPROD O O B-GENEPROD O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O B-CELL_LINE

(B) Representative flow cytometric analyses of the relocation of B220+CD11c+NK1.1+ cells in lungs that were photoconverted in TCM-stimulated KikGR mouse liver (gated in red polygonal region). Percent is shown for total cells (upper panel in B), and for gated KikGR red or KikGR green cells (lower panels in B). Region where the photoconverted cells locate is magnified in the right. Yellow arrow shows the photoconversion direction. Three independent experiments. LTCM stands for TCM derived from LLC


( A ) Representative photos of IFNγ expression in B220 + CD11c + NK1 . 1 + cells derived from TCM - stimulating mouse liver . These cells were cultured with LiCM or LuCM that had been prepared by culture with tumour - bearing - liver or lungs , respectively . NoCM stands for CM without tissues , and it was used as control ( scale bar , 10 μm )

O O O O O O B-GENEPROD B-EXP_ASSAY O B-GENEPROD O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O B-ORGANISM B-TISSUE O O O O O O B-TISSUE O B-TISSUE O O O O O O O B-DISEASE O O O B-TISSUE O B-TISSUE 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 photos of IFNγ expression in B220+CD11c+NK1.1+cells derived from TCM-stimulating mouse liver. These cells were cultured with LiCM or LuCM that had been prepared by culture with tumour-bearing- liver or lungs, respectively. NoCM stands for CM without tissues, and it was used as control (scale bar,10 μm)


( B ) Immunohistochemical quantifications of IFNγ in B220 + CD11c + NK1 . 1 + cells . Cells derived from liver and spleen were primed with LiCM or LuCM . Shown are averages ( N = 6 / group ) with SEM and one - way ANOVA

O O O B-EXP_ASSAY O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O B-TISSUE O B-TISSUE O O O B-TISSUE O B-TISSUE O O O O O O O O O O O O O O O O O O

(B) Immunohistochemical quantifications of IFNγ in B220+CD11c+NK1.1+ cells. Cells derived from liver and spleen were primed with LiCM or LuCM. Shown are averages (N = 6/group) with SEM and one-way ANOVA


( C ) Flow cytometric analysis of living Zombie - tumour cells with B220 + CD11c + NK1 . 1 + cells that were primed by NoCM or LuCM . NoCM : N = 3 , LuCM : N = 4 . Shown are averages with SEM . Welch " s t - test . Rhodamin + tumour cells were counted as viable cells

O O O B-EXP_ASSAY I-EXP_ASSAY O O B-EXP_ASSAY B-SMALL_MOLECULE O B-DISEASE O O B-GENEPROD O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O B-TISSUE O O O O O O O B-TISSUE O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O B-DISEASE O O B-EXP_ASSAY O B-EXP_ASSAY O

(C) Flow cytometric analysis of living Zombie- tumour cells with B220+CD11c+NK1.1+ cells that were primed by NoCM or LuCM. NoCM: N=3, LuCM: N=4. Shown are averages with SEM. Welch"s t-test. Rhodamin+ tumour cells were counted as viable cells


( D ) Representative immunohistochemical stainings of rhodamin + ( red ) Zombie + ( green ) tumour cells ( scale bar , 10 μm )

O O O O B-EXP_ASSAY I-EXP_ASSAY O B-SMALL_MOLECULE O O O O B-SMALL_MOLECULE O O O O B-DISEASE O O O O O O O O

(D) Representative immunohistochemical stainings of rhodamin+ (red) Zombie+ (green) tumour cells (scale bar, 10 μm)


( E ) Ratios of living tumour cells after co - culture with B220 + CD11c + NK1 . 1 + cells that had been primed with lung - or liver - CM . Shown are averages ( N = 6 / group ) with SEM and one - way ANOVA

O O O O O B-EXP_ASSAY B-DISEASE O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O B-TISSUE O O B-TISSUE O O O O O O O O O O O O O O O O O O O O

(E) Ratios of living tumour cells after co-culture with B220+CD11c+NK1.1+cells that had been primed with lung- or liver-CM. Shown are averages (N = 6/group) with SEM and one-way ANOVA


( A ) Western blotting of fibrinogen in tumour - bearing and no tumour - bearing livers

O O O B-EXP_ASSAY I-EXP_ASSAY O O O B-DISEASE O O O O B-DISEASE O O B-TISSUE

(A) Western blotting of fibrinogen in tumour-bearing and no tumour-bearing livers


( C ) IP - Western blot analysis shows protein - protein interactions between fibrinogen and Vtn / TSP

(C) IP-Western blot analysis shows protein-protein interactions between fibrinogen and Vtn/TSP


( D ) Quantification of fibrinogen deposition areas after injection of CD45 + cells derived from the liver in tumour - bearing mice . Shown are averages ( N = 7 / group ) with SEM . 1 - way ANOVA

O O O O O B-SUBCELLULAR O O O O O B-GENEPROD O O O O O B-TISSUE O B-DISEASE O O B-ORGANISM O O O O O O O O O O O O O O O O O O

(D) Quantification of fibrinogen deposition areas after injection of CD45+ cells derived from the liver in tumour-bearing mice. Shown are averages (N = 7/ group) with SEM. 1-way ANOVA


( E ) Quantifications of fibrinogen deposition areas in TCM - stimulating wild - type mouse lungs after injection of Vtn + HepELs . Vtn + cells were isolated from E0771 - bearing wild - type or FX - / - mouse liver by using anti - Vtn beads ( 1 x 106 cells / mouse ) . Shown are averages ( N = 5 / group , 3 fields / sample ) with SEM . Welch " s t - test

O O O O O B-SUBCELLULAR O O O O O O O O O B-ORGANISM B-TISSUE O O O B-GENEPROD O O O B-GENEPROD O O O O O O O O O O O O B-GENEPROD O O O B-ORGANISM B-TISSUE O O O O B-GENEPROD O O O O O O O B-ORGANISM O O 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) Quantifications of fibrinogen deposition areas in TCM-stimulating wild-type mouse lungs after injection of Vtn+ HepELs. Vtn+ cells were isolated from E0771-bearing wild-type or FX-/-mouse liver by using anti-Vtn beads (1 x 106 cells /mouse). Shown are averages (N=5/ group, 3 fields/ sample) with SEM. Welch"s t-test


( F ) Separation of Vtn + / CD45 + HepELs from tumour - bearing FX + / - and FX - / - mouse livers by a cell sorter ( Moflo Astrios ) . Sorted Vtn + CD45 + HepELs cells obtained from a littermate ( 3 animals each ) were pooled , and 8 x 103 Vtn + CD45 + HepELs cells were i . v . injected into a tumour - bearing mouse . Right graph shows fibrinogen depositions in the lung . Shown are averages ( N = 3 / group , 9 fields / sample ) with SEM . 1 - way

O O O O O B-GENEPROD O O B-GENEPROD O O O B-DISEASE O O B-GENEPROD O O O O B-GENEPROD O O O B-ORGANISM B-TISSUE O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O B-ORGANISM O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O B-DISEASE O O B-ORGANISM O O O O B-SUBCELLULAR O O O B-TISSUE O O O O O O O O O O O O O O O O O O O O O O

(F) Separation of Vtn+/ CD45+ HepELs from tumour-bearing FX+/- and FX-/- mouse livers by a cell sorter (Moflo Astrios). Sorted Vtn+CD45+HepELs cells obtained from a littermate (3 animals each) were pooled, and 8 x 103 Vtn+CD45+HepELs cells were i.v. injected into a tumour-bearing mouse. Right graph shows fibrinogen depositions in the lung. Shown are averages (N = 3/ group, 9 fields/ sample) with SEM. 1-way ANOVA


( A ) Representative immunostaining of fibrinogen depositions in tumour - bearing mouse lungs received an injection of rhodamine - labelled B220 + CD11c + NK1 . 1 + HepELs derived from tumour - bearing - FX + / − or FX − / − mice ( scale bar , 20 μm )

O O O O B-EXP_ASSAY O B-SUBCELLULAR O O B-DISEASE O O B-ORGANISM B-TISSUE O O O O B-SMALL_MOLECULE O O B-GENEPROD O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-DISEASE O O O B-GENEPROD O O O O B-GENEPROD O O O B-ORGANISM O O O O O O O

(A) Representative immunostaining of fibrinogen depositions in tumour-bearing mouse lungs received an injection of rhodamine-labelled B220+CD11c+NK1.1+HepELs derived from tumour-bearing- FX+/− or FX−/− mice (scale bar, 20 μm)


( B ) Immunohistochemical quantifications of fibrinogen depositions in tumour - bearing mouse lung after an injection of B220 + CD11c + NK1 . 1 + cells derived from tumour - bearing - FX + / − or FX − / − mouse liver . Sorted B220 + CD11c + NK1 . 1 + cells were prepared from three mice per each genotype , and were i . v . injected . Shown are averages ( N = 6 / group , 4 sections / mouse ) with SEM and Welch " s t - test

O O O B-EXP_ASSAY O O B-SUBCELLULAR O O B-DISEASE O O B-ORGANISM B-TISSUE O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-DISEASE O O O B-GENEPROD O O O O B-GENEPROD O O O B-ORGANISM B-TISSUE O O B-GENEPROD O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O B-ORGANISM O 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-ORGANISM O O O O O O O O O O

(B) Immunohistochemical quantifications of fibrinogen depositions in tumour-bearing mouse lung after an injection of B220+CD11c+NK1.1+cells derived from tumour-bearing- FX+/− or FX−/− mouse liver. Sorted B220+CD11c+NK1.1+cells were prepared from three mice per each genotype, and were i.v. injected. Shown are averages (N = 6/group, 4 sections/mouse) with SEM and Welch"s t-test


( B ) Relative mRNA levels of FX , Vtn and TSP1 in liver B220 + CD11c + NK1 . 1 + cells stimulated with LiCM or LuCM . Shown are averages ( N = 6 / group ) with SEM and one - way ANOVA

O O O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-TISSUE B-GENEPROD O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-TISSUE O B-TISSUE O O O O O O O O O O O O O O O O O O

(B) Relative mRNA levels of FX, Vtn and TSP1 in liver B220+CD11c+NK1.1+cells stimulated with LiCM or LuCM. Shown are averages (N = 6/group) with SEM and one-way ANOVA


( C ) Relative mRNA levels of FX in liver B220 + CD11c + NK1 . 1 + cells stimulated with various factors . Shown are averages ( N = 4 ) with SEM and one - way ANOVA

O O O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O B-TISSUE B-GENEPROD O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O O O

(C) Relative mRNA levels of FX in liver B220+CD11c+NK1.1+cells stimulated with various factors. Shown are averages (N = 4) with SEM and one-way ANOVA


( D ) In vitro adhesion assay . Liver B220 + CD11c + NK1 . 1 + cells were seeded on a fibrinogen plate . Cells attached to the plate were counted . Twentyμg / mL of anti - Vtn Ab or isotype control IgG were used to block the adhesion . Shown are averages ( N = 6 / group ) with SEM and one - way ANOVA

O O O O O B-EXP_ASSAY I-EXP_ASSAY O B-TISSUE B-GENEPROD O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O B-SUBCELLULAR O O O O O O O O B-EXP_ASSAY O O O O O O O B-GENEPROD O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O

(D) In vitro adhesion assay. Liver B220+CD11c+NK1.1+cells were seeded on a fibrinogen plate. Cells attached to the plate were counted. Twentyμg/mL of anti-Vtn Ab or isotype control IgG were used to block the adhesion. Shown are averages (N = 6/group) with SEM and one-way ANOVA


( E ) Lung B220 + CD11c + NK1 . 1 + cells were seeded on ECM coated ( fibrinogen , fibronectin , or collagen I ) plates . Shown are averages ( N = 5 / group ) with SEM and one - way ANOVA

O O O B-TISSUE B-GENEPROD O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O B-SUBCELLULAR O O B-SUBCELLULAR O B-GENEPROD O O B-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O

(E) Lung B220+CD11c+NK1.1+cells were seeded on ECM coated (fibrinogen, fibronectin, or collagen I) plates. Shown are averages (N = 5/group) with SEM and one-way ANOVA


( B ) Tumour cell homing in the lungs pre - treated with LiCM - primed B220 + CD11c + NK1 . 1 + cells from tumour - bearing WT or FX − / − mouse liver . Representative photos of homing of rhodamine - labelled tumour cells ( upper , scale bar , 20 μm ) . Number of homing tumour cells are shown ( lower ) . Shown are averages ( N = 60 sections , 5 / group , all field count / section , 12 sections / sample ) with SEM and Welch " s t - test

O O O B-DISEASE O O O O B-TISSUE O O O O B-TISSUE O O B-GENEPROD O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-DISEASE O O O O B-GENEPROD O O O B-ORGANISM B-TISSUE O O O O O O B-SMALL_MOLECULE O O B-DISEASE O O O O O O O O O O O B-EXP_ASSAY O O B-DISEASE O O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O

(B) Tumour cell homing in the lungs pre-treated with LiCM-primed B220+CD11c+NK1.1+cells from tumour-bearing WT or FX−/− mouse liver. Representative photos of homing of rhodamine-labelled tumour cells (upper, scale bar, 20 μm). Number of homing tumour cells are shown (lower). Shown are averages (N = 60 sections, 5/group, all field count/section, 12 sections/sample) with SEM and Welch"s t-test


( C ) Tumour cell homing in the lungs pre - treated with activated FX - overexpressed B220 + CD11c + NK1 . 1 + cells ( FX - OE - HepELs ) from tumour - bearing lungs . Number of homing tumour cells after an injection of lung HepELs or FX - OE - HepELs are shown . Shown here are averages ( N = 24 sections , 4 / group , all field count / section , 6 sections / sample ) SEM and one - way ANOVA

O O O B-DISEASE O O O O B-TISSUE O O O O O B-GENEPROD O O B-GENEPROD O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-GENEPROD O O O O O O B-DISEASE O O B-TISSUE O B-EXP_ASSAY O O B-DISEASE O O O O O B-TISSUE O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O

(C) Tumour cell homing in the lungs pre-treated with activated FX-overexpressed B220+CD11c+NK1.1+cells (FX-OE-HepELs) from tumour-bearing lungs. Number of homing tumour cells after an injection of lung HepELs or FX-OE-HepELs are shown. Shown here are averages (N = 24 sections, 4/group, all field count/section, 6 sections/sample) SEM and one-way ANOVA


( A - B ) Representative Hematoxylin and Eosin ( H & E ) staining , and immunohistochemistry of phosphorylated FAK on tyrosine ( Y ) 397 ( pY397 FAK ) , alpha - Smooth Muscle Actin ( α - SMA ) , and Fibroblast Activated Protein alpha ( FAP - α ) in serial sections of human normal pancreas ( Ct1 ) , of human normal adjacent PDAC tissue ( Ct2 ) and of human PDAC tissues ( PDAC1 - 3 ) ; scale bar , 100 μm ( A ) , and 300 µm ( B ) .

O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-EXP_ASSAY O B-EXP_ASSAY B-GENEPROD O B-SMALL_MOLECULE O O O O O O B-GENEPROD O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O B-ORGANISM O B-TISSUE O O O O O B-ORGANISM O O B-DISEASE O O O O O O B-ORGANISM B-DISEASE O O B-DISEASE O O O O O O O O O O O O O O O O O O O O

(A-B) Representative Hematoxylin and Eosin (H&E) staining, and immunohistochemistry of phosphorylated FAK on tyrosine (Y) 397 (pY397 FAK), alpha-Smooth Muscle Actin (α-SMA), and Fibroblast Activated Protein alpha (FAP-α) in serial sections of human normal pancreas (Ct1), of human normal adjacent PDAC tissue (Ct2) and of human PDAC tissues (PDAC1-3); scale bar, 100 μm (A), and 300 µm (B).


( C ) Quantification of pY397 FAK level in cancer - associated fibroblasts from human PDAC microarray : 40 different PDAC patient samples and 10 normal human pancreas have been stained for pY397 FAK and double blinded quantified ( based on intensity and positive cell number ) on a scale between 0 ( no staining ) and 6 ( Y397 FAK highly positive ) in whole cell ( left ) , cytoplasm ( middle ) and nucleus ( right ) .

O O O O O O B-GENEPROD O O B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE I-CELL_TYPE O B-ORGANISM B-DISEASE B-EXP_ASSAY O O O B-DISEASE B-ORGANISM O O O O B-ORGANISM B-TISSUE O O O O O B-GENEPROD O O 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-GENEPROD O O O O O O O O O O B-SUBCELLULAR O O O O B-SUBCELLULAR O O O O

(C) Quantification of pY397 FAK level in cancer-associated fibroblasts from human PDAC microarray: 40 different PDAC patient samples and 10 normal human pancreas have been stained for pY397 FAK and double blinded quantified (based on intensity and positive cell number) on a scale between 0 (no staining) and 6 (Y397 FAK highly positive) in whole cell (left), cytoplasm (middle) and nucleus (right).


( A ) Representative images of Definiens ® Tissue studio multi - resolution segmentation algorithm on two PDAC TMA examples ( X , Y ) . From left to right : IHC for pY397 FAK ; ROI detection of tumour cells ( dark blue ) , stroma ( orange ) and other ( light blue / grey ) ; Nuclear / cytoplasm detection in blue and green respectively ; IHC staining classification for pY397 FAK intensity signal ( dark red for high signal , orange for medium signal , yellow for low signal and white for negative signal ) .

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(A) Representative images of Definiens® Tissue studio multi-resolution segmentation algorithm on two PDAC TMA examples (X, Y). From left to right: IHC for pY397 FAK; ROI detection of tumour cells (dark blue), stroma (orange) and other (light blue/grey); Nuclear/ cytoplasm detection in blue and green respectively; IHC staining classification for pY397 FAK intensity signal (dark red for high signal, orange for medium signal, yellow for low signal and white for negative signal).


( B - C ) Disease Free Survival ( B ) and Overall Survival ( C ) from 120 patient cohort according to pY397 FAK expression in CAFs ; HR : Hazard Ratio . Survival curves were estimated with the Kaplan - Meier method and compared using log - rank test , non - parametric tests ( chi2 - test ) to compare independent groups for categorical data .

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(B-C) Disease Free Survival (B) and Overall Survival (C) from 120 patient cohort according to pY397 FAK expression in CAFs; HR: Hazard Ratio. Survival curves were estimated with the Kaplan-Meier method and compared using log-rank test, non-parametric tests (chi2-test) to compare independent groups for categorical data.


( D - E ) Disease Free Survival ( D ) and Overall Survival ( E ) from 120 patient cohort according to pY397 FAK expression in tumour cells . Survival curves were estimated with the Kaplan - Meier method and compared using log - rank test , non - parametric tests ( chi2 - test ) to compare independent groups for categorical data .

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(D-E) Disease Free Survival (D) and Overall Survival (E) from 120 patient cohort according to pY397 FAK expression in tumour cells. Survival curves were estimated with the Kaplan-Meier method and compared using log-rank test, non-parametric tests (chi2-test) to compare independent groups for categorical data.


Tumour cells were syngeneically and orthotopically co - grafted with mouse FAK - WT or FAK - KD fibroblasts . Thirty - eight days after injection , mice were euthanized . ( A ) Primary tumour mass quantification . Values are means ± SEM from at least 7 mice per group .

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Tumour cells were syngeneically and orthotopically co-grafted with mouse FAK-WT or FAK-KD fibroblasts. Thirty-eight days after injection, mice were euthanized. (A) Primary tumour mass quantification. Values are means ± SEM from at least 7 mice per group.


( B ) Representative images from primary tumour stained for H & E , cytokeratin 19 ( CK19 ) , α - SMA , Sirius Red and polarized Sirius Red . Scale bar , 100 μm .

O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD I-GENEPROD O B-GENEPROD O O B-GENEPROD I-GENEPROD I-GENEPROD O B-SMALL_MOLECULE I-SMALL_MOLECULE O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O

(B) Representative images from primary tumour stained for H&E, cytokeratin 19 (CK19), α-SMA, Sirius Red and polarized Sirius Red. Scale bar, 100 μm.


( C ) Quantification of fibrillar collagen I ( Yellow - Orange ) and III ( Green ) based on polarized Sirius Red images from B . Each dot represents a mouse ( n = 11 per group ) .

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(C) Quantification of fibrillar collagen I (Yellow-Orange) and III (Green) based on polarized Sirius Red images from B. Each dot represents a mouse (n=11 per group).


IHC representative images ( left ) and quantification ( right ) the expression of CD31 ( D ) from primary tumour ; scale bar , 100 μm .

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IHC representative images (left) and quantification (right) the expression of CD31 (D) from primary tumour; scale bar, 100 μm.


IHC representative images ( left ) and quantification ( right ) the expression of two hypoxia markers CaIX and HIF1α ( E ) from primary tumour ; scale bar , 100 μm .

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IHC representative images (left) and quantification (right) the expression of two hypoxia markers CaIX and HIF1α (E) from primary tumour; scale bar, 100 μm.


( F ) Representative images of H & E , CK19 and α - SMA IHC staining performed on lung sections . Arrow identified lung metastasis . ( G ) Quantification of lung metastasis size and number based on CK19 positive staining using NanoZoomer software . Values are means ± SEM of 6 mice per group .

O O O O B-EXP_ASSAY O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD B-EXP_ASSAY I-EXP_ASSAY O O B-TISSUE O O O O B-TISSUE B-EXP_ASSAY O O O O O O B-TISSUE B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD O B-EXP_ASSAY O O O O O O O O O O O B-ORGANISM O O O

(F) Representative images of H&E, CK19 and α-SMA IHC staining performed on lung sections. Arrow identified lung metastasis. (G) Quantification of lung metastasis size and number based on CK19 positive staining using NanoZoomer software. Values are means ± SEM of 6 mice per group.


( A - B ) Relative frequencies of tumour - infiltrating M1 macrophages and M2 macrophages analyzed by flow cytometry at 21 days ( A ) and 38 days ( B ) after grafting .

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(A-B) Relative frequencies of tumour-infiltrating M1 macrophages and M2 macrophages analyzed by flow cytometry at 21 days (A) and 38 days (B) after grafting.


( D ) Impact of conditioned media ( CM ) secreted by activated mouse fibroblasts expressing FAK - WT or FAK - KD on mouse BMDM - derived macrophage polarization . Relative frequencies of macrophages expressing CD80 , CD86 , Nos2 , CD206 , CMH2 , Dectin , and CD163 markers acquired by flow cytometry and processed using FlowJo software .

O O O O O O O O O O O O O B-ORGANISM B-CELL_TYPE B-EXP_ASSAY B-GENEPROD O O O B-GENEPROD O O O B-ORGANISM B-CELL_TYPE O O B-CELL_TYPE O O O O O B-CELL_TYPE B-EXP_ASSAY B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O B-GENEPROD O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O

(D) Impact of conditioned media (CM) secreted by activated mouse fibroblasts expressing FAK-WT or FAK-KD on mouse BMDM-derived macrophage polarization. Relative frequencies of macrophages expressing CD80, CD86, Nos2, CD206, CMH2, Dectin, and CD163 markers acquired by flow cytometry and processed using FlowJo software.


( E ) Impact of soluble factors secreted by CAFs on macrophage migration . Left : schematic of BMDM derived M1 or M2 - like macrophages . Right : Representative pictures and quantification of M1 and M2 macrophage migration at 24 and 48h towards CM secreted by CAFs ( previously treated or not with FAK inhibitor for 48h ) . Cells were stained using Cristal Violet and percentage of cristal violet positive area quantified using Cell Observer videomicroscope ( Zeiss ) and analyzed with ImageJ .

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(E) Impact of soluble factors secreted by CAFs on macrophage migration. Left: schematic of BMDM derived M1 or M2-like macrophages. Right: Representative pictures and quantification of M1 and M2 macrophage migration at 24 and 48h towards CM secreted by CAFs (previously treated or not with FAK inhibitor for 48h). Cells were stained using Cristal Violet and percentage of cristal violet positive area quantified using Cell Observer videomicroscope (Zeiss) and analyzed with ImageJ.


( F ) Quantitative real - time PCR conducted with gene - specific primers ( CCL2 , CCL5 and M - CSF ) on four different FAK - I - treated ( 16h ) and non - treated CAFs for gene expression . The normalized fold change ± SEM was calculated using the delta - delta Ct method with RSP16 as a control gene ; values are mean + / - SEM . Each individual patient - derived activated fibroblasts was assigned to a specific

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(F) Quantitative real-time PCR conducted with gene-specific primers (CCL2, CCL5 and M-CSF) on four different FAK-I-treated (16h) and non-treated CAFs for gene expression. The normalized fold change ± SEM was calculated using the delta-delta Ct method with RSP16 as a control gene; values are mean +/- SEM. Each individual patient-derived activated fibroblasts was assigned to a specific symbol


( G ) Elisa quantification of CCL2 ( MCP1 ) concentration ( µg / ml ) in conditioned medium from hCAFs treated or not with FAK - I during 72h . * p < 0 . 05 , by paired t - test ; values are mean + / - SEM . Each individual patient - derived activated fibroblasts was assigned to a specific

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(G) Elisa quantification of CCL2 (MCP1) concentration (µg/ml) in conditioned medium from hCAFs treated or not with FAK-I during 72h. * p <0.05, by paired t-test; values are mean +/- SEM. Each individual patient-derived activated fibroblasts was assigned to a specific symbol


( H ) Left : Representative immunohistochemistry pictures of phosphorylated FAK on tyrosine ( Y ) 397 and of CD206 in serial sections of human PDAC tissues . Scale bar : 200µm Middle : Correlation plot between fibroblastic pY397 FAK score and CD45 or CD206 + cell number within human PDAC samples . Fibroblastic pY397 FAK score and percentage of CD206 positive cell quantifications were performed on a 47 patient cohort based on IHC staining . Spearman r = 0 . 4145 , p = 0 . 0019 . Right : Percentage of CD206 positive cells in PDAC patient expressing either low or high fibroblastic FAK activity score .

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(H) Left: Representative immunohistochemistry pictures of phosphorylated FAK on tyrosine (Y) 397 and of CD206 in serial sections of human PDAC tissues. Scale bar: 200µm Middle: Correlation plot between fibroblastic pY397 FAK score and CD45 or CD206+ cell number within human PDAC samples. Fibroblastic pY397 FAK score and percentage of CD206 positive cell quantifications were performed on a 47 patient cohort based on IHC staining. Spearman r = 0.4145, p=0.0019. Right: Percentage of CD206 positive cells in PDAC patient expressing either low or high fibroblastic FAK activity score.


( A ) Representative images of three independent scratch wound assays of co - cultivated red - labelled pancreatic tumour cells with either FAK - WT ( wild - type ) or FAK - KD ( Kinase - dead ) green - labelled fibroblasts at time zero and 46 hours ( corresponding to the wound closure of co - cultured FAK - WT fibroblasts with tumour cells ) .

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(A) Representative images of three independent scratch wound assays of co-cultivated red-labelled pancreatic tumour cells with either FAK-WT (wild-type) or FAK-KD (Kinase-dead) green-labelled fibroblasts at time zero and 46 hours (corresponding to the wound closure of co-cultured FAK-WT fibroblasts with tumour cells).


Cell migration ( has been analyzed in detail by tracking the location of cells ( n > 16 cells per condition ) over time for fibroblasts or tumour cells ( B ) ,

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Cell migration (has been analyzed in detail by tracking the location of cells (n >16 cells per condition) over time for fibroblasts or tumour cells (B),


measuring fibroblast velocity ( C ) , directionality ( a cell with a directionality equal to 1 is moving straightforward ) ( D ) and migration distance ( E ) or tumour cell velocity ( F ) , directionality ( G ) and distance ( H ) . Values are means ± SEM of 16 tracked cells from two independent experiments .

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measuring fibroblast velocity (C), directionality (a cell with a directionality equal to 1 is moving straightforward) (D) and migration distance (E) or tumour cell velocity (F), directionality (G) and distance (H). Values are means ± SEM of 16 tracked cells from two independent experiments.


( I ) 3D reconstitution of cell invasion into collagen matrix from spheroid composed of green - labelled FAK - WT or FAK - KD fibroblasts plus red - labelled pancreatic tumour cells ( ratio 1 / 1 ) . Shown are representative 3D reconstitution of spheroid invasion into 2mg / ml collagen I matrix obtained using Zen software . ( J - K ) Quantification of fibroblast ( J ) or tumour cell ( K ) invasion within collagen matrix . Values are means ± SEM of 60 tracked cells from three independent experiments .

O O O O O O B-EXP_ASSAY I-EXP_ASSAY O B-SUBCELLULAR O O O O O O O O B-GENEPROD O O O B-GENEPROD O O B-CELL_TYPE O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O O O O B-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O O O O O B-CELL_TYPE O O O O O O O O O B-EXP_ASSAY O B-SUBCELLULAR O O O O O O O O O O O O O O O O

(I) 3D reconstitution of cell invasion into collagen matrix from spheroid composed of green-labelled FAK-WT or FAK-KD fibroblasts plus red-labelled pancreatic tumour cells (ratio 1/1). Shown are representative 3D reconstitution of spheroid invasion into 2mg/ml collagen I matrix obtained using Zen software. (J-K) Quantification of fibroblast (J) or tumour cell (K) invasion within collagen matrix. Values are means ± SEM of 60 tracked cells from three independent experiments.


( A ) Representative images of CK19 and α - SMA immunohistochemistry and Sirius red staining in serial sections of primary mouse tumour ( graft of FAK - WT or FAK - KD fibroblasts + tumours cells at 38 days after injection ) at the invasive front . Scale bar , 100 µm . Bottom : schematic organization of the cells ( tumour cells in brown , CAFs in yellow and non - transformed epithelial cells in beige ) .

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(A) Representative images of CK19 and α-SMA immunohistochemistry and Sirius red staining in serial sections of primary mouse tumour (graft of FAK-WT or FAK-KD fibroblasts + tumours cells at 38 days after injection) at the invasive front. Scale bar, 100 µm. Bottom: schematic organization of the cells (tumour cells in brown, CAFs in yellow and non-transformed epithelial cells in beige).


( B ) Representative images of ECM deposition ( stained using Alexa Fluor 488 succinimidyl ester ( NHS ) ) generated by FAK - WT or - KD activated fibroblasts during migration .

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(B) Representative images of ECM deposition (stained using Alexa Fluor 488 succinimidyl ester (NHS)) generated by FAK-WT or -KD activated fibroblasts during migration.


( C ) Quantification of collagen I gel contraction ( calculated based on photographed gels using the formula 100 * ( well diameter - gel diameter ) / well diameter ) induced by fibroblasts in presence or not of tumour cells , or by hCAF in presence or not of FAK inhibitor ( FAK - I , 1µM )

O O O O O B-SUBCELLULAR I-SUBCELLULAR B-EXP_ASSAY I-EXP_ASSAY 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-CELL_TYPE O O O O O O O O O O B-CELL_TYPE O O O O O B-GENEPROD O O B-GENEPROD O O O O O

(C) Quantification of collagen I gel contraction (calculated based on photographed gels using the formula 100*(well diameter - gel diameter)/well diameter) induced by fibroblasts in presence or not of tumour cells , or by hCAF in presence or not of FAK inhibitor (FAK-I, 1µM)


( D ) Quantification of the percentage change of Y397 FAK , total FAK , collagen I ( coll I ) , collagen III ( coll III ) , collagen IV ( coll IV ) , LOXL2 , periostin ( POSTN ) , osteopontin ( OPN ) expression by human CAFs treated or not ( NT ) with FAK inhibitor ( 1µM ) , based on immunofluorescence analyses . Values are means ± SEM obtained from at least 5 primary hCAFs , isolated from 5 to 12 different fresh human PDAC samples .

O O O O O O O O O O B-GENEPROD O O B-GENEPROD O B-SUBCELLULAR I-SUBCELLULAR O B-SUBCELLULAR I-SUBCELLULAR O O B-SUBCELLULAR I-SUBCELLULAR O B-SUBCELLULAR I-SUBCELLULAR O O B-SUBCELLULAR I-SUBCELLULAR O B-SUBCELLULAR I-SUBCELLULAR O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O B-GENEPROD O B-GENEPROD O B-EXP_ASSAY O B-ORGANISM B-CELL_TYPE O O O O O O O B-GENEPROD O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O B-CELL_TYPE O O O O O O O O B-ORGANISM B-DISEASE O O

(D) Quantification of the percentage change of Y397 FAK, total FAK, collagen I (coll I), collagen III (coll III), collagen IV (coll IV), LOXL2, periostin (POSTN), osteopontin (OPN) expression by human CAFs treated or not (NT) with FAK inhibitor (1µM), based on immunofluorescence analyses. Values are means ± SEM obtained from at least 5 primary hCAFs, isolated from 5 to 12 different fresh human PDAC samples.


( E ) Collagen I , III and IV and periostin deposition are shown using heat map of the IF staining , and cell shapes are identified by white line .

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(E) Collagen I, III and IV and periostin deposition are shown using heat map of the IF staining, and cell shapes are identified by white line.


Left : Normalized peptide abundance for collagens from hCAFs ( each CAF duplicate is represented , CAF1 in blue , 2 in red and 3 in black ) treated or not with FAK inhibitor . Right : Heatmap of the collagen modifications in the 3 hCAFs upon FAK inhibition in hCAFs . The value of log2 fold - change between FAK inhibitor treated CAFs and non - treated CAFs for each protein was indicated by the colored scale , with red indicating increased expression while blue implies decreased expression . For each CAFs and each protein , log2 fold - change was calculated based on 2 technical replicates per

O O O B-EXP_ASSAY I-EXP_ASSAY O B-SUBCELLULAR O B-CELL_TYPE O O B-CELL_TYPE O O O O B-CELL_TYPE O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O B-SUBCELLULAR O O O O B-CELL_TYPE O B-GENEPROD O O B-CELL_TYPE O O O O O O O O O B-GENEPROD O O B-CELL_TYPE O O O O B-CELL_TYPE O O O O O O O O O O O O O O B-EXP_ASSAY O O O O B-EXP_ASSAY O O O B-CELL_TYPE O O O O O O O O O O O O O O O O

Left: Normalized peptide abundance for collagens from hCAFs (each CAF duplicate is represented, CAF1 in blue, 2 in red and 3 in black) treated or not with FAK inhibitor. Right: Heatmap of the collagen modifications in the 3 hCAFs upon FAK inhibition in hCAFs. The value of log2 fold-change between FAK inhibitor treated CAFs and non-treated CAFs for each protein was indicated by the colored scale, with red indicating increased expression while blue implies decreased expression. For each CAFs and each protein, log2 fold-change was calculated based on 2 technical replicates per condition


Left : Normalized peptide abundance for proteoglycans ( H ) from hCAFs ( each CAF duplicate is represented , CAF1 in blue , 2 in red and 3 in black ) treated or not with FAK inhibitor . Right : Heatmap of the proteoglycan ( H ) modifications in the 3 hCAFs upon FAK inhibition in hCAFs . The value of log2 fold - change between FAK inhibitor treated CAFs and non - treated CAFs for each protein was indicated by the colored scale , with red indicating increased expression while blue implies decreased expression . For each CAFs and each protein , log2 fold - change was calculated based on 2 technical replicates per

O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O B-CELL_TYPE O O B-CELL_TYPE O O O O B-CELL_TYPE O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O B-CELL_TYPE O B-GENEPROD O O B-CELL_TYPE O O O O O O O O O B-GENEPROD O O B-CELL_TYPE O O O O B-CELL_TYPE O O O O O O O O O O O O O O B-EXP_ASSAY O O O O B-EXP_ASSAY O O O B-CELL_TYPE O O O O O O O O O O O O O O O O

Left: Normalized peptide abundance for proteoglycans (H) from hCAFs (each CAF duplicate is represented, CAF1 in blue, 2 in red and 3 in black) treated or not with FAK inhibitor. Right: Heatmap of the proteoglycan (H) modifications in the 3 hCAFs upon FAK inhibition in hCAFs. The value of log2 fold-change between FAK inhibitor treated CAFs and non-treated CAFs for each protein was indicated by the colored scale, with red indicating increased expression while blue implies decreased expression. For each CAFs and each protein, log2 fold-change was calculated based on 2 technical replicates per condition


( I ) Normalized peptide abundance for glycoproteins from hCAFs treated or not with FAK - I .

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(I) Normalized peptide abundance for glycoproteins from hCAFs treated or not with FAK-I.


( J ) Heatmap of the glycoprotein modifications upon FAK - I in hCAFs .

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(J) Heatmap of the glycoprotein modifications upon FAK-I in hCAFs.


( K ) Normalized peptide abundance for " matrisome - associated " protein division : ECM - affiliated proteins , Secreted factors , and ECM regulators from hCAFs treated or not with FAK inhibitor .

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(K) Normalized peptide abundance for "matrisome-associated" protein division: ECM-affiliated proteins, Secreted factors, and ECM regulators from hCAFs treated or not with FAK inhibitor.


( L ) Left : Representative pictures of β1 integrin activation ( red ) of tumour cells , upon adhesion to either ECM deposited from non - treated CAFs or treated with FAK - I for 7 days , analyzed by IF . Merge : activated β1 integrin ( red ) , phalloidin ( green ) , dapi ( blue ) . Right : quantification of the number of integrin β1 activated cluster per cells and size of those clusters .

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(L) Left: Representative pictures of β1 integrin activation (red) of tumour cells, upon adhesion to either ECM deposited from non-treated CAFs or treated with FAK-I for 7 days, analyzed by IF. Merge: activated β1 integrin (red), phalloidin (green), dapi (blue). Right: quantification of the number of integrin β1 activated cluster per cells and size of those clusters.


Flow cytometric analysis of splenocytes of mice immunized with cInsulin or control immunization on day 26 . Left and middle panel showing germinal center ( GC ) B cells ( GL - 7 + ) pre - gated on B cells ( CD19 + B220 + ) . Right panel showing insulin - reactive GC B cells pre - gated on GL - 7 + cells in a histogram . Data are representative of two independent experiments with n = 4 / group .

B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-CELL_TYPE O B-ORGANISM O O B-SMALL_MOLECULE O O O O O O O O O O O O B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE I-CELL_TYPE I-CELL_TYPE I-CELL_TYPE I-CELL_TYPE O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O B-CELL_TYPE I-CELL_TYPE O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O O O O B-SMALL_MOLECULE O O B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O

Flow cytometric analysis of splenocytes of mice immunized with cInsulin or control immunization on day 26. Left and middle panel showing germinal center (GC) B cells (GL-7+) pre-gated on B cells (CD19+ B220+). Right panel showing insulin-reactive GC B cells pre-gated on GL-7+ cells in a histogram. Data are representative of two independent experiments with n=4/group.


Coomassie stained SDS - page showing purified serum IgG of cInsulin immunized ( red ) and CI mice ( grey ) under reducing ( + β - ME ) , and non - reducing conditions . Representative of two independent experiments .

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Coomassie stained SDS-page showing purified serum IgG of cInsulin immunized (red) and CI mice (grey) under reducing (+ β-ME), and non-reducing conditions. Representative of two independent experiments.


Blood glucose levels of intravenously ( i . v . ) injected wild - type mice at indicated hours post injection . 20 µg of purified serum IgG from cInsulin immunized mice ( red , n = 6 ) or CI mice ( grey , n = 5 ) was used for injection . Dots represent individual mice . Mean ± SD , statistical significance was calculated by using repeated measure ANOVA test , * * * P < 0 . 001 , * * * * P < 0 . 0001 .

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Blood glucose levels of intravenously (i.v.) injected wild-type mice at indicated hours post injection. 20 µg of purified serum IgG from cInsulin immunized mice (red, n=6) or CI mice (grey, n=5) was used for injection. Dots represent individual mice. Mean ± SD, statistical significance was calculated by using repeated measure ANOVA test, ***P < 0.001, ****P < 0.0001.


Serum anti - Insulin - immunoglobulin titers of mice immunized with InsA - peptide ( red : 0 : 1 , n = 10 and green : 100 : 1 , n = 5 ) and control - immunization ( CI : CpG only , grey , n = 3 ) measured by ELISA at indicated days ( coating : Insulin ) . Dots represent individual mice . Mean ± SD , statistical significance was calculated by using Kruskal - Wallis test , * P < 0 . 05 , * * * P < 0 . 001 .

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Serum anti-Insulin-immunoglobulin titers of mice immunized with InsA-peptide (red: 0:1, n=10 and green: 100:1, n=5) and control-immunization (CI: CpG only, grey, n=3) measured by ELISA at indicated days (coating: Insulin). Dots represent individual mice. Mean ± SD, statistical significance was calculated by using Kruskal-Wallis test, *P < 0.05, ***P < 0.001.


Western blot analysis of insulin - specific serum IgG derived from InsA - peptide immunized mice . Top panel ( green ) : 100 : 1 serum , lower panel ( red ) : 0 : 1 serum ( sInsA : cInsA ) . Serum of mice was used as primary antibody and detection was done with anti - mouse - IgG - HRP . Black filled arrow : Proinsulin ( 12 kD ) , Black non - filled arrow : Insulin ( 6 kD ) , β - actin ( 42 kD , loading control ) . Representative of two independent experiments .

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Western blot analysis of insulin-specific serum IgG derived from InsA-peptide immunized mice. Top panel (green): 100:1 serum, lower panel (red): 0:1 serum (sInsA:cInsA). Serum of mice was used as primary antibody and detection was done with anti-mouse-IgG-HRP. Black filled arrow: Proinsulin (12 kD), Black non-filled arrow: Insulin (6 kD), β-actin (42 kD, loading control). Representative of two independent experiments.


Blood glucose levels of InsA - peptide immunized ( red : 0 : 1 , n = 5 for E and n = 10 for F ; green : 100 : 1 , n = 5 and n = 4 for F , d30 , d32 ) and CI ( grey , n = 5 for E and n = 10 for F ) mice were assessed at indicated days . Dots represent individual mice . Mean ± SD , statistical significance was calculated by using Kruskal - Wallis test ( E ) and repeated measure ANOVA test ( F ) , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 .

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Blood glucose levels of InsA-peptide immunized (red: 0:1, n=5 for E and n=10 for F; green: 100:1, n=5 and n=4 for F, d30, d32) and CI (grey, n=5 for E and n=10 for F) mice were assessed at indicated days. Dots represent individual mice. Mean ± SD, statistical significance was calculated by using Kruskal-Wallis test (E) and repeated measure ANOVA test (F), *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.


Urine glucose levels of InsA peptide immunized ( red : n = 10 for d28 , d32 and n = 8 for d30 ; green : n = 5 for d28 , n = 4 for d30 , n = 3 for d32 ) and CI ( grey , n = 3 ) mice were monitored at indicated days post immunization . Dots represent individual mice . For 100 : 1 : representative data are shown for two independent experiments with total n = 10 . Mean ± SD , statistical significance was calculated by using repeated measure ANOVA test , * * * P < 0 . 001 .

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Urine glucose levels of InsA peptide immunized (red: n=10 for d28, d32 and n=8 for d30; green: n=5 for d28, n=4 for d30, n=3 for d32) and CI (grey, n=3) mice were monitored at indicated days post immunization. Dots represent individual mice. For 100:1: representative data are shown for two independent experiments with total n=10. Mean ± SD, statistical significance was calculated by using repeated measure ANOVA test, ***P < 0.001.


( C , F ) Urine glucose levels of InsA peptide immunized ( n = 3 ) and CI ( n = 3 ) mice were monitored at indicated days post immunization . Dots represent individual mice . Mean ± SD , statistical significance was calculated by using repeated measure ANOVA test , * P < 0 . 05 . ( D , E ) Serum anti - Insulin - immunoglobulin titers of mice immunized with InsA - peptide ( day 59 , day 78 : n = 3 , day 49 : n = 7 ) and control - immunization ( CI : CpG only , n = 3 ) measured by ELISA at indicated days ( coating : Insulin ) . Dots represent individual mice . Mean ± SD , statistical significance was calculated by using Mann - Whitney - U test ( D ) and repeated measure ANOVA test ( E ) . * P < 0 . 05 , * * P < 0 . 01 .

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(C, F) Urine glucose levels of InsA peptide immunized (n=3) and CI (n=3) mice were monitored at indicated days post immunization. Dots represent individual mice. Mean ± SD, statistical significance was calculated by using repeated measure ANOVA test, *P < 0.05. (D, E) Serum anti-Insulin-immunoglobulin titers of mice immunized with InsA-peptide (day 59, day 78: n=3, day 49: n=7) and control-immunization (CI: CpG only, n=3) measured by ELISA at indicated days (coating: Insulin). Dots represent individual mice. Mean ± SD, statistical significance was calculated by using Mann-Whitney-U test (D) and repeated measure ANOVA test (E). *P < 0.05, **P < 0.01.


Anti - Insulin affinity of PR - IgM ( blue ) , primary IgM ( red ) and isotype control ( black ) measured by bio - layer interferometry . IgM binding to insulin was acquired in pm and used to calculate the dissociation constant shown in the graph ( Kd = 1 / Ka ) . Graph is showing antigen - antibody association phase . Data are representative for three independent experiments .

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Anti-Insulin affinity of PR-IgM (blue), primary IgM (red) and isotype control (black) measured by bio-layer interferometry. IgM binding to insulin was acquired in pm and used to calculate the dissociation constant shown in the graph (Kd = 1/Ka). Graph is showing antigen-antibody association phase. Data are representative for three independent experiments.


Blood glucose levels of intravenously injected mice with purified IgM from control immunization ( CI , n = 5 ) , complex InsA peptide ( cInsA ) immunization day 7 ( total IgM d7 , n = 4 ) and day 85 ( total IgM d85 , n = 5 ) . Mean ± SD , statistical significance was calculated by using repeated measure ANOVA test showing comparison of red to black line , * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 .

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Blood glucose levels of intravenously injected mice with purified IgM from control immunization (CI, n=5), complex InsA peptide (cInsA) immunization day 7 (total IgM d7, n=4) and day 85 (total IgM d85, n=5). Mean ± SD, statistical significance was calculated by using repeated measure ANOVA test showing comparison of red to black line, **P < 0.01, ***P < 0.001, ****P < 0.0001.


Anti - nuclear - IgM ( ANA ) of control - immunized ( CI , n = 3 ) and complex InsA peptide ( cInsA ) immunized mice on day 7 ( n = 3 ) and day 85 ( n = 3 ) analyzed by indirect immunofluorescence . We used ( E ) total serum ( dilutions : 1 : 20 ) or ( G ) Insulin - specific IgM ( concentration : 500 ng / mL ) purified from serum at indicated days on commercial HEp - 2 slides . Scale bar : 10 µm . Green fluorescence indicates IgM bound to nuclear structures . Data are representative for three independent experiments with isotype control : n = 3 , day 7 : n = 3 , day 85 : n = 3 .

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Anti-nuclear-IgM (ANA) of control-immunized (CI, n=3) and complex InsA peptide (cInsA) immunized mice on day 7 (n=3) and day 85 (n=3) analyzed by indirect immunofluorescence. We used (E) total serum (dilutions: 1:20) or (G) Insulin-specific IgM (concentration: 500 ng/mL) purified from serum at indicated days on commercial HEp-2 slides. Scale bar: 10 µm. Green fluorescence indicates IgM bound to nuclear structures. Data are representative for three independent experiments with isotype control: n=3, day 7: n=3, day 85: n=3.


Coomassie stained SDS - page showing primary ( cInsA d7 ) and memory ( cInsA d85 ) Insulin - specific purified IgM pre - incubated with Insulin and calf thymus dsDNA . Samples were loaded onto the gel after size exclusion with a cut - off at 10 . 000 kD ( fractions referring to > / < 104 kD ) . IgM heavy chain ( HC ) : 69 kD , IgM light chain ( LC ) : 25 kD , J - chain : 15 kD . Data shown are representative of three independent experiments .

B-SMALL_MOLECULE B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O B-SMALL_MOLECULE O O O B-SUBCELLULAR O O O O B-SMALL_MOLECULE O B-ORGANISM B-TISSUE O O O O O O O O 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-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O B-SUBCELLULAR O O O O O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O B-SUBCELLULAR O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O

Coomassie stained SDS-page showing primary (cInsA d7) and memory (cInsA d85) Insulin-specific purified IgM pre-incubated with Insulin and calf thymus dsDNA. Samples were loaded onto the gel after size exclusion with a cut-off at 10.000 kD (fractions referring to >/< 104 kD). IgM heavy chain (HC): 69 kD, IgM light chain (LC): 25 kD, J-chain: 15 kD. Data shown are representative of three independent experiments.


Sashimi plots of representative SRSF3 - dependent exons . C2C12 cells were treated with control siRNA ( siCont ) , Srsf3 siRNA ( siSrsf3 ) , Hnrnpk siRNA ( siHnrnpk ) , or both Srsf3 and Hnrnpk siRNAs ( siSrsf3 + siHnrnpk ) , and RNA - seq was performed . Lines indicate junction - spanning reads . Gene structures are shown below the Sashimi plots .

O O O O B-GENEPROD O O O O B-CELL_LINE O O O O O O O O O O B-GENEPROD O O O O O B-GENEPROD O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O

Sashimi plots of representative SRSF3-dependent exons. C2C12 cells were treated with control siRNA (siCont), Srsf3 siRNA (siSrsf3), Hnrnpk siRNA (siHnrnpk), or both Srsf3 and Hnrnpk siRNAs (siSrsf3+siHnrnpk), and RNA-seq was performed. Lines indicate junction-spanning reads. Gene structures are shown below the Sashimi plots.


Volcano plot showing fold changes versus P - values of the normalized MASCOT scores ( see Materials and Methods ) of identified proteins between 3XFLAG - SRSF3 - IP and Cont - IP . Each experiment was triplicated . The members of the representative families of splicing factors are highlighted by colors ( right upper box ) . Volcano plot showing fold changes versus P - values of the normalized MASCOT scores of identified proteins between 3XFLAG - HNRNPK - IP and Cont - IP . Each experiment was triplicated . The members of the representative families of splicing factors are highlighted by colors ( right upper box ) .

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Volcano plot showing fold changes versus P-values of the normalized MASCOT scores (see Materials and Methods) of identified proteins between 3XFLAG-SRSF3-IP and Cont-IP. Each experiment was triplicated. The members of the representative families of splicing factors are highlighted by colors (right upper box).   Volcano plot showing fold changes versus P-values of the normalized MASCOT scores of identified proteins between 3XFLAG-HNRNPK-IP and Cont-IP. Each experiment was triplicated. The members of the representative families of splicing factors are highlighted by colors (right upper box).


mRNA expression levels of seven CPSF factors in C2C12 cells treated with the indicated siRNA normalized for those treated with the control siRNA . * * p < 0 . 01 by CuffDiff ( Trapnell et all . , 2010 ) .

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mRNA expression levels of seven CPSF factors in C2C12 cells treated with the indicated siRNA normalized for those treated with the control siRNA. **p < 0.01 by CuffDiff (Trapnell et all., 2010).


Western blots showing protein expression levels of CPSF6 and β - actin ( ACTB ) . Whole cell lysates were harvested 42 h after siRNA transfection .

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Western blots showing protein expression levels of CPSF6 and β-actin (ACTB). Whole cell lysates were harvested 42 h after siRNA transfection.


SRSF3 - responsive alternative 3 ′ ends of the Med1 gene . ( Top panel ) Disorder analysis for MED1 using PONDR VL - XT ( deep blue ) and IUPred2 ( light blue ) . The scores in the ordinate indicate disordered tendencies between 0 and 1 ( a score of more than 0 . 5 indicates disordered ) . ( Middle panel ) Protein and gene structures of MED1 . The protein structure was segmented into individual exonic regions . The SRSF3 - responsive last exon is indicated in pink . ( Bottom panel ) Sashimi plots of RNA - seqs from Srsf3 - and / or Hnrnpk - silenced cells .

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SRSF3-responsive alternative 3′ ends of the Med1 gene. (Top panel) Disorder analysis for MED1 using PONDR VL-XT (deep blue) and IUPred2 (light blue). The scores in the ordinate indicate disordered tendencies between 0 and 1 (a score of more than 0.5 indicates disordered). (Middle panel) Protein and gene structures of MED1. The protein structure was segmented into individual exonic regions. The SRSF3-responsive last exon is indicated in pink. (Bottom panel) Sashimi plots of RNA-seqs from Srsf3- and/or Hnrnpk-silenced cells.


Immunofluorescence images of MED1 in C2C12 cells treated with siCont ( C ) , siSrsf3 ( S ) , siHnrnpk ( K ) , or siSrsf3 + siHnrnpk ( S + K ) . A white dotted contour outlines the nucleus . Box plots ( right ) show quantification of nuclear ( NUC ) and cytoplasmic ( CYT ) localization of MED1 . The average intensity of one nucleus or a cytoplasmic segment is individually plotted .

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Immunofluorescence images of MED1 in C2C12 cells treated with siCont (C), siSrsf3 (S), siHnrnpk (K), or siSrsf3+siHnrnpk (S+K). A white dotted contour outlines the nucleus. Box plots (right) show quantification of nuclear (NUC) and cytoplasmic (CYT) localization of MED1. The average intensity of one nucleus or a cytoplasmic segment is individually plotted.


The intrinsically disordered region ( IDR ) in MED4 . Disorder analysis , protein and gene structures , and Sashimi plots are shown in ( B ) . Splicing of these genes was not affected by the siRNA treatment .

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The intrinsically disordered region (IDR) in MED4. Disorder analysis, protein and gene structures, and Sashimi plots are shown in (B). Splicing of these genes was not affected by the siRNA treatment.


Immunofluorescence images of MED4 in C2C12 cells treated with the indicated siRNAs . Box plots show quantification of nuclear localization of MED4 . Each dot represents one nucleus .

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Immunofluorescence images of MED4 in C2C12 cells treated with the indicated siRNAs. Box plots show quantification of nuclear localization of MED4. Each dot represents one nucleus.


Immunofluorescence images of MED1 and MED4 in C2C12 cells The arrows denote representative puncta , including both MED1 and MED4 .

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Immunofluorescence images of MED1 and MED4 in C2C12 cells The arrows denote representative puncta, including both MED1 and MED4.


The number of MED1 puncta ( left ) and MED4 puncta ( right ) per nucleus in cells treated with the indicated siRNA .

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The number of MED1 puncta (left) and MED4 puncta (right) per nucleus in cells treated with the indicated siRNA.


MED1 signals on MED4 puncta normalized to nuclear MED1 signals . MED1 signal intensity on each MED4 punctum was individually plotted .

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MED1 signals on MED4 puncta normalized to nuclear MED1 signals. MED1 signal intensity on each MED4 punctum was individually plotted.


( B ) Expression of S protein on the plasma membrane of Jurkat - S cells assessed by surface biotinylation and followed by immunoprecipitation with anti - T2A , SDS - PAGE under reducing conditions and western blot with streptavidin - peroxidase . Biotinylation of the parental Jurkat cells was carried out in parallel as a negative control . The arrow indicates the position of the reduced S protein . ( C ) Expression of the S protein in native form was assessed by surface biotinylation of Jurkat - S and Jurkat cells followed by immunoprecipitation with anti - T2A followed by SDS - PAGE under non - denaturing conditions ( i . e . without reducing agents and without boiling ) . The nitrocellulose membrane was blotted with streptavidin - peroxidase . Arrows point at different oligomerization forms of the S protein .

O O O B-EXP_ASSAY O B-GENEPROD I-GENEPROD O O B-SUBCELLULAR I-SUBCELLULAR O B-CELL_LINE O O O O O O O O O O B-EXP_ASSAY O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O B-EXP_ASSAY I-EXP_ASSAY O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O B-CELL_LINE O O O O O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD O O O O B-EXP_ASSAY O O B-GENEPROD I-GENEPROD O O O O O O O O O B-CELL_LINE O O O B-CELL_LINE O O O B-EXP_ASSAY O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O B-GENEPROD I-GENEPROD O

(B) Expression of S protein on the plasma membrane of Jurkat-S cells assessed by surface biotinylation and followed by immunoprecipitation with anti-T2A, SDS-PAGE under reducing conditions and western blot with streptavidin-peroxidase. Biotinylation of the parental Jurkat cells was carried out in parallel as a negative control. The arrow indicates the position of the reduced S protein. (C) Expression of the S protein in native form was assessed by surface biotinylation of Jurkat-S and Jurkat cells followed by immunoprecipitation with anti-T2A followed by SDS-PAGE under non-denaturing conditions (i.e. without reducing agents and without boiling). The nitrocellulose membrane was blotted with streptavidin-peroxidase. Arrows point at different oligomerization forms of the S protein.


( B ) Expression of S protein on the plasma membrane of Jurkat - S cells assessed by surface biotinylation and followed by immunoprecipitation with anti - T2A , SDS - PAGE under reducing conditions and western blot with streptavidin - peroxidase . Biotinylation of the parental Jurkat cells was carried out in parallel as a negative control . The arrow indicates the position of the reduced S protein .

O O O B-EXP_ASSAY O B-GENEPROD I-GENEPROD O O B-SUBCELLULAR I-SUBCELLULAR O B-CELL_LINE O O O O O O O O O O B-EXP_ASSAY O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O B-EXP_ASSAY I-EXP_ASSAY O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O B-CELL_LINE O O O O O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD O

(B) Expression of S protein on the plasma membrane of Jurkat-S cells assessed by surface biotinylation and followed by immunoprecipitation with anti-T2A, SDS-PAGE under reducing conditions and western blot with streptavidin-peroxidase. Biotinylation of the parental Jurkat cells was carried out in parallel as a negative control. The arrow indicates the position of the reduced S protein.