( 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 .

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

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

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O 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 ) of 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 " division : ECM - affiliated , Secreted factors , 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 , 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 .

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

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 .

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

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 .

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

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 .

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

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 .

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

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 .

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

(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 .

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

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 .

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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 .

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

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

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 .

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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 .

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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 normalized to MED1 signals . MED1 signal 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 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 ) .

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(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 .

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(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 ) .

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(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 .

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(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 .

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(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 .

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(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 .

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(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 .

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(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 .

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(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 .

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(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 .

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(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 .

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(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 .

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(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.


( F ) unwinding assays upon Ago3 - piRISC - dependent target cleavage as in ( D ) . Recombinant DDX43 was added incubated in reaction mixture upon cleavage for 0 min ( i . e . , no cleavage ) , 15 min , 30 min , 60 min , materials were separated using a magnet to supernatant ( Sup . ) bead ( Beads ) fractions . RNAs by Ago3 are shown as " " . DDX43 fails to unwind RNAs from Siwi - piRISC . − DDX43 means no DDX43 added in reaction mixture .

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(F) RNA unwinding assays upon Ago3-piRISC-dependent target RNA cleavage as in (D). Recombinant DDX43 was added and incubated in the reaction mixture upon cleavage for 0 min (i.e., no cleavage), 15 min, 30 min, and 60 min, 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 fails to unwind cleaved RNAs from Siwi-piRISC. − DDX43 means no DDX43 added in the reaction mixture.


( C ) Immunoprecipitation of Ago3 with Flag - tagged DDX43 WT its D399A E400Q mutants . Ago3 DDX43 were detected by western blotting using anti - Ago3 anti - Flag - antibodies , respectively . Flag - EGFP was used as a negative control . E400Q mutation in DDX43 impairs interaction between Ago3 DDX43 . IgG h . c . : IgG heavy chain .

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(C) Immunoprecipitation of Ago3 with Flag-tagged DDX43 WT and its D399A and E400Q mutants. Ago3 and DDX43 were detected by western blotting using anti-Ago3 and anti-Flag-antibodies, respectively. Flag-EGFP was used as a negative control. The E400Q mutation in DDX43 impairs the interaction between Ago3 and DDX43. IgG h.c.: IgG heavy chain.


( F ) unwinding assays upon Ago3 - piRISC - dependent target cleavage . Unwinding activities of DDX43 WT its D399A , E400Q , ∆ KH mutants are shown . RNAs by Ago3 are shown as " " .

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(F) RNA unwinding assays upon Ago3-piRISC-dependent target RNA cleavage. Unwinding activities of DDX43 WT and its D399A, E400Q, and ∆KH mutants are shown. RNAs cleaved by Ago3 are shown as "Cleaved RNA".


( A ) Masson ' s trichrome representative of pulmonary fibrosis .

(A) Masson's trichrome representative images of pulmonary fibrosis and quantification.


( B ) Relative transcript expression of Fibronectin , ( C ) Collagen I ( D ) Collagen III to GAPDH .

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(B) Relative transcript expression of Fibronectin, (C) Collagen I and (D) Collagen III to GAPDH.


( E ) Masson ' s trichrome representative of vascular wall thickening .

(E) Masson's trichrome representative images of vascular wall thickening and quantification.


( F ) Masson ' s trichrome representative correlation between lung fibrosis vascular wall thickness in PF PF - PH . Out of 14 PF PF - PH , 3 of them had no non - fibrotic therefore were excluded for comparison between non - fibrotic fibrotic areas ( n = 11 / group ; Ashcroft score non - fibrosis : 0 - 2 ; mild - fibrosis : 3 - 5 ; severe - fibrosis > 5 ) ( * compared to non - fibrotic areas of same group of ) .

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(F) Masson's trichrome representative images and correlation between lung fibrosis and vascular wall thickness in PF and PF-PH patients. Out of the 14 PF and PF-PH patients, 3 of them had no non-fibrotic area and therefore were excluded for comparison between non-fibrotic and fibrotic areas (n=11/group; Ashcroft score non-fibrosis: 0-2; mild-fibrosis: 3-5; severe-fibrosis >5) (* compared to non-fibrotic areas of the same group of patients).


( G ) Masson ' s trichrome representative of vascular wall thickening in non - fibrotic ( Ashcroft score 0 - 2 ) fibrotic ( Ashcroft score > 2 ) in PF PF - PH . number of samples per group for each experiment are written within each bar graph .

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(G) Masson's trichrome representative images of vascular wall thickening in non-fibrotic area (Ashcroft score 0-2) and fibrotic area (Ashcroft score >2) and quantification in PF and PF-PH. The number of samples per group for each experiment are written within each bar graph.


( A ) Representative of Ki67 - positive pulmonary vascular endothelial ( EC , vWF in green ) smooth muscle ( SMC , aSMA in ) ( * vs PF , * p < 0 . 05 , * * * p < 0 . 001 ) .

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(A) Representative images of Ki67-positive pulmonary vascular endothelial cells (EC, vWF in green) and smooth muscle cells (SMC, aSMA in white) and quantification (* vs PF, *p<0.05, ***p<0.001).


Relative expression of Slug mRNA ( B ) normalized to GAPDH in PF PF - PH .

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Relative expression of Slug mRNA (B) normalized to GAPDH in PF and PF-PH patients.


Relative expression of Slug ( C ) normalized to GAPDH in PF PF - PH .

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Relative expression of Slug protein (C) normalized to GAPDH in PF and PF-PH patients.


( D ) Representative of Slug ( red ) expression in bronchial epithelial ( EpC ) , fibroblasts ( Fib ) ( vimentin in green ) macrophages ( Mф , CD68 in green ) pulmonary vascular endothelial ( EC , von Willebrand Factor in green ) pulmonary vascular smooth muscle ( SMC , aSMA in ) ; .

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(D) Representative images of Slug (red) expression in bronchial epithelial cells (EpC), fibroblasts (Fib) (vimentin in green) macrophages (Mф, CD68 in green) pulmonary vascular endothelial cells (EC, von Willebrand Factor in green) and pulmonary vascular smooth muscle cells (SMC, aSMA in white); and quantification.


( E ) Relative CD68 mRNA expression normalized to

(E) Relative CD68 mRNA expression normalized to GAPDH


( F ) Representative of Slug ( red ) expression in macrophages ( Mф , CD68 in green ) in non - fibrotic fibrotic areas of lung in PF PF - PH . number of samples per group for each experiment are included within each bar graph .

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(F) Representative images of Slug (red) expression in macrophages (Mф , CD68 in green) in non-fibrotic and fibrotic areas of the lung in PF and PF-PH patients and quantification. The number of samples per group for each experiment are included within each bar graph.


( A ) of 20 genes up - regulated in GSE24988 microarray known to be Slug transcriptionnal target .

(A) Heatmap of the 20 genes up-regulated in GSE24988 microarray and known to be Slug transcriptionnal target.


( C ) of seven transcriptional targets of Slug known to promote proliferation to be extracellular .

(C) Heatmap of the seven transcriptional targets of Slug known to promote proliferation and to be extracellular.


Relative expression of Prolactin - induced ( PIP ) mRNA ( D ) normalized to GAPDH in PF PF - PH .

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Relative expression of Prolactin-induced protein (PIP) mRNA (D) normalized to GAPDH in PF and PF-PH patients.


Relative expression of Prolactin - induced ( PIP ) expression normalized to GAPDH in PF PF - PH .

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Relative expression of Prolactin-induced protein (PIP) expression normalized to GAPDH in PF and PF-PH patients.


( F ) Representative of PIP immunohistochemistry showing its extracellular localization .

(F) Representative images of PIP immunohistochemistry showing its extracellular localization and quantification.


Proliferation on healthy fibroblast ( G ) Pulmonary arterial EC ( H ) on healthy pulmonary arterial SMC ( I in presence or absence of PIP . number of samples per group for each experiment are included within each bar graph .

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Proliferation assay on healthy fibroblast (G) Pulmonary arterial EC (H) and on healthy pulmonary arterial SMC (I in the presence or absence of PIP protein. The number of samples per group for each experiment are included within each bar graph.


Proliferation on healthy pulmonary arterial SMC J ) in presence or absence of PIP . number of samples per group for each experiment are included within each bar graph .

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Proliferation assay on healthy pulmonary arterial SMC J) in the presence or absence of PIP protein. The number of samples per group for each experiment are included within each bar graph.


( A ) Masson ' s trichrome representative of lung fibrosis two after PBS or bleomycin instillation .

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(A) Masson's trichrome representative images of lung fibrosis and quantification two weeks after PBS or bleomycin instillation.


of ( B ) fibronectin , ( C ) Collagen I ( D ) Collagen III mRNA .

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Quantification of (B) fibronectin, (C) Collagen I and (D) Collagen III mRNA.


( F ) Right ventricular systolic pressure ( RVSP ) ( G ) Right ventricular hypertrophy index following experimental protocol .

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(F) Right ventricular systolic pressure (RVSP) and (G) Right ventricular hypertrophy index following the experimental protocol.


( H ) Masson ' s trichrome representative of Ashcroft score in experimental model .

(H) Masson's trichrome representative images and quantification of Ashcroft score in the experimental model.


Relative expression of fibronectin ( I ) , collagen I ( J ) collagen III ( K ) mRNA normalized to GAPDH .

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Relative expression of fibronectin (I), collagen I (J) and collagen III (K) mRNA normalized to GAPDH.


( L ) Masson ' s trichrome representative of vascular remodeling .

(L) Masson's trichrome representative images and quantification of vascular remodeling.


( M ) Masson ' s trichrome representative of vascular remodeling between fibrotic non - fibrotic areas in experimental model . number of samples per group for each experiment are included within each bar graph .

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(M) Masson's trichrome representative images and quantification of the vascular remodeling between fibrotic and non-fibrotic areas in the experimental model. The number of samples per group for each experiment are included within each bar graph.


( A ) Representative of Ki67 - positive ( red ) pulmonary vascular endothelial ( EC , vWF in green ) smooth muscle ( SMC ) in experimental model .

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(A) Representative images and quantification of Ki67-positive (red) pulmonary vascular endothelial cells (EC, vWF in green) and smooth muscle cells (SMC) in the experimental model.


Relative expression of Slug mRNA ( B ) normalized to GAPDH .

Relative expression of Slug mRNA (B) normalized to GAPDH.


Relative expression of Slug ( C ) normalized to GAPDH .

Relative expression of Slug protein (C) normalized to GAPDH.


( D ) Representative of Slug ( red ) expression in bronchial epithelial ( EpC ) , fibroblasts ( Fib , vimentin in green ) macrophages ( Mф , CD68 in green ) pulmonary vascular endothelial ( EC , vWF ) smooth muscle ( SMC , aSMA in ) ; . ( * vs Bleo , p < 0 . 05 )

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(D) Representative images of Slug (red) expression in bronchial epithelial cells (EpC), fibroblasts (Fib, vimentin in green) macrophages (Mф, CD68 in green) pulmonary vascular endothelial cells (EC, vWF) and smooth muscle cells (SMC, aSMA in white); and quantification. (* vs Bleo, p<0.05)


(E) CD68 mRNA quantification in Bleo and Bleo-MCT rats


( F ) Representative of Slug ( red ) expression in macrophages ( Mф , CD68 in green ) in non - fibrotic fibrotic areas of lung in in Bleo Bleo - MCT rats . number of samples per group for each experiment are included within each bar graph .

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(F) Representative images of Slug (red) expression in macrophages (Mф , CD68 in green) in non-fibrotic and fibrotic areas of the lung in in Bleo and Bleo-MCT rats and quantification. The number of samples per group for each experiment are included within each bar graph.


Relative expression of PIP mRNA ( A ) normalized to GAPDH .

Relative expression of PIP mRNA (A) normalized to GAPDH.


Relative expression of PIP ( B ) normalized to GAPDH .

Relative expression of PIP protein (B) normalized to GAPDH.


( C ) Representative PIP . number of samples per group for each experiment are included within each bar graph .

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(C) Representative images and PIP quantification. The number of samples per group for each experiment are included within each bar graph.


Relative expression of Slug ( B ) normalized to GAPDH .

Relative expression of Slug protein (B) normalized to GAPDH.


Relative expression of Slug mRNA ( C ) normalized to GAPDH .

Relative expression of Slug mRNA (C) normalized to GAPDH.


( D ) right ventricular systolic pressure ( RVSP ) ( E ) right ventricular hypertrophy index in PF - PH rats treated with Si - Slug compared to Si - Scrm .

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(D) right ventricular systolic pressure (RVSP) and (E) right ventricular hypertrophy index in PF-PH rats treated with Si-Slug compared to Si-Scrm.


Relative expression of fibronectin ( F ) , Collagen I ( G ) Collagen III ( H ) normalized to GAPDH .

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Relative expression of fibronectin (F), Collagen I (G) and Collagen III (H) normalized to GAPDH.


( I ) Masson ' s trichrome representative of Ashcroft score . ( J ) Masson ' s trichrome representative of vascular wall thickness .

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(I) Masson's trichrome representative images and quantification of the Ashcroft score. (J) Masson's trichrome representative images and quantification of vascular wall thickness.


( K ) Masson ' s trichrome representative of vascular remodeling between non - fibrotic fibrotic areas of lung .

(K) Masson's trichrome representative images and quantification of the vascular remodeling between non-fibrotic and fibrotic areas of the lung.


( L ) Representative of Ki67 - positive ( red ) pulmonary vascular endothelial ( EC , vWF in green ) smooth muscle ( SMC , aSMA in ) . number of samples per group for each experiment are included within each bar graph .

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(L) Representative images and quantification of Ki67-positive (red) pulmonary vascular endothelial cells (EC, vWF in green) and smooth muscle cells (SMC, aSMA in white). The number of samples per group for each experiment are included within each bar graph.


( A ) Representative of Slug - positive macrophages ( slug , red ; CD68 green ) .

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(A) Representative images and quantification of Slug-positive macrophages (slug, red; CD68 green).


( B ) Relative expression of CD68 mRNA normalized to GAPDH .

(B) Relative expression of CD68 mRNA normalized to GAPDH.


( C ) Representative of Slug - positive macrophages between non - fibrotic fibrotic areas of lung .

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(C) Representative images and quantification of Slug-positive macrophages between non-fibrotic and fibrotic areas of the lung.


Relative expression of PIP mRNA ( D ) normalized to GAPDH .

Relative expression of PIP mRNA (D) normalized to GAPDH.


Relative expression of PIP ( E ) normalized to GAPDH .

Relative expression of PIP protein (E) normalized to GAPDH.


( F ) Representative of PIP immunohistochemistry .

(F) Representative images and quantification of PIP immunohistochemistry.


E ) H2AQ105me H3K56ac in deletions of genes important for H3K56ac

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E) H2AQ105me and H3K56ac levels in deletions of genes important for H3K56ac deposition


G - J ) ChIP - qPCR for indicated antibodies after treatment with 5nM rapamycin for indicated time points . Samples were either normalized to input or to histone . n = 3 , biological replicates , error bars are standard error of mean . * , p < 0 . 05 , relative to t = 0 , on unpaired , 2 - t -

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G-J) ChIP-qPCR for indicated antibodies after treatment with 5nM rapamycin for the indicated time points. Samples were either normalized to input or to the core histone. n=3, biological replicates, error bars are standard error of the mean. *, p<0.05, relative to t=0, based on unpaired, 2-tailed t-test


B ) Volcano plot of quantitative SILAC experiments . Left upper quadrant indicates significantly , 2 - fold enriched bound specifically to H2AQ105me . Members of FACT ( SPT16 / POB3 ) are highlighted in red .

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B) Volcano plot of quantitative SILAC experiments. Left upper quadrant indicates significantly, 2-fold enriched proteins bound specifically to H2AQ105me. Members of the FACT complex (SPT16/POB3) are highlighted in red.


C ) Peptide pulldown using recombinantly purified Nhp2 . Eluted peptides were slot blotted detected using streptavidine - HRP .

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C) Peptide pulldown using recombinantly purified Nhp2. Eluted peptides were slot blotted and detected using streptavidine-HRP.


E ) ChIP - seq profile of Nhp2 ( orange ) its respective input ( black ) across entire chromosome XII of yeast . H2A - normalised H2AQ105me ( grey ) was directly taken as comparison . Magnification shows two annotated rDNA loci in yeast genome indicated by black bars below tracks . Scale bar is for magnification .

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E) ChIP-seq profile of Nhp2 (orange) and its respective input (black) across the entire chromosome XII of yeast. H2A-normalised H2AQ105me (grey) was directly taken as comparison. Magnification shows the two annotated rDNA loci in the yeast genome indicated by the black bars below the tracks. Scale bar is for the magnification.


D ) Proteomic of Nhp2 interactors . Highlighted in red are known members of H / ACA pseudouridylation . red dashed indicates significance

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D) Proteomic analysis of Nhp2 interactors. Highlighted in red are the known members of the H/ACA pseudouridylation complex. The red dashed line indicates significance cutoff


G ) Peptide pulldowns from human ( HEK293T ) to for conservation of reported interactions between Nhp2 members of SSU processome on H2AQ105me . Dyskering ( Cbf5 homolog ) was probed to for presence of members of H / ACA . Pulldown efficiency was analyzed by western blotting using indicated antibodies .

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G) Peptide pulldowns from human cells (HEK293T) to test for conservation of the reported interactions between Nhp2 and members of the SSU processome complex on H2AQ105me. Dyskering (Cbf5 homolog) was probed to test for the presence of members of the H/ACA complex. Pulldown efficiency was analyzed by western blotting using the indicated antibodies.


I ) of Northern Blot results . Normalised to GAPDH , n = 3 , error bars are standard error of mean . Asterix indicates p - value ( * , p < 0 . 05 , on unpaired , 2 - t - ) .

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I) Quantification of Northern Blot results. Normalised to GAPDH, n=3, error bars are standard error of the mean. Asterix indicates p-value (*, p<0.05, based on unpaired, 2-tailed t-test).


E . Normalized ThT kinetics of 10μM Biot - Aβ1 - 42 with timepoints of samples that incubated with Aβ1 - 42 membranes .

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E. Normalized ThT kinetics of 10μM Biot-Aβ1-42 with timepoints of samples that incubated with Aβ1-42 membranes.


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I. ThT kinetics of Biot-Aβ1-42 seeding. 10μM of Biot-Aβ1-42 incubated with 0.5 or 1 μM of Biot-Aβ1-42 seeds.


D . Binding of Biot - Aβ1 - 42 to homologue peptides derived from ~ 520 randomly selected .

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D. Binding of Biot-Aβ1-42 to homologue peptides derived from ~520 randomly selected proteins.


E . Summary of Biot - Aβ1 - 42 binding throughout 8 membranes , color indicates mean between membranes of outline standard deviation .

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E. Summary of Biot-Aβ1-42 binding throughout 8 membranes, color indicates the mean between membranes and the size of the outline the standard deviation.


A - C . ThT kinetics of 10μM rAβ1 - 42 alone or in presence ( 1 : 1 ) of 3 homologue peptides ( full screen on Appendix Figure S3 , S4 , n = 2 independent experiments with 4 repeats ) . Reused in Appendix figure

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A-C. ThT kinetics of 10μM rAβ1-42 alone or in presence (1:1) of 3 homologue peptides (full screen on Appendix Figure S3,S4, n= 2 independent experiments with 4 repeats). Reused images in Appendix figure S3


I . Representative AFM height of Aβ fibrils alone or in a 1 : 1 mixture with P3 , P5 , P8 P12 peptides are show in top row . boxes indicate magnified regions shown in second row . Arrows indicate locations of representative fibrils shown in magnified detail , each shown as a 200 nm digitally straightened segment a 100 nm segment of corresponding 3D surface envelope model that was calculated from image data . scale bar for each row are shown to left , with both 3D model straightened image data representing 10 nm . colour scale of 3D models from blue to yellow indicate ( from low to high ) between fibril surface fibril centre axis to demonstrate their twist patterns . average fibril height distribution of around 80 manually selected filaments per sample that showed twist patterns characteristic of single , not fragmented , amyloid fibrils are shown in bottom row .

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I. Representative AFM height images of Aβ fibrils alone or in a 1:1 mixture with P3, P5, P8 and P12 peptides are show in the top row. The boxes indicate the magnified regions shown in the second row. Arrows indicate the locations of representative individual fibrils shown in magnified detail, each shown as a 200 nm digitally straightened segment and a 100 nm segment of the corresponding 3D surface envelope model that was calculated from the image data. The scale bar for each row are shown to the left, with both the 3D model and the straightened image data representing 10 nm. The colour scale of the 3D models from blue to yellow indicate the distance (from low to high) between the fibril surface and fibril centre axis to demonstrate their twist patterns. The average fibril height distribution of around 80 manually selected filaments per sample that showed twist patterns characteristic of single, not fragmented, amyloid fibrils are shown in the bottom row.


A . Overrepresentation of Aβ APRs in amyloid plaques of AD brains ( statistics : hypergeometric with Bonferroni correction ) * P ≤ 0 . 05 , * * * P ≤ 0 . 001 , * * * * P ≤ 0 . 0001 . original data was taken from Xiong et al included three biological replicates .

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A. Overrepresentation of Aβ APRs in amyloid plaques of AD brains (statistics: hypergeometric test with Bonferroni correction) * P ≤0.05, *** P≤0.001, **** P≤0.0001. The original data was taken from Xiong et al and included three biological replicates.


G . Overrepresentation of Aβ APRs in amyloid plaques of nonAD brains . ( statistics : hypergeometric with Bonferroni correction ) . * P ≤ 0 . 05 , * * P ≤ 0 . 01 original data was taken from Xiong et al included three biological replicates .

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G. Overrepresentation of Aβ APRs in amyloid plaques of nonAD brains.(statistics: hypergeometric test with Bonferroni correction). * P ≤0.05, ** P≤0.01 The original data was taken from Xiong et al and included three biological replicates.