B C . HeLa were transfected with indicated si - RNAs for 24 h then exposed to hypoxia for 24 h . were then treated with or without 50 nM BAF A1 ( Bafilomycin A1 ) for 6 h before harvesting . lysates were immunoblotted with 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 E . of TIM23 : Actin TOM20 : Actin ratios from ( B C ) . 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 were transfected by different truncated FUNDC1 - MYC . About 24h post - transfection , 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 of FX in CD45 + leukocytes in peripheral blood of E0771 ( abbreviated to E ) or LLC tumour - bearing mice . mean sizes of E0771 LLC tumours were 9 . 8 mm 9 . 5 mm , respectively . Shown are averages ( N = 8 mice / group ) with 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 of FX in CD45 + leukocytes in various organs such as lung ( Lu ) , Liver ( Li ) , spleen ( Sp ) , bone marrow ( BM ) lymph node ( Lymph ) derived from no tumour - bearing or E0771 - bearing mice . " Tumour " stands for mRNA of FX in E0771 tumours . Shown are averages ( N = 6 : organs from no tumour - bearing mice , N = 6 : organs from E0771 - bearing mice ) with one - way

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(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 liver lungs of no tumour - bearing or E0771 - bearing mice . Shown are averages ( N = 6 ) with Welch " s t -

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(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 - tumour - bearing - mouse lungs . Shown are averages ( N = 7 / group , 14 random fields ) with . Welch " s t -

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(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 + fibrinogen deposition in tumour - bearing mouse lungs . Circles show high fibrinogen deposition areas ( scale bar , 50 μm )

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(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 were detected in TCM - stimulated - liver lungs after liver exposure to violet ( arrow ) . taken from with no exposure were also shown ( scale bar , 100 μm )

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(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 lungs . were isolated 72 h after photoconversion . was calculated as number of photoconverted ( KikGR redin comparison with number of liver or lung pre - sorted with CD45 - beads . Shown are averages ( N = 5 ) with Welch " s t -

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(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 . Vertical axes represent of each marker + / photoconverted KikGR . Shown are averages ( conCM , control : N = 4 , TCM : N = 9 ) with Welch " s t -

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(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 of FX expression in NK1 . 1 + , CD11c + , B220 + CD4 + in tumour - bearing lungs ( scale bar , 10 μm )

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(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 of B220 + CD11c + NK1 . 1 + in lung of TCM - stimulated mice . Values in dot plots present ratios for total gated KikGR red KikGR green

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

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(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 + derived from TCM - stimulating mouse liver . These 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 , it was used as control ( scale bar , 10 μm )

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(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 + . derived from liver spleen were primed with LiCM or LuCM . Shown are averages ( N = 6 / group ) with one - way ANOVA

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

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(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 ( scale bar , 10 μm )

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


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

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(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 no tumour - bearing livers

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


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

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


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

(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 + were isolated from E0771 - bearing wild - type or FX - / - mouse liver by using anti - Vtn beads ( 1 x 106 / mouse ) . Shown are averages ( N = 5 / group , 3 fields / sample ) with . Welch " s t -

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

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(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 )

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(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 + derived from tumour - bearing - FX + / − or FX − / − mouse liver . Sorted B220 + CD11c + NK1 . 1 + were prepared from three mice per each genotype , were i . v . injected . Shown are averages ( N = 6 / group , 4 sections / mouse ) with Welch " s t -

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

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(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 of FX in liver B220 + CD11c + NK1 . 1 + stimulated with various factors . Shown are averages ( N = 4 ) with 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


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

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(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 + were seeded on ECM coated ( fibrinogen , fibronectin , or collagen I ) plates . Shown are averages ( N = 5 / group ) with 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


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

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

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

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

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(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 of Definiens ® Tissue studio multi - resolution segmentation algorithm on two PDAC TMA examples ( X , Y ) . From left to right : for pY397 FAK ; ROI detection of tumour ( blue ) , stroma ( orange ) other ( blue / grey ) ; / cytoplasm detection in blue green respectively ; classification for pY397 FAK signal ( red for high signal , orange for medium signal , yellow for low signal 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 ) Overall Survival ( C ) from 120 cohort according to pY397 FAK expression in CAFs ; HR : Hazard . Survival curves were estimated with Kaplan - Meier method compared using log - rank , non - parametric ( chi2 - ) 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 ) Overall Survival ( E ) from 120 cohort according to pY397 FAK expression in tumour . Survival curves were estimated with Kaplan - Meier method compared using log - rank , non - parametric ( chi2 - ) 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.


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

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

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(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 ) of fibrillar collagen I ( Yellow - Orange ) III ( Green ) on polarized Sirius Red 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).


representative ( left ) ( right ) expression of CD31 ( D ) from 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.


representative ( left ) ( right ) expression of two hypoxia markers CaIX HIF1α ( E ) from 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 of H & E , CK19 α - SMA performed on lung sections . Arrow identified lung metastasis . ( G ) of lung metastasis number on CK19 positive using NanoZoomer software . Values are means ± of 6 mice per group .

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(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 M2 macrophages analyzed by flow cytometry at 21 days ( A ) 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 , CD163 markers acquired by flow cytometry processed using FlowJo software .

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(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 of M1 M2 macrophage migration at 24 48h towards CM secreted by CAFs ( previously treated or not with FAK inhibitor for 48h ) . were stained using Cristal Violet of cristal violet positive quantified using Observer videomicroscope ( Zeiss ) 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 M - CSF ) on four different FAK - I - treated ( 16h ) non - treated CAFs for gene expression . normalized fold change ± was calculated using delta - delta Ct method with RSP16 as a control gene ; values are mean + / - . Each - 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 of CCL2 ( MCP1 ) ( µg / ml ) in conditioned medium from hCAFs treated or not with FAK - I during 72h . * p < 0 . 05 , by paired t - ; values are mean + / - . Each - 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 of CD206 in serial sections of human PDAC tissues . Scale bar : 200µm Middle : Correlation plot between fibroblastic pY397 FAK score CD45 or CD206 + number within human PDAC samples . Fibroblastic pY397 FAK score of CD206 positive quantifications were performed on a 47 cohort on . Spearman r = 0 . 4145 , p = 0 . 0019 . Right : of CD206 positive in PDAC 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 of three independent scratch wound assays of co - cultivated red - labelled pancreatic tumour with either FAK - WT ( wild - type ) or FAK - KD ( Kinase - dead ) green - labelled fibroblasts at time zero 46 hours ( corresponding to wound closure of co - cultured FAK - WT fibroblasts with tumour ) .

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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).


migration ( has been analyzed in detail by tracking location of ( n > 16 per condition ) over time for fibroblasts or tumour ( 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 with a directionality equal to 1 is moving straightforward ) ( D ) migration ( E ) or tumour velocity ( F ) , directionality ( G ) ( H ) . Values are means ± of 16 tracked 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 invasion into collagen matrix from spheroid composed of green - labelled FAK - WT or FAK - KD fibroblasts plus red - labelled pancreatic tumour ( 1 / 1 ) . Shown are representative 3D reconstitution of spheroid invasion into 2mg / ml collagen I matrix obtained using Zen software . ( J - K ) of fibroblast ( J ) or tumour ( K ) invasion within collagen matrix . Values are means ± of 60 tracked from three independent experiments .

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(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 of CK19 α - SMA immunohistochemistry Sirius red in serial sections of mouse tumour ( graft of FAK - WT or FAK - KD fibroblasts + tumours at 38 days after injection ) at invasive front . Scale bar , 100 µm . Bottom : schematic organization of ( tumour in brown , CAFs in yellow non - transformed epithelial 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 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 ) of collagen I gel contraction ( calculated on photographed gels using formula 100 * ( well diameter - gel diameter ) / well diameter ) induced by fibroblasts in presence or not of tumour , or by hCAF in presence or not of FAK inhibitor ( FAK - I , 1µM )

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(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 ) of 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 ) , on immunofluorescence analyses . Values are means ± obtained from at least 5 hCAFs , isolated from 5 to 12 different fresh human PDAC samples .

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(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 IV periostin deposition are shown using heat map of IF , shapes are identified by .

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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 3 in black ) treated or not with FAK inhibitor . Right : of collagen modifications in 3 hCAFs upon FAK inhibition in hCAFs . value of log2 fold - change between FAK inhibitor treated CAFs non - treated CAFs for each was indicated by colored scale , with red indicating increased expression while blue implies decreased expression . For each CAFs each , log2 fold - change was calculated on 2 technical replicates per

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 3 in black ) treated or not with FAK inhibitor . Right : of proteoglycan ( H ) modifications in 3 hCAFs upon FAK inhibition in hCAFs . value of log2 fold - change between FAK inhibitor treated CAFs non - treated CAFs for each was indicated by colored scale , with red indicating increased expression while blue implies decreased expression . For each CAFs each , log2 fold - change was calculated on 2 technical replicates per

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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 ) of glycoprotein modifications upon FAK - I in hCAFs .

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


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

(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 , 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 : of number of integrin β1 activated cluster per 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 of splenocytes of mice immunized with cInsulin or control immunization on day 26 . Left middle panel showing germinal center ( GC ) B ( GL - 7 + ) pre - gated on B ( CD19 + B220 + ) . Right panel showing insulin - reactive GC B pre - gated on GL - 7 + in a histogram . Data are representative of two independent experiments with n = 4 / group .

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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 ) CI mice ( grey ) under reducing ( + β - ME ) , 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 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 mice . Mean ± SD , statistical significance was calculated by using repeated measure ANOVA , * * * 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 green : 100 : 1 , n = 5 ) control - immunization ( CI : CpG only , grey , n = 3 ) measured by ELISA at indicated days ( coating : Insulin ) . Dots represent mice . Mean ± SD , statistical significance was calculated by using Kruskal - Wallis , * 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 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 antibody 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 of InsA - peptide immunized ( red : 0 : 1 , n = 5 for E n = 10 for F ; green : 100 : 1 , n = 5 n = 4 for F , d30 , d32 ) CI ( grey , n = 5 for E n = 10 for F ) mice were assessed at indicated days . Dots represent mice . Mean ± SD , statistical significance was calculated by using Kruskal - Wallis ( E ) repeated measure ANOVA ( 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 of InsA peptide immunized ( red : n = 10 for d28 , d32 n = 8 for d30 ; green : n = 5 for d28 , n = 4 for d30 , n = 3 for d32 ) CI ( grey , n = 3 ) mice were monitored at indicated days post immunization . Dots represent 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 , * * * 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 of InsA peptide immunized ( n = 3 ) CI ( n = 3 ) mice were monitored at indicated days post immunization . Dots represent mice . Mean ± SD , statistical significance was calculated by using repeated measure ANOVA , * 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 ) control - immunization ( CI : CpG only , n = 3 ) measured by ELISA at indicated days ( coating : Insulin ) . Dots represent mice . Mean ± SD , statistical significance was calculated by using Mann - Whitney - U ( D ) repeated measure ANOVA ( 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 ) , IgM ( red ) isotype control ( black ) measured by bio - layer interferometry . IgM binding to insulin was acquired in pm used to calculate dissociation constant shown in 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 of intravenously injected mice with purified IgM from control immunization ( CI , n = 5 ) , InsA peptide ( cInsA ) immunization day 7 ( total IgM d7 , n = 4 ) day 85 ( total IgM d85 , n = 5 ) . Mean ± SD , statistical significance was calculated by using repeated measure ANOVA showing comparison of red to black , * * 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 - - IgM ( ANA ) of control - immunized ( CI , n = 3 ) InsA peptide ( cInsA ) immunized mice on day 7 ( n = 3 ) day 85 ( n = 3 ) analyzed by indirect immunofluorescence . We used ( E ) total serum ( dilutions : 1 : 20 ) or ( G ) Insulin - specific IgM ( : 500 ng / mL ) purified from serum at indicated days on commercial HEp - 2 slides . Scale bar : 10 µm . Green indicates IgM bound to 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 ( cInsA d7 ) memory ( cInsA d85 ) Insulin - specific purified IgM pre - incubated with Insulin calf thymus dsDNA . Samples were loaded onto gel after exclusion with a cut - off at 10 . 000 kD ( fractions referring to > / < 104 kD ) . IgM heavy chain ( HC ) : 69 kD , IgM chain ( LC ) : 25 kD , J - chain : 15 kD . Data shown are representative of three independent experiments .

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 O O O O O O O O O O O O O O O O O O O O O O O O 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 were treated with control siRNA ( siCont ) , Srsf3 siRNA ( siSrsf3 ) , Hnrnpk siRNA ( siHnrnpk ) , or both Srsf3 Hnrnpk siRNAs ( siSrsf3 + siHnrnpk ) , - seq was performed . Lines indicate junction - spanning reads . Gene structures are shown below Sashimi plots .

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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 normalized MASCOT scores ( see Materials Methods ) of identified between 3XFLAG - SRSF3 - IP Cont - IP . Each experiment was triplicated . members of representative families of splicing factors are highlighted by colors ( right upper box ) . Volcano plot showing fold changes versus P - values of normalized MASCOT scores of identified between 3XFLAG - HNRNPK - IP Cont - IP . Each experiment was triplicated . members of 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 of seven CPSF factors in C2C12 treated with indicated siRNA normalized for those treated with control siRNA . * * p < 0 . 01 by CuffDiff ( Trapnell et . , 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 expression of CPSF6 β - actin ( ACTB ) . Whole 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 Med1 gene . ( Top panel ) Disorder for MED1 using PONDR VL - XT ( deep blue ) IUPred2 ( blue ) . scores in ordinate indicate disordered tendencies between 0 1 ( a score of more than 0 . 5 indicates disordered ) . ( Middle panel ) gene structures of MED1 . structure was segmented into exonic regions . SRSF3 - responsive last exon is indicated in pink . ( Bottom panel ) Sashimi plots of - seqs from Srsf3 - / or Hnrnpk - silenced .

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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 of MED1 in C2C12 treated with siCont ( C ) , siSrsf3 ( S ) , siHnrnpk ( K ) , or siSrsf3 + siHnrnpk ( S + K ) . A dotted contour outlines nucleus . Box plots ( right ) show of ( NUC ) ( CYT ) localization of MED1 . average of one nucleus or a 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.


intrinsically disordered region ( IDR ) in MED4 . Disorder , gene structures , Sashimi plots are shown in ( B ) . Splicing of these genes was not affected by 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 of MED4 in C2C12 treated with indicated siRNAs . Box plots show of 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 of MED1 MED4 in C2C12 arrows denote representative , including both MED1 MED4 .

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


number of MED1 ( left ) MED4 ( right ) per nucleus in treated with indicated siRNA .

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


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

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


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

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

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(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.


( D ) Formation of syncytia between CTFR - labelled ACE2 + human CFSE - labelled Jurkat - S was measured by flow cytometry by analyzing of that become double positive for CTFR CFSE markers . Bar plot to right shows effect of different doses of HepG2 on formation of syncytia with a fixed number of Jurkat . Parental Jurkat ( not expressing S ) are considered negative controls . Data represent mean ± SD of triplicated datasets .

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(D) Formation of syncytia between the CTFR-labelled ACE2+ human cell line and the CFSE-labelled Jurkat-S cells was measured by flow cytometry by analyzing the percentage of cells that become double positive for CTFR and CFSE markers. The Bar plot to the right shows the effect of different doses of HepG2 cells on the formation of syncytia with a fixed number of Jurkat. Parental Jurkat cells (not expressing S protein) are considered negative controls. Data represent the mean±SD of triplicated datasets.


( E ) Formation of syncytia between CTFR - labelled ACE2 + human CFSE - labelled Jurkat - S , in an experiment as in D , was confirmed by confocal microscopy after sorting double positive for CTFR CFSE . Nuclei were identified by DAPI . Red arrowhead in DAPI image indicates a nucleus of Hep - G2 origin ; green arrowhead nucleus of Jurkat - S origin .

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(E) Formation of syncytia between the CTFR-labelled ACE2+ human cell line and the CFSE-labelled Jurkat-S cells, in an experiment as in D, was confirmed by confocal microscopy after sorting cells double positive for CTFR and CFSE. Nuclei were identified by DAPI staining. Red arrowhead in the DAPI image indicates a nucleus of Hep-G2 origin; the green arrowhead the nucleus of Jurkat-S origin.


( F ) Overlay plot of Jurkat - S that were incubated with anti - EGFR mAb conjugated to Bv421 serum from either donor # 15 or from a pre - COVID - 19 donor followed by a secondary anti - human IgG1 antibody conjugated to PE .

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(F) Overlay plot of Jurkat-S cells that were incubated with anti-EGFR mAb conjugated to Bv421 and serum from either donor #15 or from a pre-COVID-19 donor and followed by a secondary anti-human IgG1 antibody conjugated to PE.


( A ) Overlay histograms of Jurkat - S with different human sera diluted 1 : 50 . A pre - COVID19 serum sample is taken as negative control ( grey histogram ) .

(A) Overlay histograms of Jurkat-S staining with different human sera diluted 1:50. A pre-COVID19 serum sample is taken as negative control (grey histogram).


( B ) Bar plots of flow cytometry data generated with Jurkat - S panel of serum samples of Table EV1 classified according to their result in indicated commercial : green , positive for ; orange , weak or unclear ; magenta , negative samples . Flow cytometry data is expressed as between MFI of antibody anti - S MFI for EGFR . Negative values for flow cytometry are those with a S / EGFR MFI lower than 0 . 5 . This was set in order to fit most of data negative for other serological ( pink triangles ) under that threshold . Data represent mean ± s . e . m . datapoints are shown .

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(B) Bar plots of flow cytometry data generated with Jurkat-S cells and the panel of serum samples of Table EV1 classified according to their result in the indicated commercial tests: green, positive for the test; orange, weak or unclear; magenta, negative samples. Flow cytometry data is expressed as the ratio between the MFI of the antibody anti-S and the MFI for EGFR. Negative values for the flow cytometry test are those with a S/EGFR MFI ratio lower than 0.5. This ratio was set in order to fit most of the data negative for the other serological tests (pink triangles) under that threshold.Data represent the mean±s.e.m. All datapoints are shown.


( A ) Box whiskers plot of MFI absorbance data in human sera according to their clinical classification of COVID - 19 symptoms . Clinical classification was done according to following parameters : Asymptomatic , no symptoms ; Mild , 3 or more of following symptoms : non - productive cough , hyperthermia , headache , odynophagia , dyspnea , asthenia , myalgia , ageusia , anosmia , cutaneous involvement ; Moderate , 3 or more of above symptoms plus gastrointestinal symptoms , or more than 3 of above for 7 or more days ; Moderate - Severe , 3 or more of above symptoms plus pneumonia . Box whiskers are shown to represent minimum maximum values as well as median . datapoints are shown .

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(A) Box and whiskers plot of MFI and absorbance data in human sera according to their clinical classification of COVID-19 symptoms. Clinical classification was done according to the following parameters: Asymptomatic, no symptoms; Mild, 3 or more of the following symptoms: non-productive cough, hyperthermia, headache, odynophagia, dyspnea, asthenia , myalgia, ageusia, anosmia, cutaneous involvement; Moderate, 3 or more of the above symptoms plus gastrointestinal symptoms, or more than 3 of the above for 7 or more days; Moderate-Severe, 3 or more of the above symptoms plus pneumonia. Box and whiskers are shown to represent the minimum and maximum values as well as the median. All datapoints are shown.


( B ) Comparison of MFI versus absorbance data generated by flow cytometry ELISA for human sera . A lineal regression curve was adjusted , with a 95 % confidence interval , to data with R2 values indicated in plots . # 5Selected samples of outliers ( # 4 , # 5 , # 8 , # 31 , # 46 , # 48 , # 49 # 58 ) as well as samples close to a diagonal ( # 36 , # 52 # 15 ) are indicated .

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(B) Comparison of MFI versus absorbance data generated by flow cytometry and ELISA for all human sera. A lineal regression curve was adjusted, with a 95% confidence interval, to all data with the R2 values indicated in the plots. #5Selected samples of outliers (#4, #5, #8, #31, #46, #48, #49 and #58) as well as samples close to a diagonal (#36, #52 and #15) are indicated.


( C ) Titration of selected human sera by ELISA using S1 by flow cytometry with Jurkat - S . Samples with antibodies detected by flow cytometry not by ELISA are indicated ( # 8 , # 46 , # 48 , # 49 ) . Data represent mean ± SD of duplicates . An unpaired two - t - was carried out to compare results of 1 : 50 , 1 : 150 1 : 450 dilutions of sera # 8 , # 46 , # 48 # 49 by FC to pre - COVID - 1 sample as a negative control . * , p < 0 . 05 ; * * , p < 0 . 005 ; * * * , p < 0 . 0005 .

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(C) Titration of selected human sera by ELISA using the S1 protein and by flow cytometry with Jurkat-S cells. Samples with antibodies detected by flow cytometry and not by ELISA are indicated (#8, #46, #48, #49). Data represent the mean±SD of duplicates. An unpaired two-tailed t-test was carried out to compare the results of 1:50, 1:150 and 1:450 dilutions of sera #8, #46, #48 and #49 by FC to the pre-COVID-1 sample as a negative control. *, p<0.05; **, p<0.005; ***, p<0.0005.


( B ) Validation of neutralization method was carried out with HEK - 293T culture supernatants containing lentiviral particles pseudotyped with either S or with VSV G . supernatants were mixed with sera from donor # 66 at indicated dilutions or from a pre - COVID sample as a control before addition onto HEK - 293T - ACE2 + cultures . No serum , refers to a control containing no human sera . Data represent mean ± SD of triplicates .

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(B) Validation of the neutralization method was carried out with cell HEK-293T cell culture supernatants containing lentiviral particles pseudotyped with either the S protein or with the VSV G protein. The supernatants were mixed with sera from donor #66 at the indicated dilutions or from a pre-COVID sample as a control before addition onto HEK-293T-ACE2+ cell cultures. No serum, refers to a control containing no human sera. Data represent the mean±SD of triplicates.


( C ) presence of neutralizing antibodies in sera from donors # 46 # 48 , found negative by ELISA positive by flow cytometry ( Fig . 3C ) was demonstrated as in panel B . Data represent mean ± SD of triplicates . * , p < 0 . 05 ; * * , p < 0 . 005 ; * * * * , p < 0 . 00005 ( Paired two - t - comparing serum dilutions to pre - COVID sample ) .

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(C) The presence of neutralizing antibodies in sera from donors #46 and #48, found negative by ELISA and positive by flow cytometry (Fig. 3C) was demonstrated as in panel B. Data represent the mean±SD of triplicates. *, p<0.05; **, p<0.005; ****, p<0.00005 (Paired two-tailed t-test comparing all serum dilutions to the pre-COVID sample).


( D ) Bar plot showing S / EGFR MFI determined by flow cytometry with Jurkat - S of serum samples from 30 sanitary personnel repeatedly tested as PCR negative for SARS - CoV - 2 at Ramón y Cajal Hospital of Madrid . A negative result is considered for a lower than 0 . 5 . Two clear cases of sera positive for anti - S IgG1 are indicated ( RyC52 RyC65 ) . A borderline sample just above threshold ( RyC58 ) is also indicated . Data show mean ± s . e . m . datapoints are shown .

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(D) Bar plot showing the S/EGFR MFI ratio determined by flow cytometry analysis with Jurkat-S of serum samples from 30 sanitary personnel repeatedly tested as PCR negative for SARS-CoV-2 at the Ramón y Cajal Hospital of Madrid. A negative result is considered for a ratio lower than 0.5. Two clear cases of sera positive for anti-S IgG1 are indicated (RyC52 and RyC65). A borderline sample just above the threshold line (RyC58) is also indicated. Data show the mean±s.e.m. All datapoints are shown.


( F ) Plot of S / EGFR MFI for 30 RyC samples versus a Score generated according to slope of S / EGFR shape ( see Materials Methods Fig . EV1 ) . green indicates threshold point ( 0 . 024 ) for Score above which a serum is considered positive .

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(F) Plot of the S/EGFR MFI ratio for the 30 RyC samples versus a Score generated according to the slope of S/EGFR and shape (see Materials and Methods and Fig. EV1). The green line indicates the threshold point (0.024) for the Score above which a serum is considered positive.


( G ) presence of neutralizing antibodies in sera from donors RyC46 , RyC52 , RyC56 RyC58 diluted 1 / 15 was demonstrated as in panel B . Data represent mean ± SD of triplicates . * , p < 0 . 05 ; * * , p < 0 . 005 ; * * * * , p < 0 . 00005 ( Unpaired two - t - comparing each serum to No serum sample )

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(G) The presence of neutralizing antibodies in sera from donors RyC46, RyC52, RyC56 and RyC58 diluted 1/15 was demonstrated as in panel B. Data represent the mean±SD of triplicates. *, p<0.05; **, p<0.005; ****, p<0.00005 (Unpaired two-tailed t-test comparing each serum to the No serum sample)


( A ) A total of 52 serum samples from Hospital Universitario La Princesa ( HUP ) 52 samples of pre - COVID donors stored at CBMSO were tested by flow cytometry method classified according to S / EGFR - Score . Of 52 HUP samples , a total of 40 were from donors testing positive by PCR by an ELISA , 6 were from donors testing negative by PCR but positive by ELISA , 1 from an testing negative by ELISA positive by PCR , 5 were not tested by PCR . Box whiskers are shown to represent minimum maximum values as well as median . datapoints are shown . broken indicates threshold for a Score value of 0 . 024 .

(A) A total of 52 serum samples from Hospital Universitario La Princesa (HUP) and 52 samples of pre-COVID donors stored at the CBMSO were tested by the flow cytometry method and classified according to the S/EGFR-based Score. Of the 52 HUP samples, a total of 40 were from donors testing positive by PCR and by an ELISA test, 6 were from donors testing negative by PCR but positive by ELISA, 1 from an individual testing negative by ELISA and positive by PCR, and 5 were not tested by PCR. Box and whiskers are shown to represent the minimum and maximum values as well as the median. All datapoints are shown. The broken line indicates the threshold for a Score value of 0.024.


C ) Four HUP serum samples giving discrepant results by flow cytometry method were tested in neutralization of pseudotyped lentivirus ( Fig . 4A ) . Data represent mean ± SD of duplicates . * , p < 0 . 05 ; * * , p < 0 . 005 ; * * * * , p < 0 . 00005 ; ( Paired two - t - comparing serum dilutions to pre - COVID sample )

C) Four HUP serum samples giving discrepant results by flow cytometry method were tested in the neutralization assay of pseudotyped lentivirus (Fig. 4A). Data represent the mean±SD of duplicates. *, p<0.05; **, p<0.005; ****, p<0.00005; (Paired two-tailed t-test comparing all serum dilutions to the pre-COVID sample)


( A ) A collection of serum samples from volunteers working at CBMSO were taken in months of June October 2020 tested by flow cytometry using Jurkat - S at indicated dilutions . Flow cytometry data are shown as S / EGFR MFI . Red arrows indicate a loss in titer of antibodies between June October whereas blue arrows indicate an increase in antibody titer .

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(A) A collection of serum samples from volunteers working at the CBMSO were taken in the months of June and October 2020 and tested by flow cytometry using Jurkat-S cells at the indicated dilutions. Flow cytometry data are shown as the S/EGFR MFI ratio. Red arrows indicate a loss in titer of antibodies between June and October whereas blue arrows indicate an increase in antibody titer.


( B ) A neutralization with S - pseudotyped lentivirus was carried out to resolve discrepancies . Data represent mean ± SD of duplicates . * , p < 0 . 05 ; * * , p < 0 . 005 ; * * * , p < 0 . 0005 ; * * * * * , p < 0 . 000005 ( Paired two - t - comparing serum dilutions to pre - COVID sample ) .

(B) A neutralization assay with S protein-pseudotyped lentivirus was carried out to resolve the discrepancies. Data represent the mean±SD of duplicates. *, p<0.05; **, p<0.005; ***, p<0.0005; *****, p<0.000005 (Paired two-tailed t-test comparing all serum dilutions to the pre-COVID sample).


( C ) Classification of CBMSO cohort according to Score generated from S / EGFR data . Broken indicates 0 . 024 threshold above which samples are considered positive .

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(C) Classification of the CBMSO cohort according to the Score generated from S/EGFR data. The Broken line indicates the 0.024 threshold above which samples are considered positive.


( D ) Stretching of PDMS sandwich concomitantly stretches wing disc inside microchannel ( top - down view ) ; wing disc resting on a stretching device ( anchor ) 10 min after stretch ; M = microchannel .

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(D) Stretching of the PDMS sandwich concomitantly stretches the wing disc inside the microchannel (top-down view); wing disc resting on a stretching device (anchor) and 10 min after stretch; M=microchannel.


( E ) zipper RNAi clones ( zip iR , in red ) in Arm : : GFP expressing anchored stretched third instar wing disc ; lower panel shows color coded aspect in anchored stretched discs ( bright colors indicate high elongation ) ; zip RNAi clone is outlined in .

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(E) zipper RNAi clones (zip iR, in red) in Arm::GFP expressing anchored and stretched third instar wing disc; lower panel shows color coded cell aspect ratio in anchored and stretched discs (bright colors indicate high elongation); zip RNAi clone is outlined in white.


( F ) Box plot showing distribution of aspect for control ( wild - type ) zipper RNAi in anchored stretched ( 20 min ) wing disc ; median represented by horizontal , 75th 25th centiles are represented by top bottom of boxes respectively ; n = 8 wing discs .

(F) Box plot showing distribution of cell aspect ratio for control (wild-type) and zipper RNAi cells in anchored and stretched (20 min) wing disc; median represented by horizontal line, 75th and 25th centiles are represented by top and bottom of the boxes respectively; n=8 wing discs.


( G ) Color - coded aspect in control zip RNAi ( zip RNAi clone outlined in ) subjected to anchoring , stretching ( 20 min ) relaxation ( 10 min , post stretch ) .

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(G) Color-coded aspect ratio in control and zip RNAi cells (zip RNAi clone outlined in white) subjected to anchoring, stretching (20 min) and relaxation (10 min, post stretch).


( H ) Box plot showing distribution of aspect in anchored , stretched ( 20 min ) relaxed ( 10 min ) wing disc for control ( wild - type ) zipper RNAi ; median represented by horizontal ; 75th 25th centiles are represented by top bottom of boxes respectively , n = 3 wing discs .

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(H) Box plot showing distribution of cell aspect ratio in anchored, stretched (20 min) and relaxed (10 min) wing disc for control (wild-type) and zipper RNAi cells; median represented by horizontal line; 75th and 25th centiles are represented by top and bottom of the boxes respectively, n=3 wing discs.


( I ) zip - RNAi clone in Arm : : GFP wing disc at beginning ( 5 min ) at end ( 40 min ) of stretching experiment . Yellow red arrows indicate control zip RNAi respectively .

(I) zip-RNAi clone in Arm::GFP wing disc at the beginning (5 min) and at the end (40 min) of stretching experiment. Yellow and red arrows indicate the control and zip RNAi cells respectively.


( J ) Change of zip - RNAi aspect ( a . r . ) relative to change in control aspect ( a . r . ) in course of stretch , blue shows fitted regression ; dotted represents 95 % confidence bounds of best fit ; n = 3 wing discs .

(J) Change of zip-RNAi aspect ratio (a.r.) relative to change in control cells aspect ratio (a.r.) in the course of stretch, blue line shows fitted regression; dotted line represents 95% confidence bounds of the best fit line; n=3 wing discs.


( A ) Wing disc expressing E - cad : : GFP Sqh : : mCherry prior to stretch ( anchor ) after stretch ( 15 min ) . ( A " ) , Insets of discs shown in ( A ) . ( B ) Schematics demonstrating change in Sqh before after stretch on vertical ( V ) horizontal ( H )

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(A) Wing disc expressing E-cad::GFP and Sqh::mCherry prior to stretch (anchor) and after stretch (15 min).
(A"), Insets of discs shown in (A).
(B) Schematics demonstrating change in Sqh concentration before and after stretch on vertical (V) and horizontal (H) junctions


( C ) of mean of E - cad : : GFP Sqh : : mCherry on horizontal ( H ) junctions relative to on vertical ( V ) junctions ( referred to as " polarity " ) prior to after 15 min stretch . detailed rationale of determination is described in Fig . S3 Experimental Procedures .

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(C) Quantification of mean fluorescent intensity of E-cad::GFP and Sqh::mCherry on horizontal (H) junctions relative to the intensity on vertical (V) junctions (referred to as "polarity") prior to and after 15 min stretch. The detailed rationale of intensity determination is described in Fig. S3 and Experimental Procedures.


( D - E ) , of E - cad : : GFP ( D ) Sqh : : mCherry ( E ) ( i . e . mean per junctional unit ) prior after stretch on both horizontal vertical junctions normalized to starting on unstretched ( anchored ) vertical junctions .

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(D-E), Quantification of E-cad::GFP (D) and Sqh::mCherry (E) concentration (i.e. mean intensity per junctional unit area) prior and after stretch on both horizontal and vertical junctions normalized to starting concentration on unstretched (anchored) vertical junctions.


( F ) of total amount of Sqh : : mCherry E - cad : : GFP on vertical horizontal junctions in anchored stretched discs .

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(F) Quantification of total amount of Sqh::mCherry and E-cad::GFP fluorescence on vertical and horizontal junctions in anchored and stretched discs.


( G ) Tissue expressing E - cad : : GFP Sqh : : mCherry subjected to increasing of stretch .

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(G) Tissue expressing E-cad::GFP and Sqh::mCherry subjected to increasing levels of stretch.


( H ) of Sqh : : mCherry polarity ( as in c ) of experiment described in ( G ) . level of stretch is measured as a change in tissue ( deformation relative to unstretched tissue ) .

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(H) Quantification of Sqh::mCherry polarity (as in c) of experiment described in (G). The level of stretch is measured as a change in tissue strain (deformation relative to unstretched tissue).


( I - K ) Sqh : : mCherry polarity emergence as a function of aspect change ( I ) , stress ( J ) , stress applied on average of 10 μm2 ( K ) . Yellow asterisks indicate equivalent regions in tissue . N = 8 wing discs ( A - F ) , N = 3 wing discs ( G - K ) ;

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(I-K) Sqh::mCherry polarity emergence as a function of cell aspect ratio change (I), stress (J), stress applied on average cell area of 10 μm2 (K). Yellow asterisks indicate equivalent regions in the tissue. N=8 wing discs (A- F), N=3 wing discs (G-K);


( A ) Short ( 15 min ) stretch relaxation ( 10 min ) of E - cad : : GFP Sqh : : mCherry expressing discs .

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(A) Short (15 min) stretch and relaxation (10 min) of E-cad::GFP and Sqh::mCherry expressing discs.


( B ) of Sqh : : mCherry polarity , E - cad : : GFP polarity aspect change of experiment described in ( A ) ; schematics below graph illustrates quantified changes in aspect ( yellow ) Sqh polarity ( red ) . Statistical compares Sqh polarity between anchored stretched or stretched relaxed disc .

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(B) Quantification of Sqh::mCherry polarity, E-cad::GFP polarity and aspect ratio change of experiment described in (A); schematics below the graph illustrates quantified changes in cell aspect ratio (yellow) and Sqh polarity (red). Statistical test compares Sqh polarity between anchored and stretched or stretched and relaxed disc.


( C ) Long ( 200 min ) stretch relaxation ( 10 min ) of E - cad : : GFP Sqh : : mCherry expressing discs .

(C) Long (200 min) stretch and relaxation (10 min) of E-cad::GFP and Sqh::mCherry expressing discs.


( D ) of Sqh : : mCherry polarity , E - cad : : GFP polarity aspect change of experiment described in ( C ) ; schematics below graph illustrates quantified changes in aspect ( yellow ) Sqh polarity ( red ) .

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(D) Quantification of Sqh::mCherry polarity, E-cad::GFP polarity and aspect ratio change of experiment described in (C); schematics below the graph illustrates quantified changes in aspect ratio (yellow) and Sqh polarity (red).


( A ) Overview of E - cad : : GFP disc prior to ( anchor ) after compression .

(A) Overview of the E-cad::GFP disc prior to (anchor) and after compression.


( B ) E - cad : : GFP Sqh : : mCherry localization in anchored compressed wing disc .

(B) E-cad::GFP and Sqh::mCherry localization in anchored and compressed wing disc.


( C ) cuts ( perpendicular to of stretch ) across circa 10 in E - cad : : GFP expressing wing disc tissue subjected to anchoring , short ( < 20 min ) stretching short ( < 20 min ) compression . Plot shows increase in between recoiling short axis [ μm ] of fitted ellipse ( see Fig . S5A for details ) against time [ sec . ] ; data is plotted as mean ± S . E . M , n = 7 - 10 discs / condition .

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(C) Laser cuts (perpendicular to the line of stretch) across circa 10 cells in E- cad::GFP expressing wing disc tissue subjected to anchoring, short (<20 min) stretching and short (<20 min) compression. Plot shows increase in distance between recoiling short axis [μm] of the fitted ellipse (see Fig. S5A for details) against time [sec.]; data is plotted as mean ± S.E.M, n= 7-10 discs/condition.


( D ) Activated Myosin ( anti - phosphorylated - Sqh ; in red ) anti - E - cad ( green ) in compressed discs . ( D " ) Close up of compression experiment described in ( D ) ; yellow arrow points towards an example of polarized phosphorylated - Sqh cable in compressed discs .

(D) Activated Myosin (anti-phosphorylated-Sqh; in red) and anti-E-cad (green) staining in compressed discs. (D") Close up of compression experiment described in (D); yellow arrow points towards an example of polarized phosphorylated-Sqh cable in compressed discs.


( A ) cuts ( perpendicular to of stretch ) across circa 10 in E - cad : : GFP expressing wing disc tissue subjected to anchoring , short ( < 20 min ) long ( > 120 min ) stretching stretch retraction ( tissue forced back to original , pre - stretch shape .

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(A) Laser cuts (perpendicular to the line of stretch) across circa 10 cells in E- cad::GFP expressing wing disc tissue subjected to anchoring, short (<20 min) and long (>120 min) stretching and stretch retraction (tissue forced back to the original, pre-stretch shape.


( C ) An overlay of a junction prior to ( red ) 15 . 44 sec . after ablation ( green ) .

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(C) An overlay of a junction prior to (red) and 15.44 sec. after ablation (green).


( E ) cuts ( perpendicular or parallel to of stretch ) across circa 10 in E - cad : : GFP expressing wing disc tissue subjected to anchoring or short stretching ( < 30 min ) . .

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(E) Laser cuts (perpendicular or parallel to the line of stretch) across circa 10 cells in E-cad::GFP expressing wing disc tissue subjected to anchoring or short stretching (< 30 min)..


( H ) An overlay of anchored stretched discs prior to ablation ( red ) 15 . 44 seconds post ablation ( green ) . Inset zooms in on next to ablation site ( marked with yellow asterisk ) .

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(H) An overlay of anchored and stretched discs prior to ablation (red) and 15.44 seconds post ablation (green). Inset zooms in on the area next to ablation site (marked with yellow asterisk).


( A ) RokK116a : : Venus localization in anchored , stretched , relaxed third instar imaginal discs .

(A) RokK116a::Venus localization in anchored, stretched, relaxed third instar imaginal discs.


discs . ( B ) Drok2 mitotic clones ( RFP - ) in Sqh : : GFP third instar imaginal disc . Inset demonstrates Sqh : : GFP polarity inside Drok2 clone .

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discs.
(B) Drok2 mitotic clones (RFP-) in Sqh::GFP third instar imaginal disc. Inset demonstrates Sqh::GFP polarity inside Drok2 clone.


( C ) Sqh : : GFP , enGal4 > UAS - rok RNAi , UAS - Act5CRFP discs subjected to stretching ; inset demonstrates Sqh : : GFP polarity in rok RNAi ( marked by red .

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(C) Sqh::GFP, enGal4>UAS-rok RNAi, UAS-Act5CRFP discs subjected to stretching; inset demonstrates Sqh::GFP polarity in rok RNAi cells (marked by red.


( D ) zip RNAi clones ( labeled as zip iR , indicated in red ) in stretched third instar RokK116a : : Venus discs .

(D) zip RNAi clones (labeled as zip iR, indicated in red) in stretched third instar RokK116a::Venus discs.


( A ) Discs expressing Sqh : : GFP enGal4 > UAS - dia RNAi , UAS - Act5c - RFP that were stretched ; UAS - Act5C : : RFP marks dia iR ( posterior side ) in red . ( A " ) Insets comparing Sqh : : GFP polarity in stretchedenGal4 > dia iR , UAS - Act5c - RFP discs ; panel 1 refers to anterior ( ctrl ) side ; panel 2 refers to posterior ( dia iR ) side .

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(A) Discs expressing Sqh::GFP and enGal4>UAS-dia RNAi, UAS-Act5c-RFP that were stretched; UAS-Act5C::RFP marks dia iR (posterior side) in red. (A") Insets comparing Sqh::GFP polarity in stretchedenGal4>dia iR, UAS- Act5c-RFP discs; panel 1 refers to anterior (ctrl) side; panel 2 refers to posterior (dia iR) side.


( B ) Tissue marked with E - cad : : GFP Sqh : : mCherry , expressing dia - RNAi ( dia iR ) with hhGal4 driver subjected to stretching . Dotted indicates anterior - posterior ( A - P ) compartment boundary . ( B " ) Inset demonstrates lack of Sqh : : GFP polarity in dia - RNAi .

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(B) Tissue marked with E-cad::GFP and Sqh::mCherry, expressing dia-RNAi (dia iR) with hhGal4 driver and subjected to stretching. Dotted line indicates anterior-posterior (A-P) compartment boundary. (B") Inset demonstrates lack of Sqh::GFP polarity in dia-RNAi cells.


( C - D ) , of E - cad : : GFP polarity ( C ) Sqh : : mCherry polarity ( D ) in experiment described in ( B ) ; polarity is measured as mean on horizontal junctions relative to vertical junctions ( see Experimental Procedures for details ) ; n = 4 wing discs .

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(C-D), Quantification of E-cad::GFP polarity (C) and Sqh::mCherry polarity (D) in experiment described in (B); polarity is measured as mean fluorescent intensity on horizontal junctions relative to vertical junctions (see Experimental Procedures for details); n=4 wing discs.


( E ) Stretched discs expressing Sqh : : GFP enGal4 > UAS - cofilin ( upper panel ) or UAS - AIP1 ( lower panel ) RNAi UAS - Act5c - RFP ( marks posterior side in red ) . ( E " ) , Insets comparing Sqh : : GFP polarity in stretched discs described in ( E ) ; panel 1 refers to anterior ( ctrl ) side ; panel 2 refers to posterior ( dia iR ) side .

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(E) Stretched discs expressing Sqh::GFP and enGal4>UAS-cofilin (upper panel) or UAS-AIP1 (lower panel) RNAi and UAS-Act5c-RFP (marks posterior side in red).
(E"), Insets comparing Sqh::GFP polarity in stretched discs described in (E); panel 1 refers to anterior (ctrl) side; panel 2 refers to posterior (dia iR) side.


( F ) Sqh : : GFP polarity in enGal4 > dia iR , UAS - Act5C : : RFP discs labeled with Phalloidin - 647 ; Inset ( Phall - 647 ) zooms in on shape in anterior posterior disc compartments .

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(F) Sqh::GFP polarity in enGal4> dia iR, UAS-Act5C::RFP discs labeled with Phalloidin-647; Inset (Phall-647) zooms in on cell shape in anterior and posterior disc compartments.


( G ) Discs expressing nubGal4 Cd8 : : GFP ( control ) or dia iR ( KK ) labeled with DAPI ; ellipse is fitted into pouch region ( red dotted ) aspect determined as long ellipse axis ( b ) divided by short ellipse axis ( a ) ( lines ) .

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(G) Discs expressing nubGal4 and Cd8::GFP (control) or dia iR (KK) labeled with DAPI; ellipse is fitted into the pouch region (red dotted line) and aspect ratio determined as long ellipse axis (b) divided by short ellipse axis (a) (white lines).


( H ) Box plot showing distribution of wing disc pouch aspect ratios in conditions described in ( G ) , n = 19 ( ctrl ) 16 ( dia iR ) wing pouches ; horizontal indicates median .

(H) Box plot showing distribution of wing disc pouch aspect ratios in conditions described in (G), n=19 (ctrl) and 16 (dia iR) wing pouches; horizontal line indicates median.


( C ) Co - immunoprecipitation using BmN4 transfected with Flag - tagged DEAD - box helicases identified by shotgun proteomics . Flag - tagged Ago3 were detected by western blotting . Full - Flag - tagged are indicated with red asterisks . h . c . : heavy chain .

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(C) Co-immunoprecipitation using BmN4 cells transfected with the Flag-tagged DEAD-box helicases identified by shotgun proteomics. The Flag-tagged proteins and Ago3 were detected by western blotting. Full-length Flag-tagged proteins are indicated with red asterisks. h.c.: heavy chain.


( F , G ) of Siwi - piRNA Ago3 - piRNA in Siwi Ago3 complexes with DDX43 , respectively . WB ( western blotting ) NB ( northern blotting ) signals represent PIWI piRNA , respectively ( see also Figs EV1A EV1B ) . relative ratios of piRISC were calculated on signal intensities of PIWI piRNAs .

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(F, G) Analysis of Siwi-piRNA and Ago3-piRNA levels in Siwi and Ago3 complexes with DDX43, respectively. The WB (western blotting) and NB (northern blotting) signals represent the PIWI protein and piRNA levels, respectively (see also Figs EV1A and EV1B). The relative ratios of piRISC were calculated based on the signal intensities of the PIWI proteins and piRNAs.


( B ) Immunoprecipitation western blotting showing that DDX43 associated with Vret Ago3 .

(B) Immunoprecipitation and western blotting showing that DDX43 associated with Vret and Ago3.


( C , D ) Immunoprecipitation of endogenous Siwi , DDX43 , Vret bound with Ago3 from DDX43 - depleted ( C ) or Vret - depleted ( D ) . factors were detected by western blotting . Ago3 interacts with Vret DDX43 independently of other .

(C, D) Immunoprecipitation of endogenous Siwi, DDX43, and Vret bound with Ago3 from DDX43-depleted (C) or Vret-depleted (D) cells. The factors were detected by western blotting. Ago3 interacts with Vret and DDX43 independently of the other.


( D ) unwinding assays upon Ago3 - piRISC - dependent target cleavage Recombinant Vasa or DDX43 was added incubated in reaction mixture upon cleavage materials were separated using a magnet to supernatant ( Sup . ) bead ( Beads ) fractions . RNAs by Ago3 are shown as " " . DDX43 but not Vasa released target from Ago3 - piRISC .

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(D) RNA unwinding assays upon Ago3-piRISC-dependent target RNA cleavage Recombinant Vasa or DDX43 was added and incubated in the reaction mixture upon cleavage and the materials were separated using a magnet to supernatant (Sup.) and bead (Beads) fractions. RNAs cleaved by Ago3 are shown as "Cleaved RNA". DDX43 but not Vasa released the cleaved target RNA from Ago3-piRISC.